Difference between revisions of "Carbohydrate Binding Module Family 2"

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Ligand specificities

The vast majority of family 2 CBM (CBM2) members, ~95%, are bacterial in origin. These protein modules have been shown to bind to crystalline cellulose [1], insoluble chitin [2] or xylan [3]. The xylan-specific CBM2 modules bind only to the hemicellulose, while some cellulose-binding modules also recognize chitin. The CBM2 modules from some chitinases show a strong preference for crystalline chitin [2]. Cellulose-specific CBM2 members were also shown to bind to xyloglucan, albeit with affinities ~1000x lower than for crystalline cellulose [4]. Extremely weak binding to cellulooligosaccharides was detected in cellulose binding CBM2s using NMR [5]. In contrast affinities of xylan binding CBM2s for their polysaccharide or oligosaccharide ligands with a degree of polymerization of six were similar [6].

Some enzymes contain multiple CBM2 modules that, with respect to xylan, show enhanced affinity through avidity effects [5]. Isothermal titration calorimetry showed that CBM2 modules that interact with their target cellulose or xylan ligands were mediated by entropic [7] and enthalpic [5] forces, respectively. These differences in thermodynamic forces driving ligand recognition was proposed to reflect the conformational freedom of cellulose and xylan in free solution and when bound to CBMs. Family 2 is thus highly unusual, containing both cellulose binding type A and xylan binding type B CBMs. Labelling of type A crystalline cellulose specific CBMs, including those in CBM2, which all present three aromatic planar surface residues, showed differences in specificity for pure forms of crystalline cellulose and plant cell walls [8, 9].

Structural Features

Figure 1. The fold of the CBM2 in the Cellulomonas fimi GH10 xylanase Xyn10A (also known as Cex)(see PDB ID 1exg) highlighting the three planar tryptophans that are optimised to bind to the planar surface of crystalline cellulose bound.

Figure 2. The fold of the CBM2 in the Cellulomonas fimi GH11 xylanase Xyn11D (PDB ID 2xbd) highlighting the two perpendicular tryptophans that are optimised to bind to xylose units n and n+2 in the three-fold helical structure of xylan. The tryptophan perpendicular to the surface of the CBM is mediated through a stacking interaction with a surface arginine, which is also shown.

CBM2 modules generally comprise 90 to 100 residues that adopt a canonical β-jelly-roll fold (see PDB ID 1exg for an example) [10]. The two β-sheets of these CBMs consist of four (face 1) and five (face 2) antiparallel β-strands, respectively. The structure is sometimes stabilized by a disulphide bridge between the N- and C-terminal regions, but not in all cases. In cellulose binding CBM2 members, face 1 presents a planar surface that contains three highly conserved aromatic residues (generally tryptophans although tyrosine can also be present) that are positioned to bind to glucose units in linear planar cellulose chains present in the crystalline polysaccharide (Figure 1). The corresponding aromatic residues interact with N-acetylglucosamine residues in chitin [2]. Mutagenesis and chemical modfication studies confirmed the critical role played by the aromatic residues in cellulose recognition [11, 12, 13]. The central tryptophan plays a more important role in ligand binding than the distal aromatic amino acids [12].

In xylan binding CBM2 modules (see PDB ID 2xbd for an example) face 1 displays a slightly concave surface that contains only two tryptophan residues [14]. The perpendicular orientation (with respect to each other; Figure 2) and location of these two tryptophan residues indicate that they interact with xylose residues n and n+2 in the three fold-helical structure of xylan chains. NMR titration experiments with xylooligosaccharides and mutagenesis studies showed that both aromatic residues play a critical role in xylan recognition.

An arginine residue adjacent to one of the tryptophans in the xylan binding CBM2 forces it into the perpendicular orientation with respect to the other surface aromatic residue (Figure 2). In cellulose binding CBM2s the corresponding perpendicular tryptophan (in xylan binding CBM2s) is planar with the other two surface aromatic residues (see PDB ID 1e5b). The adoption of the planar orientation was predicted to be mediated by the replacement of the arginine (in xylan binding CBM2s) with glycine in cellulose specific CBM2s. This was supported by mutagenesis studies showing that substituting the arginine with glycine in a xylan binding CBM2 created a planar orientation for both surface tryptophans (see Fig. 5 and 6 in [15]), abrogated xylan binding and introduced cellulose recognition [15]. Mutagenesis studies showed that alanine substitution of polar residues predicted to interact with CBM2 ligands did not influence affinity for xylan or cellulose, demonstrating that hydrophobic forces dominated carbohydrate recognition [12, 16].

