CAZypedia - User contributions [en-ca]

Carbohydrate Binding Module Family 63

2018-05-23T22:00:39Z

Will Chase:

{{UnderConstruction}}
* [[Author]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>

== Ligand specificities ==
In nature, CBM63s are found almost exclusively as the C-terminal domain of two-domain proteins named expansins <cite>Cosgrove2015</cite>. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, possibly a consequence of the large sequence divergence between bacterial and plant expansins, which might hinder automated sequence-based methods used to construct CBM families. However, structural analyses (see below) leave little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

The most detailed characterization of CBM63 is from the founding member found on the C-terminus of the expansin BsEXLX1 produced by the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite> (GenBank: [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). It contains ~100 amino acids with a molecular weight of ~11.5 kDa. Recombinant protein (the entire two-domain protein, without histidine tags) binds cellulose (Avicel) with an affinity (''K''<sub>D</sub>) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose <cite>Georgelis2011</cite>. Virtually identical binding parameters were found for the recombinant CBM63 alone, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay <cite>Georgelis2011</cite>. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain <cite>Georgelis2011</cite>.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (''K''<sub>D</sub>) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall <cite>Georgelis2011</cite>. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides <cite>Georgelis2011</cite>. Site-directed mutagenesis and additional binding experiments <cite>Georgelis2011</cite> show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl<sub>2</sub>, whereas binding to cellulose was reduced only ~40% in CaCl<sub>2</sub> (up to 100 mM) <cite>Georgelis2011</cite>. Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (''K''<sub>D</sub> 4.7 μM; Bmax 3.3 μmol/g) <cite>Georgelis2011</cite>. This binding was eliminated when three of the basic residues listed above were changed to Q <cite>Georgelis2011</cite>.

The above studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and matrix polysaccharides (e.g. arabinoxylan) through distinct physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials <cite>Gilbert2013 Armenta2017 Georgelis2015 McQueen-Mason1992 Shcherban1995</cite> but the contribution of the CBM63 domain was not ascertained (see review in <cite>Cosgrove2017</cite>). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) <cite>McQueen-Mason1995</cite> or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen <cite>Yennawar2006 Wang2016</cite>. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than BsEXLX1 (''K''<sub>D</sub> 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration <cite>Wang2016</cite>. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose <cite>Wang2016</cite>. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding interactions were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose ([{{PDBlink}}4FER 4FER]). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the ligand. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures of CBM63s include two bacterial expansins (BsEXLX1 <cite>Kerff2008</cite>: [{{PDBlink}}3D30 3D30] and CmEXLX1 from ''Clavibacter michiganensis'': [{{PDBlink}}4JJO 4JJO]) and two plant expansins (ZmEXPB1 <cite>Yennawar2006</cite> from ''Zea mays'': [{{PDBlink}}2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'': [{{PDBlink}}1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([{{PDBlink}}1who 1WHO]) and PhlP3 ([{{PDBlink}}3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63 <cite>Li2002 Sampedro2005</cite>, but their binding properties are unknown. The most convincing evidence that the C-terminal domain of plant expansins can be classified as CBM63 comes from structural alignments between the C-terminal of BsEXLX1 (the founding member of CBM63) and the C-terminal domain of the plant expansin ZmEXPB1. A structural superposition of these two domains showed a remarkably similar structure, with only a 1.6 Å RMSD <cite>Kerff2008</cite>.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich <cite>Kerff2008</cite>. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides <cite>Georgelis2012</cite>. Thus, CBM63 is considered a [[Carbohydrate-binding_modules#Types|type A]] CBM [11,12]. Mutagenesis of these residues reduces cellulose binding <cite>Georgelis2011</cite>. In [http://www.cazy.org/CBM63_structure.html crystal complexes with cello-oligosaccharides], BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between two CBM63s from two different protein chains in opposite polarity (Figure 1-A). The aromatic residues of the two CBM63s interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-π interactions <cite>Georgelis2012</cite>.

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) <cite>Yennawar2006</cite>, which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity and computational models suggest they have a similar 8-stranded β-sandwich fold <cite>Gaete-Eastman2015</cite>.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips but the CBM63 domain by itself lacks both of these activities <cite>Georgelis2011</cite>. In a BsEXLX1 mutant with reduced matrix binding, wall loosening increased, whereas a mutant with no binding to cellulose lost wall loosening activity, demonstrating that cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix <cite>Georgelis2011</cite>. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies <cite>Wang2013</cite> which showed that in partially-extracted cell walls from ''Arabidopsis'', BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansins to specific sites, dubbed biomechanical hotspots <cite>Yennawar2006 Wang2016</cite>, where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains <cite>Georgelis2011</cite>, but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in <cite>Georgelis2015</cite>). Certain microbial expansins are fused to other CAZy domains, including CBM1, CBM2, and GH5 (summarized in <cite>Nikolaidis2014</cite>). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases <cite>Artzi2016</cite>. Cellulosomal expansins bound microcrystalline cellulose <cite>Artzi2016</cite>, and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degradation <cite>Artzi2016 Chen2016</cite>, but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
;First Identified: The isolation <cite>McQueen-Mason1992</cite>, characterization <cite>McQueen-Mason1995</cite> and sequencing <cite>Shcherban1995</cite> of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain <cite>Cosgrove1996</cite>. <cite>Georgelis2012</cite>.

;First Structural Characterization: The crystal structures of ZmEXPB1 <cite>Yennawar2006</cite> and BsEXLX1 <cite>Kerff2008</cite> confirmed the identity of the C-terminal domain as a type-A CBM, which was later recognized as a distinct family, dubbed CBM63 based on the C-terminal domain of BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides <cite>Georgelis2012</cite>.

== References ==
<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 pmid=28886679
#Nikolaidis2014 pmid=24150040
#Georgelis2012 pmid=22927418
#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 pmid=11536663
#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 pmid=23769966
#Armenta2017 pmid=28547780
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 pmid=11538167
#Shcherban1995 pmid=7568110
#Artzi2016 pmid=26973715
#Chen2016 pmid=26521249
#Cosgrove1996 pmid=8757932
#Wang2013 pmid=24065828
#Sampedro2005 pmid=16356276
#Li2002 pmid=11891242
#Gaete-Eastman2015 pmid=25863690
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-23T21:59:27Z

Will Chase:

{{UnderConstruction}}
* [[Author]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>

== Ligand specificities ==
In nature, CBM63s are found almost exclusively as the C-terminal domain of two-domain proteins named expansins <cite>Cosgrove2015</cite>. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, possibly a consequence of the large sequence divergence between bacterial and plant expansins, which might hinder automated sequence-based methods used to construct CBM families. However, structural analyses (see below) leave little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

The most detailed characterization of CBM63 is from the founding member found on the C-terminus of the expansin BsEXLX1 produced by the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite> (GenBank: [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). It contains ~100 amino acids with a molecular weight of ~11.5 kDa. Recombinant protein (the entire two-domain protein, without histidine tags) binds cellulose (Avicel) with an affinity (''K''<sub>D</sub>) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose <cite>Georgelis2011</cite>. Virtually identical binding parameters were found for the recombinant CBM63 alone, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay <cite>Georgelis2011</cite>. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain <cite>Georgelis2011</cite>.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (''K''<sub>D</sub>) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall <cite>Georgelis2011</cite>. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides <cite>Georgelis2011</cite>. Site-directed mutagenesis and additional binding experiments <cite>Georgelis2011</cite> show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl<sub>2</sub>, whereas binding to cellulose was reduced only ~40% in CaCl<sub>2</sub> (up to 100 mM) <cite>Georgelis2011</cite>. Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (''K''<sub>D</sub> 4.7 μM; Bmax 3.3 μmol/g) <cite>Georgelis2011</cite>. This binding was eliminated when three of the basic residues listed above were changed to Q <cite>Georgelis2011</cite>.

The above studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and matrix polysaccharides (e.g. arabinoxylan) through distinct physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials <cite>Gilbert2013 Armenta2017 Georgelis2015 McQueen-Mason1992 Shcherban1995</cite> but the contribution of the CBM63 domain was not ascertained (see review in <cite>Cosgrove2017</cite>). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) <cite>McQueen-Mason1995</cite> or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen <cite>Yennawar2006 Wang2016</cite>. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than BsEXLX1 (''K''<sub>D</sub> 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration <cite>Wang2016</cite>. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose <cite>Wang2016</cite>. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding interactions were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose ([{{PDBlink}}4FER 4FER]). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the ligand. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures of CBM63s include two bacterial expansins (BsEXLX1 <cite>Kerff2008</cite>: [{{PDBlink}}3D30 3D30] and CmEXLX1 from ''Clavibacter michiganensis'': [{{PDBlink}}4JJO 4JJO]) and two plant expansins (ZmEXPB1 <cite>Yennawar2006</cite> from ''Zea mays'': [{{PDBlink}}2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'': [{{PDBlink}}1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([{{PDBlink}}1who 1WHO]) and PhlP3 ([{{PDBlink}}3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63 <cite>Li2002 Sampedro2005</cite>, but their binding properties are unknown. The most convincing evidence that domain 2 of plant expansins can be classified as CBM63 comes from structural alignments between domain 2 of BsEXLX1 (the founding member of CBM63) and domain 2 of the plant expansin ZmEXPB1. A structural superposition of these two domains showed a remarkably similar structure, with only a 1.6 Å RMSD <cite>Kerff2008</cite>.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich <cite>Kerff2008</cite>. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides <cite>Georgelis2012</cite>. Thus, CBM63 is considered a [[Carbohydrate-binding_modules#Types|type A]] CBM [11,12]. Mutagenesis of these residues reduces cellulose binding <cite>Georgelis2011</cite>. In [http://www.cazy.org/CBM63_structure.html crystal complexes with cello-oligosaccharides], BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between two CBM63s from two different protein chains in opposite polarity (Figure 1-A). The aromatic residues of the two CBM63s interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-π interactions <cite>Georgelis2012</cite>.

