Carbohydrate Binding Module Family 87

2021-03-30T18:38:59Z

Lynne Howell:

{{CuratorApproved}}
* [[Author]]: ^^^Natalie Bamford^^^
* [[Responsible Curator]]: ^^^Lynne Howell^^^
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== Ligand specificities ==
The CBM87 family was formed in 2020 after the structural and functional characterization of Agd3, a galactosaminogalactan deacetylase involved in ''Aspergillus fumigatus'' biofilm formation <cite>Bamford2020</cite>. This study also resulted in the creation of a novel carbohydrate esterase superfamily, the [[CE18]] family. Functional characterization has focused on the CBM87 module Agd3CBM87. Binding of Agd3CBM87 to soluble galactosaminogalactan from culture supernatants of ''A. fumigatus'' was demonstrated using an ELISA based assay <cite>Bamford2020</cite>. Galactosaminogalactan is a linear heteropolymer of ⍺-1,4-linked D-galactose (Gal) and partially deacetylated N-acetyl-D-galactosamine (GalNAc and GalN) <cite>Fontaine2011 Lee2016</cite>. Using electrospray ionization mass spectrometry (ESI-MS) and synthesized oligosaccharides Agd3CBM87 was found to have higher affinity for ⍺-1,4-(GalN-⍺-1,4-GalNAc)3 as compared to Gal, GalNAc, or GalN homopolymers of the same linkage <cite>Bamford2020</cite>.

== Structural Features ==
[[File:CBM87 figures-01.png|thumb|400px|right|'''Figure 1. Agd3CBM87 from ''A. fumigatus'' adopts a Rossman-like fold.
A.''' The structure of Agd3CBM87 ([{{PDBlink}}6nwz 6NWZ])consists of three ⍺-helices (H1-H3) and a central β-sheet (indicated with a grey box and numbered from N-terminus to C-terminus). A secondary β-sheet (indicated with a grey line) involves the N-terminus of the sixth and C-terminus of the fifth β-strands of the central sheet. '''B.''' Cartoon representation of the central β-sheet showing the arrangement of strands. Numbering is based on location in the primary sequences from N-terminus to C-terminus. Other secondary structure elements are not shown. '''C.''' The side view of the structure with partial transparency reveals the secondary β-sheet. The strands from the central sheet are numbered. Structure images were created in PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).]]

CBM87 members are roughly 220 residues in length and are found N-terminal to [[CE18]] domains. The structure of ''A. fumigatus'' Agd3CBM87, was determined using X-ray crystallography to 2.6 Å resolution (PDB 6NWZ) <cite>Bamford2020</cite>. The structure contains a Rossman-like fold (Figure 1A). The central β-sheet consists of seven strands, with the sixth stand running anti-parallel to the other stands (Figure 1B). The fifth and sixth strands rotate at one end to lie almost perpendicular to the primary central sheet and form the centre of a smaller secondary sheet (Figure 1C). The loops at the C-terminal end of the central β-sheet form a cleft that is predicted to be the location of GAG binding (Figure 2A). Surface exposed conserved aromatic residues (W292 and W344 in ''A. fumigatus'' Agd3) were found to contribute to GAG binding (Figure 2B). Binding studies of site directed alanine mutants of W292 and W344 had reduced affinity for GAG produced by ''A. fumigatus'' cultures, supporting this hypothesis <cite>Bamford2020</cite>. The presence of an open binding cleft suggests that CBM87s can bind within a polysaccharide chain and thus may be [[Carbohydrate-binding_modules#Types|type B]] CBMs.

== Functionalities ==
[[File:CBM87 figures-02.png|thumb|400px|'''Figure 2. Proposed carbohydrate binding site contains two conserved aromatic residues. A.''' Surface representation of the X-ray crystal structure of Agd3CBM87 with W292 and W344 shown in orange. Dotted line represents the proposed binding site for GAG. '''B.''' Sequence logo representation of a sequence alignment of the 24 family members (as of February 1, 2021). Aromatic residues are coloured in orange and all other amino acids are in black. ''A. fumigatus'' Agd3 W292 aligns with aromatic residues (71% W, 25% Y, and 4% F). Logo was created using WebLogo v3 <cite>Crooks2004</cite>. '''C.''' The CBM87 domain (orange) is C-terminal to the [[CE18]] domain (purple) and contributes to an elongated substrate binding cleft. The CBM binding subsites are coloured as in panel A, and the active site of the [[CE18]] is coloured dark blue. The active site zinc ion is coloured grey.]]

CBM87 members are found N-terminal to [[CE18]] domains ([http://www.cazy.org/CE18.html CE18 page of the CAZy Database]). The first characterized [[CE18]], Agd3, was found to be important for exopolysaccharide processing and biofilm formation in the fungus ''A. fumigatus'' <cite>Lee2016</cite>. The structure of Agd3 revealed that the CBM87 domain extends the [[CE18]] active site cleft (Figure 2C). When the CBM87 domain was removed from Agd3, the constructs could not be successfully expressed or purified suggesting that the CBM was required for stability of the catalytic domain <cite>Bamford2020</cite>. Due to this, the levels of deacetylation activity could not be compared between the [[CE18]] alone and in the presence of the CBM. Therefore, mutations were made in the CBM to disrupt predicted carbohydrate binding sites. Mutation of the conserved surface exposed aromatics, W292 or W344, to alanine led to decreased activity in a GAG deacetylation assay <cite>Bamford2020</cite>. The CBM87 domain is proposed to contribute important substrate binding sites leading to increase substrate affinity. This was confirmed using mass spectrometry of synthesized hepta-saccharide substrates which revealed a decreased rate of primary deacetylation by the W344A variant. Wild-type Agd3 is capable of deacetylating GAG polymers on multiple subsites <cite>Bamford2020</cite>. Whether other CBM87 members have similar roles in their cognate systems is still to be determined.

== Family Firsts ==
;First Identified: The first CBM87 member (Agd3CBM87) to be identified was from ''A. fumigatus'' Agd3 <cite>Bamford2020</cite>.
;First Structural Characterization: The first crystal structure of a CBM87 was Agd3CBM87 <cite>Bamford2020</cite>.

== References ==
<biblio>
#Bamford2020 pmid=32415073
#Fontaine2011 pmid=22102815
#Lee2016 pmid=27048799
#Crooks2004 pmid=15173120
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM087]]

CAZypedia - User contributions [en-ca]

Carbohydrate Binding Module Family 87