Functionalities

CBM2 modules that target cellulose as the principle ligand have been reported in cellulases [1], xylan degrading enzymes (xylanases, arabinofuranosidases, esterases) [17, 18], pectate lyases [19], mannanases [20] and lytic polysaccharide monooxygenases (LPMOs) [21, 22]. Xylan and chitin binding CBM2s are restricted to enzymes that target these polysaccharides [2, 3]. Many of these modular enzymes contain other CBM families in addition to CBM2 members [23], with CBM10-CBM2 modular motifs being particularly common in glycoside hydrolases from Cellvibrio japonicus [24] (e.g. [25]) and Saccharophagus degradans [26]. CBM2 modules are linked to catalytic modules by linker sequences that are required to be >15 residues in length to maximize the boost in activity provided by the CBM [27, 28].

CBM2 modules substantially potentiate the activity of cellulases and chitinsaes against purified insoluble or crystalline forms of cellulose and chitin, respectively [1, 2]. It has been proposed that cellulase-derived CBM2 modules enhance enzyme activity by increasing the local concentration or proximity of the enzyme in the vicinity of the substrate [5]. It has, however, also been reported that a CBM2 module was able to disrupt the structure of crystalline cellulose leading to enhanced enzyme activity [29]. Cell wall imaging studies have also shown that cellulose and xylan binding CBM2s enhance the activity of a pectate lyase, arabinofuranosidase and xylanases against plant cell walls [30]. Although CBM2 modules can bind irreversibly to crystalline cellulose [31], fluorescent bleaching experiments showed that these protein modules can migrate across the surface of the polysaccharide [32]. CBM2 protein modules have been explored as both affinity tags for purifying proteins and in the immobilization of industrially relevant enzymes to insoluble matrices [31, 33]. These modules are also deployed in the exploration of the architecture of plant cell walls [34].

Family Firsts

First Identified: A CBM2 module was first discovered in the cellulase CenA and the xylanase CexA from Cellulomonas fimi. These modules were identified through treatment of these enzymes with proteinases, which resulted in the loss of the C-terminal region comprising the CBM2 module, which reduced binding to cellulose and, in the case of CenA, activity against crystalline forms of the polysaccharide [1]. In the early literature CBM2 modules were referred to as cellulose binding domains or CBDs.
First structural characterization: The first 3D structure of a cellulose binding CBM2 module, which was determined by NMR, was the C. fimi xylanase Cex [10]. The first structure of a xylan binding CBM2 member, also elucidated by NMR, was from the C. fimi xylanase Xyn11A [14]. The first crystal structure of a member of CBM2 was that of the chitin binding module from the Pyrococcus chitinase Chi18A (see PDB ID 2cwr) [2].