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) <cite>Yennawar2006</cite>, which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity and computational models suggest they have a similar 8-stranded β-sandwich fold <cite>Gaete-Eastman2015</cite>.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips but the CBM63 domain by itself lacks both of these activities <cite>Georgelis2011</cite>. In a BsEXLX1 mutant with reduced matrix binding, wall loosening increased, whereas a mutant with no binding to cellulose lost wall loosening activity, demonstrating that cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix <cite>Georgelis2011</cite>. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies <cite>Wang2013</cite> which showed that in partially-extracted cell walls from ''Arabidopsis'', BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansins to specific sites, dubbed biomechanical hotspots <cite>Yennawar2006 Wang2016</cite>, where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains <cite>Georgelis2011</cite>, but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in <cite>Georgelis2015</cite>). Certain microbial expansins are fused to other CAZy domains, including CBM1, CBM2, and GH5 (summarized in <cite>Nikolaidis2014</cite>). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases <cite>Artzi2016</cite>. Cellulosomal expansins bound microcrystalline cellulose <cite>Artzi2016</cite>, and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degradation <cite>Artzi2016 Chen2016</cite>, but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
;First Identified: The isolation <cite>McQueen-Mason1992</cite>, characterization <cite>McQueen-Mason1995</cite> and sequencing <cite>Shcherban1995</cite> of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain <cite>Cosgrove1996</cite>. <cite>Georgelis2012</cite>.

;First Structural Characterization: The crystal structures of ZmEXPB1 <cite>Yennawar2006</cite> and BsEXLX1 <cite>Kerff2008</cite> confirmed the identity of the C-terminal domain as a type-A CBM, which was later recognized as a distinct family, dubbed CBM63 based on the C-terminal domain of BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides <cite>Georgelis2012</cite>.

== References ==
<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 pmid=28886679
#Nikolaidis2014 pmid=24150040
#Georgelis2012 pmid=22927418
#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 pmid=11536663
#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 pmid=23769966
#Armenta2017 pmid=28547780
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 pmid=11538167
#Shcherban1995 pmid=7568110
#Artzi2016 pmid=26973715
#Chen2016 pmid=26521249
#Cosgrove1996 pmid=8757932
#Wang2013 pmid=24065828
#Sampedro2005 pmid=16356276
#Li2002 pmid=11891242
#Gaete-Eastman2015 pmid=25863690
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-23T21:48:10Z

Will Chase:

{{UnderConstruction}}
* [[Author]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>

== Ligand specificities ==
In nature, CBM63s are found almost exclusively as the C-terminal domain of two-domain proteins named expansins <cite>Cosgrove2015</cite>. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, possibly a consequence of the large sequence divergence between bacterial and plant expansins, which might hinder automated sequence-based methods used to construct CBM families. However, structural analyses (see below) leave little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

The most detailed characterization of CBM63 is from the founding member found on the C-terminus of the expansin BsEXLX1 produced by the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite> (GenBank: [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). It contains ~100 amino acids with a molecular weight of ~11.5 kDa. Recombinant protein (the entire two-domain protein, without histidine tags) binds cellulose (Avicel) with an affinity (''K''<sub>D</sub>) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose <cite>Georgelis2011</cite>. Virtually identical binding parameters were found for the recombinant CBM63 alone, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay <cite>Georgelis2011</cite>. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain <cite>Georgelis2011</cite>.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (''K''<sub>D</sub>) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall <cite>Georgelis2011</cite>. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides <cite>Georgelis2011</cite>. Site-directed mutagenesis and additional binding experiments <cite>Georgelis2011</cite> show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl<sub>2</sub>, whereas binding to cellulose was reduced only ~40% in CaCl<sub>2</sub> (up to 100 mM) <cite>Georgelis2011</cite>. Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (''K''<sub>D</sub> 4.7 μM; Bmax 3.3 μmol/g) <cite>Georgelis2011</cite>. This binding was eliminated when three of the basic residues listed above were changed to Q <cite>Georgelis2011</cite>.

The above studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and matrix polysaccharides (e.g. arabinoxylan) through distinct physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials <cite>Gilbert2013 Armenta2017 Georgelis2015 McQueen-Mason1992 Shcherban1995</cite> but the contribution of the CBM63 domain was not ascertained (see review in <cite>Cosgrove2017</cite>). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) <cite>McQueen-Mason1995</cite> or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen <cite>Yennawar2006 Wang2016</cite>. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than BsEXLX1 (''K''<sub>D</sub> 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration <cite>Wang2016</cite>. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose <cite>Wang2016</cite>. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding interactions were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose ([{{PDBlink}}4FER 4FER]). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the ligand. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures of CBM63s include two bacterial expansins (BsEXLX1 <cite>Kerff2008</cite>: [{{PDBlink}}3D30 3D30] and CmEXLX1 from ''Clavibacter michiganensis'': [{{PDBlink}}4JJO 4JJO]) and two plant expansins (ZmEXPB1 <cite>Yennawar2006</cite> from ''Zea mays'': [{{PDBlink}}2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'': [{{PDBlink}}1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([{{PDBlink}}1who 1WHO]) and PhlP3 ([{{PDBlink}}3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63 <cite>Li2002 Sampedro2005</cite>, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich <cite>Kerff2008</cite>. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides <cite>Georgelis2012</cite>. Thus, CBM63 is considered a [[Carbohydrate-binding_modules#Types|type A]] CBM [11,12]. Mutagenesis of these residues reduces cellulose binding <cite>Georgelis2011</cite>. In [http://www.cazy.org/CBM63_structure.html crystal complexes with cello-oligosaccharides], BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between two CBM63s from two different protein chains in opposite polarity (Figure 1-A). The aromatic residues of the two CBM63s interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-π interactions <cite>Georgelis2012</cite>.

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) <cite>Yennawar2006</cite>, which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity and computational models suggest they have a similar 8-stranded β-sandwich fold <cite>Gaete-Eastman2015</cite>.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips but the CBM63 domain by itself lacks both of these activities <cite>Georgelis2011</cite>. In a BsEXLX1 mutant with reduced matrix binding, wall loosening increased, whereas a mutant with no binding to cellulose lost wall loosening activity, demonstrating that cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix <cite>Georgelis2011</cite>. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies <cite>Wang2013</cite> which showed that in partially-extracted cell walls from ''Arabidopsis'', BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansins to specific sites, dubbed biomechanical hotspots <cite>Yennawar2006 Wang2016</cite>, where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains <cite>Georgelis2011</cite>, but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in <cite>Georgelis2015</cite>). Certain microbial expansins are fused to other CAZy domains, including CBM1, CBM2, and GH5 (summarized in <cite>Nikolaidis2014</cite>). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases <cite>Artzi2016</cite>. Cellulosomal expansins bound microcrystalline cellulose <cite>Artzi2016</cite>, and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degradation <cite>Artzi2016 Chen2016</cite>, but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
;First Identified: The isolation <cite>McQueen-Mason1992</cite>, characterization <cite>McQueen-Mason1995</cite> and sequencing <cite>Shcherban1995</cite> of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain <cite>Cosgrove1996</cite>. <cite>Georgelis2012</cite>.

;First Structural Characterization: The crystal structures of ZmEXPB1 <cite>Yennawar2006</cite> and BsEXLX1 <cite>Kerff2008</cite> confirmed the identity of the C-terminal domain as a type-A CBM, which was later recognized as a distinct family, dubbed CBM63 based on the C-terminal domain of BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides <cite>Georgelis2012</cite>.

== References ==
<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 pmid=28886679
#Nikolaidis2014 pmid=24150040
#Georgelis2012 pmid=22927418
#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 pmid=11536663
#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 pmid=23769966
#Armenta2017 pmid=28547780
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 pmid=11538167
#Shcherban1995 pmid=7568110
#Artzi2016 pmid=26973715
#Chen2016 pmid=26521249
#Cosgrove1996 pmid=8757932
#Wang2013 pmid=24065828
#Sampedro2005 pmid=16356276
#Li2002 pmid=11891242
#Gaete-Eastman2015 pmid=25863690
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-07T19:55:17Z

Will Chase:

{{UnderConstruction}}
* [[Author]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==
In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank: [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (K<sub>D</sub>) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose <cite>Georgelis2011</cite>. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (K<sub>D</sub>) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall <cite>Georgelis2011</cite>. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments <cite>Georgelis2011</cite> show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl<sub>2</sub>, whereas binding to cellulose was reduced only ~40% in CaCl<sub>2</sub> (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (K<sub>D</sub> 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials <cite>Gilbert2013 Armenta2017 Georgelis2015 McQueen-Mason1992 Shcherban1995</cite> but the contribution of the CBM63 domain was not ascertained (see review in <cite>Cosgrove2017</cite>). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) <cite>McQueen-Mason1995</cite> or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen <cite>Yennawar2006 Wang2016</cite>. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (K<sub>D</sub> 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration <cite>Wang2016</cite>. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose <cite>Wang2016</cite>. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose ([{{PDBlink}}4FER 4FER]). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [{{PDBlink}}3D30 3D30] and CmEXLX1 from ''Clavibacter michiganensis'': [{{PDBlink}}4JJO 4JJO]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [{{PDBlink}}2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'': [{{PDBlink}}1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([{{PDBlink}}1who 1WHO]) and PhlP3 ([{{PDBlink}}3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich <cite>Kerff2008</cite>. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides <cite>Georgelis2012</cite>. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding <cite>Georgelis2011</cite>. In [http://www.cazy.org/CBM63_structure.html crystal complexes with cello-oligosaccharides], BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-π interactions <cite>Georgelis2012</cite>.

