MOLECULAR TUNING OF AN EF-HAND-LIKE CALCIUM-BINDING LOOP - CONTRIBUTIONS OF THE COORDINATING SIDE-CHAIN AT LOOP POSITION-3

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding moti f. The ion binding parameters of this motif are controlled, in part, b y the structure of its Ca2+ binding loop, termed tile EF-loop. The EF- loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular r oles. The present study uses a structurally homologous Ca2+ binding lo op, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third lo op position to intramolecular tuning. 10 different side chains are com pared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equili bria and kinetics. Substitution of an acidic Asp side chain for the na tive Asn is found to generate a 6,000-fold increase in the ion selecti vity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivi ty. Replacement of Asn by neutral side chains differing in size and sh ape each alter die ionic size selectivity in a similar manner, support ing a model in which large-ion size selectivity is controlled by compl ex interactions between multiple side chains rather than by the dimens ions of a single coordinating side chain. Finally, the pattern of pert urbations generated by side chain substitutions helps to explain the p revalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting tile unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.