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J. Biol. Chem., Vol. 280, Issue 25, 23718-23726, June 24, 2005

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Probing the Mechanism of Ligand Recognition in Family 29 Carbohydrate-binding Modules^*

James Flint, David N. Bolam, Didier Nurizzo¶, Edward J. Taylor, Michael P. Williamson||, Christopher Walters||, Gideon J. Davies, and Harry J. Gilbert**

From the Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, the Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, and the ^||Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2UH, United Kingdom

The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 Å resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both -manno- and -gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity.

Received for publication, February 10, 2005 , and in revised form, March 11, 2005.

^* This work was supported by the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Table 1S.

The atomic coordinates and structure factors (code 1W8W, 1W8Z, 1W9F, 1W90, 1W8U, 1W8T, and 1WCU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^¶ Present address: ESRF, Grenoble, BP 220, F-38043 Grenoble Cedex, France.

^** To whom correspondence should be addressed. Tel.: 44-191-222-6962; Fax: 44-191-222-7424; E-mail: H.J.Gilbert{at}ncl.ac.uk.

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