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) <cite>Yennawar2006</cite>, which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities <cite>Georgelis2011</cite>. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix <cite>Georgelis2011</cite>. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies <cite>Wang2013</cite> which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots <cite>Yennawar2006 Wang2016</cite>, where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains <cite>Georgelis2011</cite>, but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in <cite>Georgelis2015</cite>). Certain microbial expansins are fused to other CAZY domains, including CBM1, CBM2, and GH5 (summarized in <cite>Nikolaidis2014</cite>). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases <cite>Artzi2016</cite>. Cellulosomal expansins bound microcrystalline cellulose <cite>Artzi2016</cite>, and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degradation <cite>Artzi2016 Chen2016</cite>, but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation <cite>McQueen-Mason1992</cite>, characterization <cite>McQueen-Mason1995</cite> and sequencing <cite>Shcherban1995</cite> of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain <cite>Cosgrove1996</cite>. This concept was substantiated by the crystal structures of ZmEXPB1 <cite>Yennawar2006</cite> and BsEXLX1 <cite>Kerff2008</cite>, followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain <cite>Georgelis2011</cite>. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides <cite>Georgelis2012</cite>.

== References ==
<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 pmid=28886679
#Nikolaidis2014 pmid=24150040
#Georgelis2012 pmid=22927418
#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 pmid=11536663
#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 pmid=23769966
#Armenta2017 pmid=28547780
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 pmid=11538167
#Shcherban1995 pmid=7568110
#Artzi2016 pmid=26973715
#Chen2016 pmid=26521249
#Cosgrove1996 pmid=8757932
#Wang2013 pmid=24065828
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-07T16:42:08Z

Will Chase:

{{UnderConstruction}}
* [[Author]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==
In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank: [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (K<sub>D</sub>) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose <cite>Georgelis2011</cite>. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (K<sub>D</sub>) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall <cite>Georgelis2011</cite>. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments <cite>Georgelis2011</cite> show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl<sub>2</sub>, whereas binding to cellulose was reduced only ~40% in CaCl<sub>2</sub> (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (K<sub>D</sub> 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials <cite>Gilbert2013 Armenta2017 Georgelis2015 McQueen-Mason1992 Shcherban1995</cite> but the contribution of the CBM63 domain was not ascertained (see review in <cite>Cosgrove2017</cite>). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) <cite>McQueen-Mason1995</cite> or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen <cite>Yennawar2006 Wang2016</cite>. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (K<sub>D</sub> 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration <cite>Wang2016</cite>. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose <cite>Wang2016</cite>. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose ([{{PDBlink}}4FER 4FER]). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [{{PDBlink}}3D30 3D30] and CmEXLX1 from ''Clavibacter michiganensis'': [{{PDBlink}}4JJO 4JJO]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [{{PDBlink}}2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[{{PDBlink}}1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([{{PDBlink}}1who 1WHO]) and PhlP3 ([{{PDBlink}}3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich <cite>Kerff2008</cite>. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides <cite>Georgelis2012</cite>. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding <cite>Georgelis2011</cite>. In crystal complexes with cello-oligosaccharides ([http://www.cazy.org/CBM63_structure.html http://www.cazy.org/CBM63_structure.html]), BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-π interactions <cite>Georgelis2012</cite>.

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) <cite>Yennawar2006</cite>, which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities <cite>Georgelis2011</cite>. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix <cite>Georgelis2011</cite>. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies <cite>Wang2013</cite> which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots <cite>Yennawar2006 Wang2016</cite>, where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains <cite>Georgelis2011</cite>, but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in <cite>Georgelis2015</cite>). Certain microbial expansins are fused to other CAZY domains, including CBM1, CBM2, and GH5 (summarized in <cite>Nikolaidis2014</cite>). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases <cite>Artzi2016</cite>. Cellulosomal expansins bound microcrystalline cellulose <cite>Artzi2016</cite>, and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degradation <cite>Artzi2016 Chen2016</cite>, but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation <cite>McQueen-Mason1992</cite>, characterization <cite>McQueen-Mason1995</cite> and sequencing <cite>Shcherban1995</cite> of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain <cite>Cosgrove1996</cite>. This concept was substantiated by the crystal structures of ZmEXPB1 <cite>Yennawar2006</cite> and BsEXLX1 <cite>Kerff2008</cite>, followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain <cite>Georgelis2011</cite>. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides <cite>Georgelis2012</cite>.

== References ==
<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 pmid=28886679
#Nikolaidis2014 pmid=24150040
#Georgelis2012 pmid=22927418
#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 pmid=11536663
#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 pmid=23769966
#Armenta2017 pmid=28547780
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 pmid=11538167
#Shcherban1995 pmid=7568110
#Artzi2016 pmid=26973715
#Chen2016 pmid=26521249
#Cosgrove1996 pmid=8757932
#Wang2013 pmid=24065828
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-07T16:33:28Z

Will Chase:

{{UnderConstruction}}
* [[Author]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==
In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank: [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (K<sub>D</sub>) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose <cite>Georgelis2011</cite>. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (K<sub>D</sub>) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall <cite>Georgelis2011</cite>. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments <cite>Georgelis2011</cite> show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl<sub>2</sub>, whereas binding to cellulose was reduced only ~40% in CaCl<sub>2</sub> (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (K<sub>D</sub> 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials <cite>Gilbert2013 Armenta2017 Georgelis2015 McQueen-Mason1992 Shcherban1995</cite> but the contribution of the CBM63 domain was not ascertained (see review in <cite>Cosgrove2017</cite>). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) <cite>McQueen-Mason1995</cite> or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen <cite>Yennawar2006 Wang2016</cite>. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (K<sub>D</sub> 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration <cite>Wang2016</cite>. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose <cite>Wang2016</cite>. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose ([{{PDBlink}}4FER 4FER]). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [{{PDBlink}}3D30 3D30] and CmEXLX1 from ''Clavibacter michiganensis'': [{{PDBlink}}4JJO 4JJO]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [{{PDBlink}}2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[{{PDBlink}}1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([{{PDBlink}}1who 1WHO]) and PhlP3 ([{{PDBlink}}3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich <cite>Kerff2008</cite>. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides <cite>Georgelis2012</cite>. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding <cite>Georgelis2011</cite>. In crystal complexes with cello-oligosaccharides ([http://www.cazy.org/CBM63_structure.html http://www.cazy.org/CBM63_structure.html]), BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions <cite>Georgelis2012</cite>.

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) <cite>Yennawar2006</cite>, which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities <cite>Georgelis2011</cite>. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix <cite>Georgelis2011</cite>. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies <cite>Wang2013</cite> which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots <cite>Yennawar2006 Wang2016</cite>, where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains <cite>Georgelis2011</cite>, but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in <cite>Georgelis2015</cite>). Certain microbial expansins are fused to other CAZY domains, including CBM1, CBM2, and GH5 (summarized in <cite>Nikolaidis2014</cite>). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases <cite>Artzi2016</cite>. Cellulosomal expansins bound microcrystalline cellulose <cite>Artzi2016</cite>, and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degradation <cite>Artzi2016 Chen2016</cite>, but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation <cite>McQueen-Mason1992</cite>, characterization <cite>McQueen-Mason1995</cite> and sequencing <cite>Shcherban1995</cite> of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain <cite>Cosgrove1996</cite>. This concept was substantiated by the crystal structures of ZmEXPB1 <cite>Yennawar2006</cite> and BsEXLX1 <cite>Kerff2008</cite>, followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain <cite>Georgelis2011</cite>. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides <cite>Georgelis2012</cite>.

== References ==
<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 pmid=28886679
#Nikolaidis2014 pmid=24150040
#Georgelis2012 pmid=22927418
#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 pmid=11536663
#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 pmid=23769966
#Armenta2017 pmid=28547780
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 pmid=11538167
#Shcherban1995 pmid=7568110
#Artzi2016 pmid=26973715
#Chen2016 pmid=26521249
#Cosgrove1996 pmid=8757932
#Wang2013 pmid=24065828
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-07T16:32:09Z

Will Chase:

{{UnderConstruction}}
* [[Author]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==
In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank: [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (K<sub>D</sub>) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose <cite>Georgelis2011</cite>. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (K<sub>D</sub>) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall <cite>Georgelis2011</cite>. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments <cite>Georgelis2011</cite> show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl<sub>2</sub>, whereas binding to cellulose was reduced only ~40% in CaCl<sub>2</sub> (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (K<sub>D</sub> 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials <cite>Gilbert2013 Armenta2017 Georgelis2015 McQueen-Mason1992 Shcherban1995</cite> but the contribution of the CBM63 domain was not ascertained (see review in <cite>Cosgrove2017</cite>). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) <cite>McQueen-Mason1995</cite> or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen <cite>Yennawar2006 Wang2016</cite>. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (K<sub>D</sub> 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration <cite>Wang2016</cite>. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose <cite>Wang2016</cite>. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose ([{{PDBlink}}4FER 4FER]). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [{{PDBlink}}3D30 3D30] and CmEXLX1 from ''Clavibacter michiganensis'': [{{PDBlink}}4JJO 4JJO]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [{{PDBlink}}2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[{{PDBlink}}1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([{{PDBlink}}1who 1WHO]) and PhlP3 ([{{PDBlink}}3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich <cite>Kerff2008</cite>. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides <cite>Georgelis2012</cite>. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding <cite>Georgelis2011</cite>. In crystal complexes with cello-oligosaccharides (see [http://www.cazy.org/CBM63_structure.html http://www.cazy.org/CBM63_structure.html]), BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions <cite>Georgelis2012</cite>.

