Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21

TY - JOUR

T1 - Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21

AU - Vaaje-Kolstad, Gustav

AU - Houston, Douglas R.

AU - Riemen, Anna H. K.

AU - Eijsink, Vincent G. H.

AU - van Aalten, Daan M. F.

N1 - MEDLINE® is the source for the MeSH terms of this document.

PY - 2005/3/25

Y1 - 2005/3/25

N2 - 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 ß-sheets, arranged as a ß-sheet sandwich, with a 65-residue "bud" consisting of three short helices, located between ß-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 ß-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.

AB - 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 ß-sheets, arranged as a ß-sheet sandwich, with a 65-residue "bud" consisting of three short helices, located between ß-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 ß-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.

UR - https://www.scopus.com/pages/publications/15744367514

U2 - 10.1074/jbc.M407175200

DO - 10.1074/jbc.M407175200

M3 - Article

AN - SCOPUS:15744367514

SN - 0021-9258

VL - 280

SP - 11313

EP - 11319

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 12

ER -