References

All Medline abstracts: PubMed

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 == Ligand specificities ==
-The vast majority of family 2  CBMs (CBM2s), ~95%, are bacterial in origin. These protein modules have been shown to bind to crystalline cellulose <cite>Gilkes1988</cite>, insoluble chitin <cite>Nakamura2008</cite> or xylan <cite>Black1995</cite>. The xylan-specific CBM2s bind only to the hemicellulose, while some cellulose-binding modules also recognize chitin. The CBM2s from some chitinases show a strong preference for crystalline chitin <cite>Nakamura2008</cite>. Cellulose-specific CBM2s were also shown to bind to xyloglucan, albeit with affinities ~1000x lower than for crystalline cellulose <cite>Hernandez-Gomez2015 </cite>.  Extremely weak binding to cellulooligosaccharides was detected in cellulose binding CBM2s using NMR <cite>Bolam1998</cite>. In contrast affinities of xylan binding CBM2s for their polysaccharide or oligosaccharide ligands with a degree of polymerization of six were similar <cite>Bolam2001</cite>. Some enzymes contain multiple CBM2s that, with respect to xylan, show enhanced affinity through avidity effects <cite>Bolam1998</cite>. Isothermal titration calorimetry showed that CBM2 interactions with their target cellulose or xylan ligands was mediated by entropic <cite>Creagh1996</cite> and enthalpic <cite>Bolam1998</cite> forces, respectively. These differences in thermodynamic forces driving ligand recognition was proposed to reflect the conformational freedom of cellulose and xylan in free solution and when bound to CBMs. Family 2 is thus highly unusual, containing both cellulose binding type A and xylan binding type B CBMs. Labelling of type A crystalline cellulose specific CBMs, including CBM2s, which all present three aromatic planar surface residues, showed differences in specificity for pure forms of crystalline cellulose and plant cell walls <cite>Blake2006,McLean2002</cite>.
+The vast majority of family 2  CBM (CBM2) members, ~95%, are bacterial in origin. These protein modules have been shown to bind to crystalline cellulose <cite>Gilkes1988</cite>, insoluble chitin <cite>Nakamura2008</cite> or xylan <cite>Black1995</cite>. The xylan-specific CBM2 modules bind only to the hemicellulose, while some cellulose-binding modules also recognize chitin. The CBM2 modules from some chitinases show a strong preference for crystalline chitin <cite>Nakamura2008</cite>. Cellulose-specific CBM2 members were also shown to bind to xyloglucan, albeit with affinities ~1000x lower than for crystalline cellulose <cite>Hernandez-Gomez2015 </cite>.  Extremely weak binding to cellulooligosaccharides was detected in cellulose binding CBM2s using NMR <cite>Bolam1998</cite>. In contrast affinities of xylan binding CBM2s for their polysaccharide or oligosaccharide ligands with a degree of polymerization of six were similar <cite>Bolam2001</cite>.
+Some enzymes contain multiple CBM2 modules that, with respect to xylan, show enhanced affinity through avidity effects <cite>Bolam1998</cite>. Isothermal titration calorimetry showed that CBM2 modules that interact with their target cellulose or xylan ligands were mediated by entropic <cite>Creagh1996</cite> and enthalpic <cite>Bolam1998</cite> forces, respectively. These differences in thermodynamic forces driving ligand recognition was proposed to reflect the conformational freedom of cellulose and xylan in free solution and when bound to CBMs. Family 2 is thus highly unusual, containing both cellulose binding [[Carbohydrate-binding_modules#Types|type A]] and xylan binding [[Carbohydrate-binding_modules#Types|type B]] CBMs. Labelling of type A crystalline cellulose specific CBMs, including those in CBM2, which all present three aromatic planar surface residues, showed differences in specificity for pure forms of crystalline cellulose and plant cell walls <cite>Blake2006,McLean2002</cite>.
 == Structural Features ==
-[[File:CBM2-cellulosestructure.png|thumb|300px|right|'''Figure 1.'''  The fold of the CBM2 in the ''Cellulomonas fimi GH10 xylanase Xyn10A (also known as Cex)(see [{{PDBlink}}1exg PDB ID 1exg]) highlighting the three planar tryptophans that are optimised to bind to the planar surface of crystalline cellulose bound.]]
+[[File:CBM2-cellulosestructure.png|thumb|300px|right|'''Figure 1.'''  The fold of the CBM2 in the ''Cellulomonas fimi'' GH10 xylanase Xyn10A (also known as Cex)(see [{{PDBlink}}1exg PDB ID 1exg]) highlighting the three planar tryptophans that are optimised to bind to the planar surface of crystalline cellulose bound.]]