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) <cite>Yennawar2006</cite>, which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities <cite>Georgelis2011</cite>. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix <cite>Georgelis2011</cite>. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies <cite>Wang2013</cite> which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots <cite>Yennawar2006 Wang2016</cite>, where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains <cite>Georgelis2011</cite>, but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in <cite>Georgelis2015</cite>). Certain microbial expansins are fused to other CAZY domains, including CBM1, CBM2, and GH5 (summarized in <cite>Nikolaidis2014</cite>). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases <cite>Artzi2016</cite>. Cellulosomal expansins bound microcrystalline cellulose <cite>Artzi2016</cite>, and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degradation <cite>Artzi2016 Chen2016</cite>, but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation <cite>McQueen-Mason1992</cite>, characterization <cite>McQueen-Mason1995</cite> and sequencing <cite>Shcherban1995</cite> of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain <cite>Cosgrove1996</cite>. This concept was substantiated by the crystal structures of ZmEXPB1 <cite>Yennawar2006</cite> and BsEXLX1 <cite>Kerff2008</cite>, followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain <cite>Georgelis2011</cite>. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides <cite>Georgelis2012</cite>.

== References ==
<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 pmid=28886679
#Nikolaidis2014 pmid=24150040
#Georgelis2012 pmid=22927418
#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 pmid=11536663
#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 pmid=23769966
#Armenta2017 pmid=28547780
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 pmid=11538167
#Shcherban1995 pmid=7568110
#Artzi2016 pmid=26973715
#Chen2016 pmid=26521249
#Cosgrove1996 pmid=8757932
#Wang2013 pmid=24065828
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T20:02:05Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==
In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (K<sub>D</sub>) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose <cite>Georgelis2011</cite>. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (K<sub>D</sub>) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall <cite>Georgelis2011</cite>. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments <cite>Georgelis2011</cite> show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl<sub>2</sub>, whereas binding to cellulose was reduced only ~40% in CaCl<sub>2</sub> (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (K<sub>D</sub> 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials <cite>Gilbert2013 Armenta2017 Georgelis2015 McQueen-Mason1992 Shcherban1995</cite> but the contribution of the CBM63 domain was not ascertained (see review in <cite>Cosgrove2017</cite>). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) <cite>McQueen-Mason1995</cite> or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen <cite>Yennawar2006 Wang2016</cite>. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (K<sub>D</sub> 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration <cite>Wang2016</cite>. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose <cite>Wang2016</cite>. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich <cite>Kerff2008</cite>. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides <cite>Georgelis2012</cite>. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding <cite>Georgelis2011</cite>. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions <cite>Georgelis2012</cite>.

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) <cite>Yennawar2006</cite>, which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities <cite>Georgelis2011</cite>. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix <cite>Georgelis2011</cite>. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies <cite>Wang2013</cite> which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots <cite>Yennawar2006 Wang2016</cite>, where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains <cite>Georgelis2011</cite>, but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in <cite>Georgelis2015</cite>). Certain microbial expansins are fused to other CAZY domains, including CBM1, CBM2, and GH5 (summarized in <cite>Nikolaidis2014</cite>). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases <cite>Artzi2016</cite>. These cellulosomal expansins bound microcrystalline cellulose <cite>Artzi2016</cite>, and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degradation <cite>Artzi2016 Chen2016</cite>, but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation <cite>McQueen-Mason1992</cite>, characterization <cite>McQueen-Mason1995</cite> and sequencing <cite>Shcherban1995</cite> of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain <cite>Cosgrove1996</cite>. This concept was substantiated by the crystal structures of ZmEXPB1 <cite>Yennawar2006</cite> and BsEXLX1 <cite>Kerff2008</cite>, followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain <cite>Georgelis2011</cite>. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides <cite>Georgelis2012</cite>.

== References ==
<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 Cosgrove, D.J., 2017. Microbial Expansins. Annual review of microbiology, 71, pp.479-497. [https://doi.org/10.1146/annurev-micro-090816-093315 DOI: 10.1146/annurev-micro-090816-093315]
#Nikolaidis2014 Nikolaidis, N., Doran, N. and Cosgrove, D.J., 2013. Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Molecular biology and evolution, 31(2), pp.376-386. [https://doi.org/10.1093/molbev/mst206 DOI: 10.1093/molbev/mst206]
#Georgelis2012 pmid=22927418

#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 McQueen-Mason, S.J. and Cosgrove, D.J., 1995. Expansin mode of action on cell walls (analysis of wall hydrolysis, stress relaxation, and binding). Plant Physiology, 107(1), pp.87-100. [https://doi.org/10.1104/pp.107.1.87 DOI: 10.1104/pp.107.1.87]

#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 Gilbert, H.J., Knox, J.P. and Boraston, A.B., 2013. Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Current opinion in structural biology, 23(5), pp.669-677. [https://doi.org/10.1016/j.sbi.2013.05.005 DOI: 10.1016/j.sbi.2013.05.005]

#Armenta2017 Armenta, S., Moreno‐Mendieta, S., Sánchez‐Cuapio, Z., Sánchez, S. and Rodríguez‐Sanoja, R., 2017. Advances in Molecular Engineering of Carbohydrate‐Binding Modules. Proteins: Structure, Function, and Bioinformatics. [https://doi.org/10.1002/prot.25327 DOI: 10.1002/prot.25327]
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 McQueen-Mason, S., Durachko, D.M. and Cosgrove, D.J., 1992. Two endogenous proteins that induce cell wall extension in plants. The Plant Cell, 4(11), pp.1425-1433. [https://doi.org/10.1105/tpc.4.11.1425 DOI: 10.1105/tpc.4.11.1425]

#Shcherban1995 Shcherban, T.Y., Shi, J., Durachko, D.M., Guiltinan, M.J., McQueen-Mason, S.J., Shieh, M. and Cosgrove, D.J., 1995. Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants. Proceedings of the National Academy of Sciences, 92(20), pp.9245-9249. [https://doi.org/10.1073/pnas.92.20.9245 DOI: 10.1073/pnas.92.20.9245]
#Artzi2016 pmid=26973715
#Chen2016 Chen, C., Cui, Z., Song, X., Liu, Y.J., Cui, Q. and Feng, Y., 2016. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation. Applied microbiology and biotechnology, 100(5), pp.2203-2212. [https://doi.org/10.1007/s00253-015-7071-6 DOI: 10.1007/s00253-015-7071-6]
#Cosgrove1996 Cosgrove, D.J., 1996. Plant cell enlargement and the action of expansins. BioEssays, 18(7), pp.533-540 [https://doi.org/10.1002/bies.950180704 DOI: 10.1002/bies.950180704].
#Wang2013 Wang, T., Park, Y.B., Caporini, M.A., Rosay, M., Zhong, L., Cosgrove, D.J. and Hong, M., 2013. Sensitivity-enhanced solid-state NMR detection of expansin’s target in plant cell walls. Proceedings of the National Academy of Sciences, 110(41), pp.16444-16449. [https://doi.org/10.1073/pnas.1316290110 DOI: 10.1073/pnas.1316290110]
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T19:03:57Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==
In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (KD) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose <cite>Georgelis2011</cite>. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (KD) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall <cite>Georgelis2011</cite>. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments <cite>Georgelis2011</cite> show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl2, whereas binding to cellulose was reduced only ~40% in CaCl2 (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (KD 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials <cite>Gilbert2013 Armenta2017 Georgelis2015 McQueen-Mason1992 Shcherban1995</cite> but the contribution of the CBM63 domain was not ascertained (see review in <cite>Cosgrove2017</cite>). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) <cite>McQueen-Mason1995</cite> or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen <cite>Yennawar2006 Wang2016</cite>. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (KD 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration <cite>Wang2016</cite>. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose <cite>Wang2016</cite>. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich <cite>Kerff2008</cite>. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides <cite>Georgelis2012</cite>. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding <cite>Georgelis2011</cite>. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions <cite>Georgelis2012</cite>.

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) <cite>Yennawar2006</cite>, which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities <cite>Georgelis2011</cite>. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix <cite>Georgelis2011</cite>. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies <cite>Wang2013</cite> which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots <cite>Yennawar2006 Wang2016</cite>, where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains <cite>Georgelis2011</cite>, but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in <cite>Georgelis2015</cite>). Certain microbial expansins are fused to other CAZY domains, including CBMI, CBMII, and GH5 (summarized in <cite>Nikolaidis2014</cite>). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases <cite>Artzi2016</cite>. These cellulosomal expansins bound microcrystalline cellulose <cite>Artzi2016</cite>, and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degredation <cite>Artzi2016 Chen2016</cite>, but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation <cite>McQueen-Mason1992</cite>, characterization <cite>McQueen-Mason1995</cite> and sequencing <cite>Shcherban1995</cite> of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain <cite>Cosgrove1996</cite>. This concept was substantiated by the crystal structures of ZmEXPB1 <cite>Yennawar2006</cite> and BsEXLX1 <cite>Kerff2008</cite>, followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain <cite>Georgelis2011</cite>. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides <cite>Georgelis2012</cite>.

