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, fro m Cellulomonas fimi beta-1,4-glucanase CenC with calcium was investiga ted using NMR spectroscopy and calorimetry. CBDN1 binds a single calci um 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 m ol(-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). F rom an NMR line shape analysis, the rate constants for calcium associa tion 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 kine tics 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 postulat ed to form the calcium-binding site of CBDN1. Consistent with the calc ium-independent affinity of CBDN1 for cellopentaose, this exposed site is located on the face of CBDN1 opposite to that forming the oligosac charide-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 t hermodynamically linked to native-state stability.