-CBM2s generally comprise 90 to 100 residues that adopt a canonical β-jelly-roll fold (see [{{PDBlink}}1exg PDB ID 1exg] for an example)   <cite>Xu1995</cite>. The two β-sheets of these CBMs consist of four (face 1) and five (face 2) antiparallel β-strands, respectively. The structure is sometimes stabilized by a disulphide bridge between the N- and C-terminal regions, but not in all cases. In cellulose binding CBM2s, face 1 presents a planar surface that contains three highly conserved aromatic residues (generally tryptophans although tyrosine can also be present)  that are positioned to bind to glucose units in linear planar cellulose chains present in the crystalline polysaccharide (Figure 1). The corresponding aromatic residues interact with N-acetylglucosamine residues in chitin <cite>Nakamura2008</cite>. Mutagenesis and chemical modfication studies confirmed the critical role played by the aromatic residues in cellulose recognition <cite>Bray1996,McLean2000,Nagy1998</cite>. The central tryptophan plays a more important role in ligand binding than the distal aromatic amino acids <cite>McLean2000</cite>. In xylan binding CBM2s (see [{{PDBlink}}2xbd PDB ID 2xbd] for an example) face 1 displays a slightly concave surface that contains only two tryptophan residues <cite>Simpson1999</cite>. The perpendicular orientation (with respect to each other; Figure 2) and location of these two tryptophan residues indicate that they interact with xylose residues n and n+2 in the three fold-helical structure of xylan chains.  NMR titration experiments with xylooligosaccharides and mutagenesis studies showed that both aromatic residues play a critical role in xylan recognition. An arginine residue adjacent to one of the tryptophans forces it into perpendicular orientation with respect to the other surface aromatic residue (Figure 2). In cellulose binding CBM2s the corresponding perpendicular tryptophan (in xylan binding CBM2s) is planar with the other two surface aromatic residues (see [{{PDBlink}}1e5b PDB ID 1e5b]). The adoption of the planar orientation was predicted to be mediated by the replacement of the arginine (in xylan binding CBM2s) with glycine in cellulose specific CBM2s. It was proposed that this created the required space for the tryptophan to adopt the planar orientation with respect to the other aromatic residues in face 1 <cite>Simpson2000</cite>. Mutagenesis studies showed that substituting the arginine with glycine in a xylan binding CBM2 created a planar orientation for both surface tryptophans (see Fig. 5 and 6 in <cite>Simpson2000</cite>), abrogated xylan binding and introduced cellulose recognition <cite>Simpson2000</cite>. Mutagenesis studies showed that alanine substitution of polar residues predicted to interact with CBM2 ligands did not influence affinity for xylan or cellulose, demonstrating that hydrophobic forces dominated carbohydrate recognition <cite>McLean2000,Xie2001</cite>.
+[[File:CBM2-xylanstructure.png|thumb|300px|right|'''Figure 2.'''  The fold of the CBM2 in the ''Cellulomonas fimi'' GH11 xylanase Xyn11D ([{{PDBlink}}2xbd PDB ID 2xbd]) highlighting the two perpendicular tryptophans that are optimised to bind to xylose units n and n+2 in the three-fold helical structure of xylan. The tryptophan perpendicular to the surface of the CBM is mediated through a stacking interaction with a surface arginine, which is also shown.]]
-== Functionalities ==
+CBM2 modules generally comprise 90 to 100 residues that adopt a canonical β-jelly-roll fold (see [{{PDBlink}}1exg PDB ID 1exg] for an example)   <cite>Xu1995</cite>. The two β-sheets of these CBMs consist of four (face 1) and five (face 2) antiparallel β-strands, respectively. The structure is sometimes stabilized by a disulphide bridge between the N- and C-terminal regions, but not in all cases. In cellulose binding CBM2 members, face 1 presents a planar surface that contains three highly conserved aromatic residues (generally tryptophans although tyrosine can also be present)  that are positioned to bind to glucose units in linear planar cellulose chains present in the crystalline polysaccharide (Figure 1). The corresponding aromatic residues interact with N-acetylglucosamine residues in chitin <cite>Nakamura2008</cite>. Mutagenesis and chemical modfication studies confirmed the critical role played by the aromatic residues in cellulose recognition <cite>Bray1996,McLean2000,Nagy1998</cite>. The central tryptophan plays a more important role in ligand binding than the distal aromatic amino acids <cite>McLean2000</cite>.