== References ==
<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 Cosgrove, D.J., 2017. Microbial Expansins. Annual review of microbiology, 71, pp.479-497. [https://doi.org/10.1146/annurev-micro-090816-093315 DOI: 10.1146/annurev-micro-090816-093315]
#Nikolaidis2014 Nikolaidis, N., Doran, N. and Cosgrove, D.J., 2013. Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Molecular biology and evolution, 31(2), pp.376-386. [https://doi.org/10.1093/molbev/mst206 DOI: 10.1093/molbev/mst206]
#Georgelis2012 pmid=22927418

#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 McQueen-Mason, S.J. and Cosgrove, D.J., 1995. Expansin mode of action on cell walls (analysis of wall hydrolysis, stress relaxation, and binding). Plant Physiology, 107(1), pp.87-100. [https://doi.org/10.1104/pp.107.1.87 DOI: 10.1104/pp.107.1.87]

#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 Gilbert, H.J., Knox, J.P. and Boraston, A.B., 2013. Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Current opinion in structural biology, 23(5), pp.669-677. [https://doi.org/10.1016/j.sbi.2013.05.005 DOI: 10.1016/j.sbi.2013.05.005]

#Armenta2017 Armenta, S., Moreno‐Mendieta, S., Sánchez‐Cuapio, Z., Sánchez, S. and Rodríguez‐Sanoja, R., 2017. Advances in Molecular Engineering of Carbohydrate‐Binding Modules. Proteins: Structure, Function, and Bioinformatics. [https://doi.org/10.1002/prot.25327 DOI: 10.1002/prot.25327]
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 McQueen-Mason, S., Durachko, D.M. and Cosgrove, D.J., 1992. Two endogenous proteins that induce cell wall extension in plants. The Plant Cell, 4(11), pp.1425-1433. [https://doi.org/10.1105/tpc.4.11.1425 DOI: 10.1105/tpc.4.11.1425]

#Shcherban1995 Shcherban, T.Y., Shi, J., Durachko, D.M., Guiltinan, M.J., McQueen-Mason, S.J., Shieh, M. and Cosgrove, D.J., 1995. Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants. Proceedings of the National Academy of Sciences, 92(20), pp.9245-9249. [https://doi.org/10.1073/pnas.92.20.9245 DOI: 10.1073/pnas.92.20.9245]
#Artzi2016 pmid=26973715
#Chen2016 Chen, C., Cui, Z., Song, X., Liu, Y.J., Cui, Q. and Feng, Y., 2016. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation. Applied microbiology and biotechnology, 100(5), pp.2203-2212. [https://doi.org/10.1007/s00253-015-7071-6 DOI: 10.1007/s00253-015-7071-6]
#Cosgrove1996 Cosgrove, D.J., 1996. Plant cell enlargement and the action of expansins. BioEssays, 18(7), pp.533-540 [https://doi.org/10.1002/bies.950180704 DOI: 10.1002/bies.950180704].
#Wang2013 Wang, T., Park, Y.B., Caporini, M.A., Rosay, M., Zhong, L., Cosgrove, D.J. and Hong, M., 2013. Sensitivity-enhanced solid-state NMR detection of expansin’s target in plant cell walls. Proceedings of the National Academy of Sciences, 110(41), pp.16444-16449. [https://doi.org/10.1073/pnas.1316290110 DOI: 10.1073/pnas.1316290110]
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T18:50:17Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==

***TO DO: ADD REFS, ADD LINKS TO PDB, FINAL EDITS***

In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification <cite>Cosgrove2016 Cosgrove2015</cite>. They are also present in diverse microbes, including many bacterial and fungal plant pathogens <cite>Cosgrove2017</cite>. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past <cite>Nikolaidis2014</cite>. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' <cite>Georgelis2012</cite>. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins <cite>Kerff2008</cite>, including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (KD) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose [7]. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (KD) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl2, whereas binding to cellulose was reduced only ~40% in CaCl2 (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (KD 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials [11-15] but the contribution of the CBM63 domain was not ascertained (see review in [3]). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) [8] or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen [9,10]. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (KD 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration [10]. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose [10]. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich [6]. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides [5]. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding [7]. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions [5].

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) [9], which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities [7]. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix [7]. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies [8] which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots [9,10], where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains [7], but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in [13]). Certain microbial expansins are fused to other CAZY domains, including CBMI, CBMII, and GH5 (summarized in [4]). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases [16]. These cellulosomal expansins bound microcrystalline cellulose [16], and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degredation [16, 17], but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation [14], characterization [8] and sequencing [15] of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain [18]. This concept was substantiated by the crystal structures of ZmEXPB1 [9] and BsEXLX1 [6], followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain [7]. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides [5].

== References ==
TO DO

<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 Cosgrove, D.J., 2017. Microbial Expansins. Annual review of microbiology, 71, pp.479-497. [https://doi.org/10.1146/annurev-micro-090816-093315 DOI: 10.1146/annurev-micro-090816-093315]
#Nikolaidis2014 Nikolaidis, N., Doran, N. and Cosgrove, D.J., 2013. Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Molecular biology and evolution, 31(2), pp.376-386. [https://doi.org/10.1093/molbev/mst206 DOI: 10.1093/molbev/mst206]
#Georgelis2012 pmid=22927418

#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 McQueen-Mason, S.J. and Cosgrove, D.J., 1995. Expansin mode of action on cell walls (analysis of wall hydrolysis, stress relaxation, and binding). Plant Physiology, 107(1), pp.87-100. [https://doi.org/10.1104/pp.107.1.87 DOI: 10.1104/pp.107.1.87]

#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 Gilbert, H.J., Knox, J.P. and Boraston, A.B., 2013. Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Current opinion in structural biology, 23(5), pp.669-677. [https://doi.org/10.1016/j.sbi.2013.05.005 DOI: 10.1016/j.sbi.2013.05.005]

#Armenta2017 Armenta, S., Moreno‐Mendieta, S., Sánchez‐Cuapio, Z., Sánchez, S. and Rodríguez‐Sanoja, R., 2017. Advances in Molecular Engineering of Carbohydrate‐Binding Modules. Proteins: Structure, Function, and Bioinformatics. [https://doi.org/10.1002/prot.25327 DOI: 10.1002/prot.25327]
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 McQueen-Mason, S., Durachko, D.M. and Cosgrove, D.J., 1992. Two endogenous proteins that induce cell wall extension in plants. The Plant Cell, 4(11), pp.1425-1433. [https://doi.org/10.1105/tpc.4.11.1425 DOI: 10.1105/tpc.4.11.1425]

#Shcherban1995 Shcherban, T.Y., Shi, J., Durachko, D.M., Guiltinan, M.J., McQueen-Mason, S.J., Shieh, M. and Cosgrove, D.J., 1995. Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants. Proceedings of the National Academy of Sciences, 92(20), pp.9245-9249. [https://doi.org/10.1073/pnas.92.20.9245 DOI: 10.1073/pnas.92.20.9245]
#Artzi2016 pmid=26973715
#Chen2016 Chen, C., Cui, Z., Song, X., Liu, Y.J., Cui, Q. and Feng, Y., 2016. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation. Applied microbiology and biotechnology, 100(5), pp.2203-2212. [https://doi.org/10.1007/s00253-015-7071-6 DOI: 10.1007/s00253-015-7071-6]
#Cosgrove1996 Cosgrove, D.J., 1996. Plant cell enlargement and the action of expansins. BioEssays, 18(7), pp.533-540 [https://doi.org/10.1002/bies.950180704 DOI: 10.1002/bies.950180704].
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T18:47:34Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==

***TO DO: ADD REFS, ADD LINKS TO PDB, FINAL EDITS***

In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification [1,2]. They are also present in diverse microbes, including many bacterial and fungal plant pathogens [3]. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past [4]. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' [5]. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins [6], including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (KD) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose [7]. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (KD) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl2, whereas binding to cellulose was reduced only ~40% in CaCl2 (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (KD 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials [11-15] but the contribution of the CBM63 domain was not ascertained (see review in [3]). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) [8] or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen [9,10]. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (KD 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration [10]. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose [10]. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich [6]. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides [5]. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding [7]. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions [5].

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) [9], which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities [7]. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix [7]. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies [8] which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots [9,10], where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains [7], but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in [13]). Certain microbial expansins are fused to other CAZY domains, including CBMI, CBMII, and GH5 (summarized in [4]). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases [16]. These cellulosomal expansins bound microcrystalline cellulose [16], and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degredation [16, 17], but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation [14], characterization [8] and sequencing [15] of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain [18]. This concept was substantiated by the crystal structures of ZmEXPB1 [9] and BsEXLX1 [6], followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain [7]. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides [5].

== References ==
TO DO

<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 Cosgrove, D.J., 2017. Microbial Expansins. Annual review of microbiology, 71, pp.479-497. [https://doi.org/10.1146/annurev-micro-090816-093315 DOI: 10.1146/annurev-micro-090816-093315]
#Nikolaidis2014 Nikolaidis, N., Doran, N. and Cosgrove, D.J., 2013. Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Molecular biology and evolution, 31(2), pp.376-386. [https://doi.org/10.1093/molbev/mst206 DOI: 10.1093/molbev/mst206]
#Georgelis2012 pmid=22927418

#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 McQueen-Mason, S.J. and Cosgrove, D.J., 1995. Expansin mode of action on cell walls (analysis of wall hydrolysis, stress relaxation, and binding). Plant Physiology, 107(1), pp.87-100. [https://doi.org/10.1104/pp.107.1.87 DOI: 10.1104/pp.107.1.87]

#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 Gilbert, H.J., Knox, J.P. and Boraston, A.B., 2013. Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Current opinion in structural biology, 23(5), pp.669-677. [https://doi.org/10.1016/j.sbi.2013.05.005 DOI: 10.1016/j.sbi.2013.05.005]

#Armenta2017 Armenta, S., Moreno‐Mendieta, S., Sánchez‐Cuapio, Z., Sánchez, S. and Rodríguez‐Sanoja, R., 2017. Advances in Molecular Engineering of Carbohydrate‐Binding Modules. Proteins: Structure, Function, and Bioinformatics. [https://doi.org/10.1002/prot.25327 DOI: 10.1002/prot.25327]
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 McQueen-Mason, S., Durachko, D.M. and Cosgrove, D.J., 1992. Two endogenous proteins that induce cell wall extension in plants. The Plant Cell, 4(11), pp.1425-1433. [https://doi.org/10.1105/tpc.4.11.1425 DOI: 10.1105/tpc.4.11.1425]

