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J. Biol. Chem., Vol. 280, Issue 1, 530-537, January 7, 2005

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Family 6 Carbohydrate Binding Modules Recognize the Non-reducing End of -1,3-Linked Glucans by Presenting a Unique Ligand Binding Surface^*

Alicia Lammerts van Bueren, Carl Morland, Harry J. Gilbert, and Alisdair B. Boraston¶

From the Department of Biochemistry and Microbiology, University of Victoria, P. O. Box 3055 STN CSC, Victoria, British Columbia V8W 3P6, Canada and the School of Biomedical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom

Enzymes that hydrolyze insoluble complex polysaccharide structures contain non-catalytic carbohydrate binding modules (CBMS) that play a pivotal role in the action of these enzymes against recalcitrant substrates. Family 6 CBMs (CBM6s) are distinct from other CBM families in that these protein modules contain multiple distinct ligand binding sites, a feature that makes CBM6s particularly appropriate receptors for the -1,3-glucan laminarin, which displays an extended U-shaped conformation. To investigate the mechanism by which family 6 CBMs recognize laminarin, we report the biochemical and structural properties of a CBM6 (designated BhCBM6) that is located in an enzyme, which is shown, in this work, to display -1,3-glucanase activity. BhCBM6 binds -1,3-glucooligosaccharides with affinities of 1 x 10⁵ M^-1. The x-ray crystal structure of this CBM in complex with laminarihexaose reveals similarity with the structures of other CBM6s but a unique binding mode. The binding cleft in this protein is sealed at one end, which prevents binding of linear polysaccharides such as cellulose, and the orientation of the sugar at this site prevents glycone extension of the ligand and thus conferring specificity for the non-reducing ends of glycans. The high affinity for extended -1,3-glucooligosaccharides is conferred by interactions with the surface of the protein located between the two binding sites common to CBM6s and thus reveals a third ligand binding site in family 6 CBMs. This study therefore demonstrates how the multiple binding clefts and highly unusual protein surface of family 6 CBMs confers the extensive range of specificities displayed by this protein family. This is in sharp contrast to other families of CBMs where variation in specificity between different members reflects differences in the topology of a single binding site.

Received for publication, September 2, 2004 , and in revised form, October 20, 2004.

The atomic coordinates and structure factors (codes 1W9S, 1W9T, and 1W9W) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. 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.

^¶ A Canada Research Chair in Molecular Interactions. To whom correspondence should be addressed. Tel.: 250-472-4168; Fax: 250-721-8855; E-mail: boraston{at}uvic.ca.

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