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

• Published in: The Journal of biological chemistry 2005-03, Vol.280 (12), p.11313-11319
• Main Authors: Vaaje-Kolstad, Gustav, Houston, Douglas R., Riemen, Anna H.K., Eijsink, Vincent G.H., van Aalten, Daan M.F.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Chitin - metabolism; Crystallography; Dimerization; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; Protein Structure, Secondary
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M407175200

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 β-sheets, arranged as a β-sheet sandwich, with a 65-residue “bud” consisting of three short helices, located between β-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 β-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.