#Shcherban1995 Shcherban, T.Y., Shi, J., Durachko, D.M., Guiltinan, M.J., McQueen-Mason, S.J., Shieh, M. and Cosgrove, D.J., 1995. Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants. Proceedings of the National Academy of Sciences, 92(20), pp.9245-9249. [https://doi.org/10.1073/pnas.92.20.9245 DOI: 10.1073/pnas.92.20.9245]
#Artzi2016 pmid=26973715
#Chen2016 Chen, C., Cui, Z., Song, X., Liu, Y.J., Cui, Q. and Feng, Y., 2016. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation. Applied microbiology and biotechnology, 100(5), pp.2203-2212. [https://doi.org/10.1007/s00253-015-7071-6 DOI: 10.1007/s00253-015-7071-6]
#Cosgrove1996 Cosgrove, D.J., 1996. Plant cell enlargement and the action of expansins. BioEssays, 18(7), pp.533-540 [https://doi.org/10.1002/bies.950180704 DOI: 10.1002/bies.950180704].
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T18:45:43Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==

***TO DO: ADD REFS, ADD LINKS TO PDB, FINAL EDITS***

In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification [1,2]. They are also present in diverse microbes, including many bacterial and fungal plant pathogens [3]. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past [4]. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' [5]. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins [6], including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (KD) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose [7]. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (KD) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl2, whereas binding to cellulose was reduced only ~40% in CaCl2 (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (KD 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials [11-15] but the contribution of the CBM63 domain was not ascertained (see review in [3]). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) [8] or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen [9,10]. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (KD 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration [10]. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose [10]. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich [6]. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides [5]. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding [7]. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions [5].

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) [9], which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities [7]. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix [7]. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies [8] which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots [9,10], where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains [7], but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in [13]). Certain microbial expansins are fused to other CAZY domains, including CBMI, CBMII, and GH5 (summarized in [4]). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases [16]. These cellulosomal expansins bound microcrystalline cellulose [16], and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degredation [16, 17], but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation [14], characterization [8] and sequencing [15] of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain [18]. This concept was substantiated by the crystal structures of ZmEXPB1 [9] and BsEXLX1 [6], followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain [7]. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides [5].

== References ==
TO DO

<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 Cosgrove, D.J., 2017. Microbial Expansins. Annual review of microbiology, 71, pp.479-497. [https://doi.org/10.1146/annurev-micro-090816-093315 DOI: 10.1146/annurev-micro-090816-093315]
#Nikolaidis2014 Nikolaidis, N., Doran, N. and Cosgrove, D.J., 2013. Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Molecular biology and evolution, 31(2), pp.376-386. [https://doi.org/10.1093/molbev/mst206 DOI: 10.1093/molbev/mst206]
#Kerff2008 pmid=18971341
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 McQueen-Mason, S.J. and Cosgrove, D.J., 1995. Expansin mode of action on cell walls (analysis of wall hydrolysis, stress relaxation, and binding). Plant Physiology, 107(1), pp.87-100. [https://doi.org/10.1104/pp.107.1.87 DOI: 10.1104/pp.107.1.87]

#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 Gilbert, H.J., Knox, J.P. and Boraston, A.B., 2013. Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Current opinion in structural biology, 23(5), pp.669-677. [https://doi.org/10.1016/j.sbi.2013.05.005 DOI: 10.1016/j.sbi.2013.05.005]

#Armenta2017 Armenta, S., Moreno‐Mendieta, S., Sánchez‐Cuapio, Z., Sánchez, S. and Rodríguez‐Sanoja, R., 2017. Advances in Molecular Engineering of Carbohydrate‐Binding Modules. Proteins: Structure, Function, and Bioinformatics. [https://doi.org/10.1002/prot.25327 DOI: 10.1002/prot.25327]
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 McQueen-Mason, S., Durachko, D.M. and Cosgrove, D.J., 1992. Two endogenous proteins that induce cell wall extension in plants. The Plant Cell, 4(11), pp.1425-1433. [https://doi.org/10.1105/tpc.4.11.1425 DOI: 10.1105/tpc.4.11.1425]

#Shcherban1995 Shcherban, T.Y., Shi, J., Durachko, D.M., Guiltinan, M.J., McQueen-Mason, S.J., Shieh, M. and Cosgrove, D.J., 1995. Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants. Proceedings of the National Academy of Sciences, 92(20), pp.9245-9249. [https://doi.org/10.1073/pnas.92.20.9245 DOI: 10.1073/pnas.92.20.9245]
#Artzi2016 pmid=26973715
#Chen2016 Chen, C., Cui, Z., Song, X., Liu, Y.J., Cui, Q. and Feng, Y., 2016. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation. Applied microbiology and biotechnology, 100(5), pp.2203-2212. [https://doi.org/10.1007/s00253-015-7071-6 DOI: 10.1007/s00253-015-7071-6]
#Cosgrove1996 Cosgrove, D.J., 1996. Plant cell enlargement and the action of expansins. BioEssays, 18(7), pp.533-540 [https://doi.org/10.1002/bies.950180704 DOI: 10.1002/bies.950180704].
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T18:44:18Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==

***TO DO: ADD REFS, ADD LINKS TO PDB, FINAL EDITS***

In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification [1,2]. They are also present in diverse microbes, including many bacterial and fungal plant pathogens [3]. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past [4]. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' [5]. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins [6], including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (KD) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose [7]. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (KD) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl2, whereas binding to cellulose was reduced only ~40% in CaCl2 (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (KD 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials [11-15] but the contribution of the CBM63 domain was not ascertained (see review in [3]). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) [8] or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen [9,10]. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (KD 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration [10]. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose [10]. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich [6]. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides [5]. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding [7]. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions [5].

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) [9], which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities [7]. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix [7]. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies [8] which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots [9,10], where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains [7], but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in [13]). Certain microbial expansins are fused to other CAZY domains, including CBMI, CBMII, and GH5 (summarized in [4]). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases [16]. These cellulosomal expansins bound microcrystalline cellulose [16], and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degredation [16, 17], but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation [14], characterization [8] and sequencing [15] of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain [18]. This concept was substantiated by the crystal structures of ZmEXPB1 [9] and BsEXLX1 [6], followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain [7]. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides [5].

== References ==
TO DO

<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 Cosgrove, D.J., 2017. Microbial Expansins. Annual review of microbiology, 71, pp.479-497. [https://doi.org/10.1146/annurev-micro-090816-093315 DOI: 10.1146/annurev-micro-090816-093315]
#Nikolaidis2014 Nikolaidis, N., Doran, N. and Cosgrove, D.J., 2013. Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Molecular biology and evolution, 31(2), pp.376-386. [https://doi.org/10.1093/molbev/mst206 DOI: 10.1093/molbev/mst206]
#Kerff2008 pmid=22927418
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 McQueen-Mason, S.J. and Cosgrove, D.J., 1995. Expansin mode of action on cell walls (analysis of wall hydrolysis, stress relaxation, and binding). Plant Physiology, 107(1), pp.87-100. [https://doi.org/10.1104/pp.107.1.87 DOI: 10.1104/pp.107.1.87]

#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 Gilbert, H.J., Knox, J.P. and Boraston, A.B., 2013. Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Current opinion in structural biology, 23(5), pp.669-677. [https://doi.org/10.1016/j.sbi.2013.05.005 DOI: 10.1016/j.sbi.2013.05.005]

#Armenta2017 Armenta, S., Moreno‐Mendieta, S., Sánchez‐Cuapio, Z., Sánchez, S. and Rodríguez‐Sanoja, R., 2017. Advances in Molecular Engineering of Carbohydrate‐Binding Modules. Proteins: Structure, Function, and Bioinformatics. [https://doi.org/10.1002/prot.25327 DOI: 10.1002/prot.25327]
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 McQueen-Mason, S., Durachko, D.M. and Cosgrove, D.J., 1992. Two endogenous proteins that induce cell wall extension in plants. The Plant Cell, 4(11), pp.1425-1433. [https://doi.org/10.1105/tpc.4.11.1425 DOI: 10.1105/tpc.4.11.1425]

#Shcherban1995 Shcherban, T.Y., Shi, J., Durachko, D.M., Guiltinan, M.J., McQueen-Mason, S.J., Shieh, M. and Cosgrove, D.J., 1995. Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants. Proceedings of the National Academy of Sciences, 92(20), pp.9245-9249. [https://doi.org/10.1073/pnas.92.20.9245 DOI: 10.1073/pnas.92.20.9245]
#Artzi2016 pmid=26973715
#Chen2016 Chen, C., Cui, Z., Song, X., Liu, Y.J., Cui, Q. and Feng, Y., 2016. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation. Applied microbiology and biotechnology, 100(5), pp.2203-2212. [https://doi.org/10.1007/s00253-015-7071-6 DOI: 10.1007/s00253-015-7071-6]
#Cosgrove1996 Cosgrove, D.J., 1996. Plant cell enlargement and the action of expansins. BioEssays, 18(7), pp.533-540 [https://doi.org/10.1002/bies.950180704 DOI: 10.1002/bies.950180704].
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T18:36:39Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==

***TO DO: ADD REFS, ADD LINKS TO PDB, FINAL EDITS***

In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification [1,2]. They are also present in diverse microbes, including many bacterial and fungal plant pathogens [3]. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past [4]. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' [5]. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins [6], including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (KD) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose [7]. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (KD) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl2, whereas binding to cellulose was reduced only ~40% in CaCl2 (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (KD 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials [11-15] but the contribution of the CBM63 domain was not ascertained (see review in [3]). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) [8] or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen [9,10]. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (KD 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration [10]. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose [10]. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich [6]. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides [5]. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding [7]. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions [5].

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) [9], which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities [7]. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix [7]. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies [8] which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots [9,10], where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains [7], but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in [13]). Certain microbial expansins are fused to other CAZY domains, including CBMI, CBMII, and GH5 (summarized in [4]). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases [16]. These cellulosomal expansins bound microcrystalline cellulose [16], and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degredation [16, 17], but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation [14], characterization [8] and sequencing [15] of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain [18]. This concept was substantiated by the crystal structures of ZmEXPB1 [9] and BsEXLX1 [6], followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain [7]. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides [5].

