Carbohydrate Binding Module Family 62

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• Author: ^^^Cedric Montanier^^^
• Responsible Curator: ^^^Harry Gilbert^^^

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CAZy DB link

https://www.cazy.org/CBM62.html

Ligand specificities

The only known crystallographic structure of a carbohydrate-binding module of family 62 is CtCBM62. Moreover, its structure has been solved in complex with a xyloglucan oligosaccharide, a 6¹-α-D-galactosyl mannotriose (GM3) or arabinose [1]. These sugar complexed crystal structures of CtCBM62 revealed that the interactions between the protein and α-L-arabinopyranose and α-D-galactopyranose are highly conserved. Recognition of an axial O4 is thus a key determinant for the specificity of CtCBM62 for both galactose and arabinopyranose, as opposed to mannose, glucose and xylose.

Isothermal titration calorimetry revealed affinity for a wide range of galactose and/or arabinose containing polysaccharides such as galactomannan, xyloglucan, arabinogalactan and arabinan. The ligand-binding site is located in the the loops that connect the β-sheets. CtCBM62 recognises the non-reducing terminal sugars of oligosaccharides and polysaccharides.

Structural Features

The final structure of CtCBM62 corresponds to residues 739-878 of full length CtXyl5A [2]. It presents a classic β-jelly-roll fold, consisting of five major antiparallel β-strands on one face (β1, 2, 4, 5 and 7) and three antiparallel β-strands on the other face (β3, 6 and 8). Two α-helixes and five loops on the top of the β-jelly-roll complete the structure. A single structural calcium ion is found between the beginning of strand β-8 and the end of helix α-1. It displays typical hepta-coordination and is coordinated to the main-chain O of residues Lys 25, Asp 30 and Ala 130, the Oε2 of Asp 28 and Glu 131, and a bidentate interaction with both the main-chain carbonyl and Oε2 of Thr 33.

CtCBM62 displays a shallow surface groove, containing Trp 44 that runs along the whole of the concave face of the jelly-roll, which could be, by analogy with other CBMs, a ligand-binding site. However, a second tryptophan, Trp 16, is present in the loop area on the top of the β-jelly-roll where a pocket ≈ 7.5 Å wide and ≈ 5 Å deep is located, which is the ligand-binding site. CtCBM62 is thus a type C CBM.

The galactopyranose moiety of both xyloglucan oligosaccharides and GM3 is bound to CtCBM62 through hydrogen bonds with the side-chains of Asp 36, Arg 65, Tyr 68 and Arg 71 in a shallow binding pocket, while the indole side-chain of Trp 16 makes a hydrophobic interaction with the sugar ring. The OH of Tyr 68 hydrogen bonds to the O2 of Gal, while an NH2 of Arg 65 also interacts with the C2 hydroxyl of the hexose sugar. O3 of the sugar ring is hydrogen bonded to the protein by both NH2 of Arg 65 in addition to an O of Asp 36. The C4 hydroxyl is tetra coordinated by a NH2 of Arg 65, both side-chain oxygens of Asp 36 and an NH2 of Arg 71, while the second amine group of Arg 71 hydrogen bonds to the oxygen ring of Gal. The xylose moiety of xyloglucan oligosaccharide does not interact with CtCBM62, but O of the β-1,2 linkage binds to the OH of Tyr 68 and an NH2 of Arg 71. The arabinopyranose is bound to CtCBM62 in a similar way to galactose, except that the hydroxyl in C2 is hydrogen bonded only to the OH of Tyr 68.

Functionalities

CtCBM62 targets polysaccharides containing terminal D-galactose or L-arabinopyranose residues, whereas the appended catalytic domain CtXyl5A from family GH5 is an arabinoxylan-specific xylanase [2]. It is possible that the primary substrate for CtXyl5A is an arabinoxylan that also contains D-galactose side chains recognized by CtCBM62 or that arabinoxylans are in close association with polysaccharides displaying D-galactose or L-arabinofuranose.

It has been demonstrated that in the presence of calcium, CtCBM62 binds ≈ 200-fold more tightly to the galactosyl side-chains of the decorated ligands galactomannan and ≈ 100-fold xyloglucan, than to galactose. Such binding is associated with the formation of an insoluble polysaccharides lattice, which is a classic features of avidity effects [3, 4].

Family Firsts

First Identified

CtCBM62 from the Clostridium thermocellum multi-modular xylanase CtXyl5A.

First Structural Characterization

The first available crystal structure and the first complex structure of a CBM62 is from CtCBM62.

References

1. Montanier CY, Correia MA, Flint JE, Zhu Y, Baslé A, McKee LS, Prates JA, Polizzi SJ, Coutinho PM, Lewis RJ, Henrissat B, Fontes CM, and Gilbert HJ. (2011). A novel, noncatalytic carbohydrate-binding module displays specificity for galactose-containing polysaccharides through calcium-mediated oligomerization. J Biol Chem. 2011;286(25):22499-509. DOI:10.1074/jbc.M110.217372 |
2. Correia MA, Mazumder K, Brás JL, Firbank SJ, Zhu Y, Lewis RJ, York WS, Fontes CM, and Gilbert HJ. (2011). Structure and function of an arabinoxylan-specific xylanase. J Biol Chem. 2011;286(25):22510-20. DOI:10.1074/jbc.M110.217315 |
3. Boraston AB, McLean BW, Chen G, Li A, Warren RA, and Kilburn DG. (2002). Co-operative binding of triplicate carbohydrate-binding modules from a thermophilic xylanase. Mol Microbiol. 2002;43(1):187-94. DOI:10.1046/j.1365-2958.2002.02730.x |
4. Vijayan M and Chandra N. (1999). Lectins. Curr Opin Struct Biol. 1999;9(6):707-14. DOI:10.1016/s0959-440x(99)00034-2 |