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J. Biol. Chem., Vol. 280, Issue 12, 11313-11319, March 25, 2005

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Crystal Structure and Binding Properties of the Serratia marcescens Chitin-binding Protein CBP21^*

Gustav Vaaje-Kolstad, Douglas R. Houston¶, Anna H. K. Riemen, Vincent G. H. Eijsink, and Daan M. F. van Aalten||

From the Department of Chemistry, Biotechnology, and Food Science, Postbox 5003, Agricultural University of Norway, N-1432 Ås, Norway and Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom

Chitin proteins are commonly found in bacteria that utilize chitin as a source of energy. CBP21 is a chitin-binding protein from Serratia marcescens, a Gram-negative soil bacterium capable of efficient chitin degradation. When grown on chitin, S. marcescens secretes large amounts of CBP21, along with chitin-degrading enzymes. In an attempt to understand the molecular mechanism of CBP21 action, we have determined its crystal structure at 1.55 Å resolution. This is the first structure to be solved of a family 33 carbohydrate-binding module. The structure reveals a "budded" fibronectin type III fold consisting of two -sheets, arranged as a -sheet sandwich, with a 65-residue "bud" consisting of three short helices, located between -strands 1 and 2. Remarkably, conserved aromatic residues that have been suggested previously to play a role in chitin binding were mainly found in the interior of the protein, seemingly incapable of interacting with chitin, whereas the structure revealed a surface patch of highly conserved, mainly hydrophilic residues. The roles of six of these conserved surface-exposed residues (Tyr-54, Glu-55, Glu-60, His-114, Asp-182, and Asn-185) were probed by site-directed mutagenesis and subsequent binding studies. All single point mutations lowered the affinity of CBP21 for -chitin, as shown by 3–8-fold increases in the apparent binding constant. Thus, binding of CBP21 to chitin seems to be mediated primarily by conserved, solvent-exposed, polar side chains.

Received for publication, June 25, 2004 , and in revised form, November 30, 2004.

^* This work was supported in part by Norwegian Research Council Grant 140497/420. 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 atomic coordinates and structure factors (code 2BEM and 2BEN) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

^¶ Supported by BBSRC CASE studentship (Cyclacel).

^|| Supported by a Wellcome Trust Senior Research Fellowship and the EMBO Young Investigator Programme. To whom correspondence should be addressed. Fax: 44-1382-345764; E-mail: dava{at}davapc1.bioch.dundee.ac.uk.

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