-CBM2s that target cellulose as the principle ligand have been reported in cellulases <cite>Gilkes1988</cite>, xylan degrading enzymes (xylanases, arabinofuranosidases, esterases) <cite>Kellett1990,Ferreira1993</cite>, pectate lyases <cite>Brown2001</cite>, mannanases <cite>Hogg2003</cite> and lytic polysaccharide monooxygenases (LPMOs) <cite>Crouch2016,Forsberg2014</cite>. Xylan and chitin binding CBM2s are restricted to enzymes that target these polysaccharides <cite>Nakamura2008,Black1995</cite>. Many of these modular enzymes contain other CBM families in addition to CBM2 <cite>Bolam2004</cite>, with CBM10-CBM2 modular motifs being particularly common in glycoside hydrolases from ''Cellvibrio japonicus'' <cite>DeBoy2008</cite> and ''Saccharophagus degradans'' <cite>Weiner2008</cite>. CBM2s are linked to catalytic modules by linker sequences that are required to be >15 residues in length to maximize the boost in activity provided by the CBM <cite>Ferreira1990,Shen1991</cite>. CBM2s substantially potentiate the activity of cellulases and chitinsaes against purified insoluble or crystalline forms of cellulose and chitin, respectively <cite>Gilkes1988,Nakamura2008</cite>. It has been proposed that cellulase-derived CBM2s enhance enzyme activity by increasing the local concentration or proximity of the enzyme in the vicinity of the substrate <cite>Bolam1998</cite>. It has, however, also been reported that a CBM2 was able to disrupt the structure of crystalline cellulose leading to enhanced enzyme activity <cite>Din1994</cite>. Cell wall imaging studies have also shown that cellulose and xylan binding CBM2s enhance the activity of a pectate lyase, arabinofuranosidase and xylanases against plant cell walls <cite>Herve2010</cite>. Although CBM2s can bind irreversibly to crystalline cellulose <cite>Tomme1998</cite>, fluorescent bleaching experiments showed that these protein modules can migrate across the surface of the polysaccharide <cite>Jervis1997</cite>. CBM2s have been explored as both affinity tags for purifying proteins and in the immobilization of industrially relevant enzymes to insoluble matrices <cite>Tomme1998,Boraston2001</cite>.  These modules are also deployed in the exploration of the architecture of plant cell walls <cite>McCartney2006</cite>.
+In xylan binding CBM2 modules (see [{{PDBlink}}2xbd PDB ID 2xbd] for an example) face 1 displays a slightly concave surface that contains only two tryptophan residues <cite>Simpson1999</cite>. The perpendicular orientation (with respect to each other; Figure 2) and location of these two tryptophan residues indicate that they interact with xylose residues n and n+2 in the three fold-helical structure of xylan chains.  NMR titration experiments with xylooligosaccharides and mutagenesis studies showed that both aromatic residues play a critical role in xylan recognition.
-== Family Firsts ==
+An arginine residue adjacent to one of the tryptophans in the xylan binding CBM2 forces it into the perpendicular orientation with respect to the other surface aromatic residue (Figure 2). In cellulose binding CBM2s the corresponding perpendicular tryptophan (in xylan binding CBM2s) is planar with the other two surface aromatic residues (see [{{PDBlink}}1e5b PDB ID 1e5b]). The adoption of the planar orientation was predicted to be mediated by the replacement of the arginine (in xylan binding CBM2s) with glycine in cellulose specific CBM2s. This was supported by mutagenesis studies showing that substituting the arginine with glycine in a xylan binding CBM2 created a planar orientation for both surface tryptophans (see Fig. 5 and 6 in <cite>Simpson2000</cite>), abrogated xylan binding and introduced cellulose recognition <cite>Simpson2000</cite>. Mutagenesis studies showed that alanine substitution of polar residues predicted to interact with CBM2 ligands did not influence affinity for xylan or cellulose, demonstrating that hydrophobic forces dominated carbohydrate recognition <cite>McLean2000,Xie2001</cite>.
-'''First Identified:'''
-CBM2s were first discovered in the cellulase CenA and the xylanase CexA from ''Cellulomonas fimi''. These modules were identified through treatment of these enzymes with proteinases, which resulted in the loss of the C-terminal region comprising the CBM2, which reduced binding to cellulose and, in the case of CenA, activity against crystalline forms of the polysaccharide <cite>Gilkes1988</cite>. In the early literature CBM2s were referred to as cellulose binding domains or CBDs.
+== Functionalities ==
