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Calcium binding by the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC

TitleCalcium binding by the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC
Publication TypeJournal Article
Year of Publication1998
AuthorsJohnson, PE, Creagh, AL, Brun, E, Joe, K, Tomme, P, Haynes, CA, McIntosh, LP
JournalBiochemistry
Volume37
Pagination12772-12781
Date PublishedSep
Type of ArticleArticle
ISBN Number0006-2960
Keywords4-GLUCANOHYDROLASE, AFFINITY, CA2+, CRYSTAL-STRUCTURE, GLUCANASES, NUCLEAR-MAGNETIC-RESONANCE, RESOLUTION, SPECTROSCOPY, THERMODYNAMICS, THERMOSTABILITY
Abstract

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 degrees C 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 (Delta C-p = -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 +/- 7) 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 Asp 142 and a side chain carboxyl oxygen of Asp 142 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 degrees C at pH 6.0 in the presence of saturating amounts of calcium, confirming that metal ion binding is thermodynamically linked to native-state stability.

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