Calcium binding by the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC.
The interaction of the N-terminal cellulose-binding domain, CBDN1, from Cellulomonas fimi beta-1,4-glucanase CenC with calcium was investigated using NMR spectroscopy and calorimetry. CBDN1 binds a single calcium ion with an equilibrium association constant of approximately 10(5) M-1 at 35 degreesC and pH 6.0. Binding is exothermic (-42 +/- 2 kJ mol-1) under these conditions and is accompanied by a small negative change in heat capacity (DeltaCp = -0.41 +/- 0.16 kJ mol-1 K-1). From an NMR line shape analysis, the rate constants for calcium association and dissociation were found to be (5 +/- 2) x 10(7) s-1 M-1 and (4.5 +/- 0.6) x 10(2) s-1, respectively. The rapid association kinetics indicate that the calcium-binding site on CBDN1 is accessible and, to the first approximation, preformed. Based on patterns of chemical shift perturbations, and structural comparisons with the Bacillus sp. 1, 3-1,4-beta-glucanases, the backbone carbonyl oxygens of Thr8, Gly30, and Asp142 and a side chain carboxyl oxygen of Asp142 are postulated to form the calcium-binding site of CBDN1. Consistent with the calcium-independent affinity of CBDN1 for cellopentaose, this exposed site is located on the face of CBDN1 opposite to that forming the oligosaccharide-binding cleft. The midpoint denaturation temperature of CBDN1 is increased by approximately 8 degreesC at pH 6.0 in the presence of saturating amounts of calcium, confirming that metal ion binding is thermodynamically linked to native-state stability.
Johnson
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Protein Engineering Network of Centres of Excellence, The University of British Columbia, Vancouver, Canada.
Creagh
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Brun
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Joe
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Tomme
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Haynes
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McIntosh
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D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D044643:Glucan 1,4-beta-Glucosidase; D006093:Gram-Positive Asporogenous Rods / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; C110968:CipB protein, Photorhabdus luminescens; D009844:Oligosaccharides; D010446:Peptide Fragments; C019193:maltopentaose; D002482:Cellulose; D001617:beta-Glucosidase; D044643:Glucan 1,4-beta-Glucosidase; D002118:Calcium