+CBM2 modules that target cellulose as the principle ligand have been reported in cellulases <cite>Gilkes1988</cite>, xylan degrading enzymes (xylanases, arabinofuranosidases, esterases) <cite>Kellett1990,Ferreira1993</cite>, pectate lyases <cite>Brown2001</cite>, mannanases <cite>Hogg2003</cite> and lytic polysaccharide monooxygenases (LPMOs) <cite>Crouch2016,Forsberg2014</cite>. Xylan and chitin binding CBM2s are restricted to enzymes that target these polysaccharides <cite>Nakamura2008,Black1995</cite>. Many of these modular enzymes contain other CBM families in addition to CBM2 members <cite>Bolam2004</cite>, with CBM10-CBM2 modular motifs being particularly common in glycoside hydrolases from ''Cellvibrio japonicus'' <cite>DeBoy2008</cite> (e.g. <cite>Attia2016</cite>) and ''Saccharophagus degradans'' <cite>Weiner2008</cite>. CBM2 modules are linked to catalytic modules by linker sequences that are required to be >15 residues in length to maximize the boost in activity provided by the CBM <cite>Ferreira1990,Shen1991</cite>.
-'''First structural characterization:'''
+CBM2 modules substantially potentiate the activity of cellulases and chitinsaes against purified insoluble or crystalline forms of cellulose and chitin, respectively <cite>Gilkes1988,Nakamura2008</cite>. It has been proposed that cellulase-derived CBM2 modules enhance enzyme activity by increasing the local concentration or proximity of the enzyme in the vicinity of the substrate <cite>Bolam1998</cite>. It has, however, also been reported that a CBM2 module was able to disrupt the structure of crystalline cellulose leading to enhanced enzyme activity <cite>Din1994</cite>. Cell wall imaging studies have also shown that cellulose and xylan binding CBM2s enhance the activity of a pectate lyase, arabinofuranosidase and xylanases against plant cell walls <cite>Herve2010</cite>. Although CBM2 modules can bind irreversibly to crystalline cellulose <cite>Tomme1998</cite>, fluorescent bleaching experiments showed that these protein modules can migrate across the surface of the polysaccharide <cite>Jervis1997</cite>. CBM2 protein modules have been explored as both affinity tags for purifying proteins and in the immobilization of industrially relevant enzymes to insoluble matrices <cite>Tomme1998,Boraston2001</cite>.  These modules are also deployed in the exploration of the architecture of plant cell walls <cite>McCartney2006</cite>.
-The first 3D structure of a cellulose binding CBM2, which was determined by NMR, was the ''C. fimi'' xylanase Cex <cite>Xu1995</cite>. The first structure of a xylan binding CBM2, also elucidated by NMR, was from the ''C. fimi'' xylanase Xyn11A <cite>Simpson1999</cite>. The first crystal structure of a CBM2 was that of the chitin binding module from the ''Pyrococcus'' chitinase Chi18A (see [{{PDBlink}}2cwr PDB ID 2cwr]) <cite>Nakamura2008</cite>.
+== Family Firsts ==
+;First Identified: A CBM2 module was first discovered in the cellulase CenA and the xylanase CexA from ''Cellulomonas fimi''. These modules were identified through treatment of these enzymes with proteinases, which resulted in the loss of the C-terminal region comprising the CBM2 module, which reduced binding to cellulose and, in the case of CenA, activity against crystalline forms of the polysaccharide <cite>Gilkes1988</cite>. In the early literature CBM2 modules were referred to as cellulose binding domains or CBDs.
+;First structural characterization: The first 3D structure of a cellulose binding CBM2 module, which was determined by NMR, was the ''C. fimi'' xylanase Cex <cite>Xu1995</cite>. The first structure of a xylan binding CBM2 member, also elucidated by NMR, was from the ''C. fimi'' xylanase Xyn11A <cite>Simpson1999</cite>. The first crystal structure of a member of CBM2 was that of the chitin binding module from the ''Pyrococcus'' chitinase Chi18A (see [{{PDBlink}}2cwr PDB ID 2cwr]) <cite>Nakamura2008</cite>.
 == References ==
 <biblio>
 #Gilkes1988 pmid=3134347
 #Nakamura2008 pmid=18582475
 #Black1995 pmid=7717975
 #Hernandez-Gomez2015 pmid=26193423
 #Bolam1998 pmid=9560304
 #Bolam2001 pmid=11327868
 #Creagh1996 pmid=8901562
 #Blake2006 pmid=16844685
 #McLean2002 pmid=12191997
 #Xu1995 pmid=7766609
 #Bray1996 pmid=8931149
 #McLean2000 pmid=11161112
 #Nagy1998 pmid=9662439
 #Simpson1999 pmid=10425686
 #Simpson2000 pmid=10973978
 #Xie2001 pmid=11341835
 #Kellett1990 pmid=2125205
 #Ferreira1993 pmid=8373350
 #Brown2001 pmid=11256960
 #Hogg2003 pmid=12523937
 #Crouch2016 pmid=26801613
 #Forsberg2014 pmid=24912171
 #Bolam2004 pmid=15004012
 #DeBoy2008 pmid=18556790
 #Weiner2008 pmid=18516288
 #Ferreira1990 pmid=2115772
 #Shen1991 pmid=1904063
 #Din1994 pmid=7972069
 #Herve2010 pmid=20696902
 #Tomme1998 pmid=9792516
 #Jervis1997 pmid=9295354
 #Boraston2001 pmid=11281716
 #McCartney2006 pmid=16537424
+#Attia2016 pmid=26929175
 </biblio>
 [[Category:Carbohydrate Binding Module Families|CBM002]]