== References ==
TO DO

<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 Cosgrove DJ: Plant expansins: diversity and interactions with plant cell walls. Curr Opin Plant Biol. 2015; 25:162-172. DOI: 10.1016/j.pbi.2015.05.014
#Nikolaidis2014 Nikolaidis N, Doran N, Cosgrove DJ: Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Mol Biol Evol. 2014; 31:376-386. DOI: 10.1093/molbev/mst206
#Kerff2008 pmid=22927418
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 McQueen-Mason SJ, Cosgrove DJ: Expansin mode of action on cell walls. Analysis of wall hydrolysis, stress relaxation, and binding. Plant Physiol. 1995; 107:87-100. DOI: 10.1104/pp.107.1.87

#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 Gilbert HJ, Knox JP, Boraston AB: Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Curr Opin Struct Biol. 2013; 23:669-677. DOI: 10.1016/j.sbi.2013.05.005

#Armenta2017 Armenta S, Moreno-Mendieta S, Sanchez-Cuapio Z, Sanchez S, Rodriguez-Sanoja R: Advances in molecular engineering of carbohydrate-binding modules. Proteins-Structure Function and Bioinformatics. 2017; 85:1602-1617. DOI: 10.1002/prot.25327
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 McQueen-Mason S, Durachko DM, Cosgrove DJ: Two endogenous proteins that induce cell wall extension in plants. Plant Cell. 1992; 4:1425-1433. DOI: 10.1105/tpc.4.11.1425

#Shcherban1995 Shcherban, T.Y., Shi, J., Durachko, D.M., Guiltinan, M.J., McQueen-Mason, S.J., Shieh, M. and Cosgrove, D.J., 1995. Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants. Proceedings of the National Academy of Sciences, 92(20), pp.9245-9249. [https://doi.org/10.1073/pnas.92.20.9245 DOI: 10.1073/pnas.92.20.9245]
#Artzi2016 pmid=26973715
#Chen2016 Chen, C., Cui, Z., Song, X., Liu, Y.J., Cui, Q. and Feng, Y., 2016. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation. Applied microbiology and biotechnology, 100(5), pp.2203-2212.
#Cosgrove1996 Cosgrove, D.J., 1996. Plant cell enlargement and the action of expansins. BioEssays, 18(7), pp.533-540 [https://doi.org/10.1002/bies.950180704 DOI: 10.1002/bies.950180704].
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T18:32:07Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==

***TO DO: ADD REFS, ADD LINKS TO PDB, FINAL EDITS***

In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification [1,2]. They are also present in diverse microbes, including many bacterial and fungal plant pathogens [3]. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past [4]. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' [5]. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins [6], including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (KD) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose [7]. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (KD) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl2, whereas binding to cellulose was reduced only ~40% in CaCl2 (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (KD 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials [11-15] but the contribution of the CBM63 domain was not ascertained (see review in [3]). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) [8] or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen [9,10]. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (KD 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration [10]. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose [10]. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich [6]. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides [5]. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding [7]. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions [5].

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) [9], which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities [7]. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix [7]. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies [8] which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots [9,10], where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains [7], but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in [13]). Certain microbial expansins are fused to other CAZY domains, including CBMI, CBMII, and GH5 (summarized in [4]). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases [16]. These cellulosomal expansins bound microcrystalline cellulose [16], and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degredation [16, 17], but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation [14], characterization [8] and sequencing [15] of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain [18]. This concept was substantiated by the crystal structures of ZmEXPB1 [9] and BsEXLX1 [6], followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain [7]. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides [5].

== References ==
TO DO

<biblio>
#Cosgrove2016 pmid=26918182
#Cosgrove2015 pmid=26057089
#Cosgrove2017 Cosgrove DJ: Plant expansins: diversity and interactions with plant cell walls. Curr Opin Plant Biol. 2015; 25:162-172. DOI: 10.1016/j.pbi.2015.05.014
#Nikolaidis2014 Nikolaidis N, Doran N, Cosgrove DJ: Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Mol Biol Evol. 2014; 31:376-386. DOI: 10.1093/molbev/mst206
#Kerff2008 pmid=22927418
#Georgelis2011 pmid=21454649
#McQueen-Mason1995 McQueen-Mason SJ, Cosgrove DJ: Expansin mode of action on cell walls. Analysis of wall hydrolysis, stress relaxation, and binding. Plant Physiol. 1995; 107:87-100. DOI: 10.1104/pp.107.1.87

#Yennawar2006 pmid=16984999
#Wang2016 pmid=27729469
#Gilbert2013 Gilbert HJ, Knox JP, Boraston AB: Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Curr Opin Struct Biol. 2013; 23:669-677. DOI: 10.1016/j.sbi.2013.05.005

#Armenta2017 Armenta S, Moreno-Mendieta S, Sanchez-Cuapio Z, Sanchez S, Rodriguez-Sanoja R: Advances in molecular engineering of carbohydrate-binding modules. Proteins-Structure Function and Bioinformatics. 2017; 85:1602-1617. DOI: 10.1002/prot.25327
#Georgelis2015 pmid=25833181
#McQueen-Mason1992 McQueen-Mason S, Durachko DM, Cosgrove DJ: Two endogenous proteins that induce cell wall extension in plants. Plant Cell. 1992; 4:1425-1433. DOI: 10.1105/tpc.4.11.1425

#Shcherban1995 Shcherban, T.Y., Shi, J., Durachko, D.M., Guiltinan, M.J., McQueen-Mason, S.J., Shieh, M. and Cosgrove, D.J., 1995. Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants. Proceedings of the National Academy of Sciences, 92(20), pp.9245-9249.
#Artzi2016 pmid=26973715
#Chen2016 Chen, C., Cui, Z., Song, X., Liu, Y.J., Cui, Q. and Feng, Y., 2016. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation. Applied microbiology and biotechnology, 100(5), pp.2203-2212.
#Cosgrove1996 Cosgrove, D.J., 1996. Plant cell enlargement and the action of expansins. BioEssays, 18(7), pp.533-540.
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T03:02:29Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==

***TO DO: ADD REFS, ADD LINKS TO PDB, FINAL EDITS***

In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification [1,2]. They are also present in diverse microbes, including many bacterial and fungal plant pathogens [3]. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past [4]. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' [5]. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins [6], including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (KD) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose [7]. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (KD) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl2, whereas binding to cellulose was reduced only ~40% in CaCl2 (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (KD 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials [11-15] but the contribution of the CBM63 domain was not ascertained (see review in [3]). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) [8] or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen [9,10]. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (KD 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration [10]. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose [10]. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich [6]. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides [5]. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding [7]. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions [5].

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) [9], which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities [7]. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix [7]. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies [8] which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots [9,10], where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains [7], but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in [13]). Certain microbial expansins are fused to other CAZY domains, including CBMI, CBMII, and GH5 (summarized in [4]). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases [16]. These cellulosomal expansins bound microcrystalline cellulose [16], and the inclusion of expansin in native and designer cellulosomes enhanced cellulose degredation [16, 17], but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation [14], characterization [8] and sequencing [15] of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain [18]. This concept was substantiated by the crystal structures of ZmEXPB1 [9] and BsEXLX1 [6], followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain [7]. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides [5].

== References ==
TO DO

<biblio>

</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T01:53:45Z

Will Chase:

{{UnderConstruction}}
* [[Authors]]: ^^^Will Chase^^^
* [[Responsible Curator]]: ^^^Daniel Cosgrove^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM63.html
|}
</div>


==Introduction==

***TO DO: ADD REFS, ADD LINKS TO PDB, FINAL EDITS***

In nature, CBM63 is found almost exclusively as the C-terminal domain of the two-domain protein named expansin. Expansins are ubiquitous in plants where they function in cell growth and other developmental processes that involve plant cell wall modification [1,2]. They are also present in diverse microbes, including many bacterial and fungal plant pathogens [3]. Microbes may have acquired expansin genes via horizontal gene transfer(s) from plants in the distant past [4]. The founding member of CBM63 is based on the C-terminal domain of the expansin designated BsEXLX1 from the soil bacterium ''Bacillus subtilis'' [5]. It contains ~100 amino acids with a total relative MW of ~11.5 kDa. In the current CAZypedia database, plant sequences are not included in the CBM63 listings, a consequence of the large sequence divergence of expansins and limitations of automated sequence-based methods used to construct CBM families. However, structural analysis leaves little doubt of the homology between plant and bacterial expansins [6], including the C-terminal domain identified as CBM63 in numerous microbial expansins.

== Ligand specificities ==
The most detailed characterization of CBM63 is from BsEXLX1 (GenBank [https://www.ncbi.nlm.nih.gov/protein/AAB84448.1 AAB84448.1]). Recombinant protein (the entire two-domain protein, without histidne tags) binds cellulose (Avicel) with an affinity (KD) of 2.1 μM and binding capacity (Bmax) of 0.3 μmol/g of cellulose [7]. Virtually identical binding parameters were found for the recombinant CBM63 domain by itself, whereas binding of the N-terminal domain was below the detection limit of the depletion isotherm assay. Evidently BsEXLX1 binding to cellulose is solely determined by the CBM63 domain.

