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Importance of hydrophobic and polar residues in ligand binding in the family 15 carbohydrate-binding module from Cellvibrio japonicus Xyn10C

Lookup NU author(s): Gavin Pell, Huamao Du, Professor Harry Gilbert, Dr David Bolam

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Abstract

Modular glycoside hydrolases that degrade the plant cell wall often contain noncatalytic carbohydrate-binding modules (CBMs) that interact with specific polysaccharides within this complex macromolecule. CBMs, by bringing the appended catalytic module into intimate and prolonged association with the substrate, increase the rate at which these enzymes are able to hydrolyze glycosidic bonds. Recently, the crystal structure of the family 15 CBM (CBM15) from Cellvibrio japonicus (formerly Pseudomonas cellulosa) Xyn10C was determined in complex with the ligand xylopentaose. In this report we have used a rational design approach, informed by the crystal structure of the CBM15 - ligand complex, to probe the importance of hydrophobic stacking interactions and both direct and water-mediated hydrogen bonds in the binding of this protein to xylan and xylohexaose. The data show that replacing either Trp 171 or Trp 186, which stack against xylose residues n and n + 2 in xylopentaose, with alanine abolished ligand binding. Similarly, replacing Asn 106, Gln 171, and Gln 217, which make direct hydrogen bonds with xylopentaose, with alanine greatly reduced the affinity of the protein for its saccharide ligands. By contrast, disrupting water-mediated hydrogen bonds between CBM15 and xylopentaose by introducing the mutations S108A, Q167A, Q221A, and K223A had little effect on the affinity of the protein for xylan or xylohexaose. These data indicate that CBM15 binds xylan and xylooligosaccharides via the same interactions and provide clear evidence that direct hydrogen bonds are a key determinant of affinity in a type B CBM. The generic importance of these data is discussed.


Publication metadata

Author(s): Pell G, Williamson M, Walters C, Du H, Gilbert HJ, Bolam DN

Publication type: Article

Publication status: Published

Journal: Biochemistry

Year: 2003

Volume: 42

Issue: 31

Pages: 9316-9323

ISSN (print): 0006-2960

ISSN (electronic): 1943-295X

Publisher: American Chemical Society

URL: http://dx.doi.org/10.1021/bi0347510

DOI: 10.1021/bi0347510

PubMed id: 12899618


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