The two-domain BsEXLX1 protein also binds to whole cell walls from wheat coleoptiles, primarily via the CBM63 domain, with an affinity (KD) of 1.79 μM and the remarkably high binding capacity of 30 μmol/g (0.345 g/g) of cell wall. Hence, whole cell walls have 100-fold greater binding capacity for CBM63 compared to Avicel, on a dry weight basis. This high capacity may be partly the result of differences in cellulose structure, aggregation and accessibility, but more likely the higher capacity results primarily from abundant interactions with the matrix, including electrostatic interactions of this basic protein (pI ~ 9.7) with pectins and other acidic matrix polysaccharides. Site-directed mutagenesis and additional binding experiments show that the high binding capacity of wheat cell walls depends on electrostatic interactions between basic residues (K145, K171, R173, K180, K183, K188) on the ‘back side’ of the CBM63 domain (the ‘front side’ is defined as the surface that binds cellulose; see figure 1) and matrix polysaccharides (predominantly glucuronoarabinoxylan and pectins). Consistent with this conclusion, binding to coleoptile cell wall was reduced 95% in the presence of 10 mM CaCl2, whereas binding to cellulose was reduced only ~40% in CaCl2 (up to 100 mM). Moreover, wild-type BsEXLX1 binds insoluble arabinoxylan from wheat flour (KD 4.7 μM; Bmax 3.3 μmol/g). This binding is eliminated when three of the basic residues listed above are changed to Q.

These studies show that CBM63 from BsEXLX1 has separate surfaces that bind cellulose and arabinoxylan by distinctive physical interactions. In other studies, whole expansins from various microbial sources were similarly found to bind to various forms of cellulose, xylan and lignocellulosic materials [11-15] but the contribution of the CBM63 domain was not ascertained (see review in [3]). Studies of native expansins from plant sources document binding to disordered cellulose by an α-expansin from cucumber hypocotyls (CsEXPA1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/KGN50732.1 KGN50732.1]) [8] or to xylans by a β-expansin (ZmEXPB1, GenBank: [https://www.ncbi.nlm.nih.gov/protein/NP_001288510.1 NP_001288510.1]) from maize pollen [9,10]. Binding of ZmEXPB1 to cellulose (Avicel) in isolation is weaker than of BsEXLX1 (KD 5.8 μM, Bmax 0.4 μmol/g; ratio: 0.07 L/g versus 0.14 L/g for BsEXLX1), whereas it shows stronger binding to whole maize cell walls with complex kinetics that depend on buffer concentration [10]. NMR analysis of ZmEXPB1 targeting in whole maize cell walls shows that it interacts with matrix polysaccharides rather than cellulose [10]. This result is likely due to its greater affinity for the matrix components and to limited accessibility of cellulose in the complex cell wall. The specific contributions of the CBM63 domain to these binding characteristics were not determined.

== Structural Features ==
[[File:Cbm63_figure.jpg|thumb|500px|right|'''Figure 1''' The crystal structure of BsEXLX1 in complex with cellohexaose (4FER). (A) Cellohexaose is shown sandwiched between two expansins, with the planar binding face of the CBM63 domains (colored blue) facing the substrate. Aromatic binding residues are shown in red; positively charged residues on the 'back face' are colored green. (B) The twisting of the bound cellohexaose (colored magenta) is evident from this view.]]
Crystal structures with CBM63 domains include two bacterial expansins (BsEXLX1: [http://www.rcsb.org/structure/2BH0 2BH0], [http://www.rcsb.org/structure/3D30 3D30], [http://www.rcsb.org/structure/4FER 4FER], [http://www.rcsb.org/structure/4FFT 4FFT], [http://www.rcsb.org/structure/4FG2 4FG2], [http://www.rcsb.org/structure/4FG4 4FG4]; and CmEXLX1 from ''Clavibacter michiganensis'': [http://www.rcsb.org/structure/4JCW 4JCW], [http://www.rcsb.org/structure/4JJO 4JJO], [http://www.rcsb.org/structure/4JS7 4JS7], [http://www.rcsb.org/structure/4L48 4L48]) and two plant expansins (ZmEXPB1 from ''Zea mays'': [https://www.rcsb.org/structure/2hcz 2HCZ]; and PhlP1 from ''Phleum pratense'':[https://www.rcsb.org/structure/1n10 1N10]). In addition, the ''Phleum'' pollen allergens PhlP2 ([https://www.rcsb.org/structure/1who 1WHO]) and PhlP3 ([https://www.rcsb.org/structure/3ft9 3FT9]) are evolutionarily derived from plant β-expansins and are homologous to CBM63, but their binding properties are unknown.

In BsEXLX1 the CBM63 fold consists of two sets of four anti-parallel β-strands that form a β-sandwich [6]. This fold forms a planar binding surface populated by three co-linear aromatic amino acids (W125, W126, Y157) which mediate hydrophobic binding interactions with cellohexaose and related cellulose-like oligosaccharides [5]. Thus, CBM63 is considered a Type A CBM [11,12]. Mutagenesis of these residues reduces cellulose binding [7]. In crystal complexes with cello-oligosaccharides, BsEXLX1 forms an unusual structure in which a single glucan chain is sandwiched between the CBM63 domains of two proteins, in opposite polarity (Figure 1-A). The aromatic residues from the CBM63 domains of two proteins interact with alternating glucose residues, always with the more hydrophobic face that is populated by three –CH groups. Hydrogen bonds are formed between the K119 residues of both proteins and several hydroxyl groups of cellohexaose. The cellohexaose bound in the sandwich structure displays a twist (Figure 1-B). Binding to cellulose is entropically driven due to the exclusion of bound water during the formation of CH-pi interactions [5].

In the plant β-expansin ZmEXPB1, there are only two aromatic residues on the CBM63 surface (Y158, W192) [9], which may account for its weaker binding to cellulose. There are no crystal structures for alpha expansins, but sequence similarity suggests they have a similar 8-stranded β-sandwich fold.

== Functionalities ==
BsEXLX1 induces creep of plant cell walls and reduces the breaking strength of filter paper strips, but the CBM63 domain by itself lacks these activities [7]. Cell wall loosening activity of BsEXLX1 depends on binding to cellulose rather than the matrix [7]. Hence, matrix binding appears to be nonproductive for wall loosening. This conclusion is also supported by solid-state NMR studies [8] which showed that in partially-extracted cell walls from Arabidopsis, BsEXLX1 binds to a minor cellulose component with a chemical shift slightly different from the bulk of the cellulose. The implication of these results is that CBM63 targets expansin to specific sites, dubbed biomechanical hotspots [9,10], where cell wall loosening occurs. It has been proposed that wall loosening requires the cooperative action of both expansin domains [7], but this idea needs additional testing. BsEXLX1 and other bacterial expansins have been tested for their ability to enhance cellulase action, but with mixed results (reviewed in [13]). Certain microbial expansins are fused to other CAZY domains, including CBMI, CBMII, and GH5 (summarized in [4]). These chimeric proteins presumably facilitate plant-microbe association (whether pathogenic or commensal), but the exact function and contribution of the CBM63 domain is unknown. Likewise, dockerin-fused expansins have been documented in ''Clostridium clariflavum'' (GenBank: [https://www.ncbi.nlm.nih.gov/protein/WP_014255055.1 WP_014255055.1]), where they integrate into the cellulosome complex alongside other CBMs and endoglucanases [16]. It was shown that cellulosomal expansin bound microcrystalline cellulose [16], and the inclusion of expansin in native and designer cellulosomes enhanced cellulose [16, 17], but again the exact contribution of CBM63 is unknown.

== Family Firsts ==
The isolation [14], characterization [8] and sequencing [15] of α-expansin led to speculation that the C-terminal region of expansin was a CBM-like domain [18]. This concept was substantiated by the crystal structures of ZmEXPB1 [9] and BsEXLX1 [6], followed by site-directed mutagenesis of BsEXLX1 to test the functionalities of the CBM63 domain [7]. Crystal structural analysis of BsEXLX1 complexed with various cellulose-like oligosaccharides yielded the first crystal structure of a Type A CBM complexed with cellulose-like oligosaccharides [5].

== References ==
TO DO

<biblio>

</biblio>

[[Category:Carbohydrate Binding Module Families|CBM063]]

Carbohydrate Binding Module Family 63

2018-05-04T01:36:53Z

Will Chase:

Carbohydrate Binding Module Family 63

2018-05-04T00:14:18Z

Will Chase:

Carbohydrate Binding Module Family 63

2018-05-04T00:12:25Z

Will Chase:

Carbohydrate Binding Module Family 63

2018-05-04T00:11:18Z

Will Chase:

File:Cbm63 figure.jpg

2018-05-04T00:04:50Z

Will Chase: BsEXLX1 crystal structure.

BsEXLX1 crystal structure.

Carbohydrate Binding Module Family 63

2018-05-03T23:43:19Z

Will Chase:

Carbohydrate Binding Module Family 63

2018-05-03T23:40:47Z

Will Chase:

User:Will Chase

2018-05-01T19:55:21Z

Will Chase:

User:Will Chase

2018-05-01T19:55:02Z

Will Chase:

[[Image: Will_Chase.jpg|200px|right]]

I study the structure of the plant cell wall and the enzymes that modify that structure. I received my B.S. from Penn State in 2016 and now work in Daniel Cosgrove's lab. My work focuses on the structure, function, and evolution of expansin proteins, particularly expansins found in microbes.

* See [[User:Gerlind_Sulzenbacher]] for an example. You may copy text from this example by opening the page in another browser window and clicking the "Edit" tab.
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----

<biblio>
#Gilbert2008 pmid=18430603

</biblio>


[[Category:Contributors|Chase,Will]]

File:Will Chase.jpg

2018-05-01T19:51:40Z

Will Chase: Will's picture

Will's picture

User:Will Chase

2018-05-01T19:49:50Z

Will Chase:

[[Image: Will_monkeys.jpg|200px|right]]
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#Gilbert2008 pmid=18430603

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[[Category:Contributors|Chase,Will]]

User:Will Chase

2018-05-01T19:49:01Z

Will Chase:

[[Image: will_monkeys.jpg|200px|right]]
'''This is an empty template to help you get started with composing your User page.'''

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<biblio>
#Gilbert2008 pmid=18430603

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[[Category:Contributors|Chase,Will]]