CHARACTERIZATION OF THE N-TERMINAL HALF-SATURATED STATE OF CALBINDIN D-9K - NMR-STUDIES OF THE N56A MUTANT

Calbindin D-9k is a small EF-hand protein that binds two calcium ions with positive cooperativity. The molecular basis of cooperativity for the binding pathway where the first ion binds in the N-terminal site ( I) is investigated by NMR experiments on the half-saturated state of t he N56A mutant, which exhibits sequential yet cooperative binding (Lin se S, Chazin WJ, 1995, Protein Sci 4:1038-1044). Analysis of calcium-i nduced changes in chemical shifts, amide proton exchange rates, and NO Es indicates that ion binding to the N-terminal binding loop causes si gnificant changes in conformation and/or dynamics throughout the prote in. In particular, all three parameters indicate that the hydrophobic core undergoes a change in packing to a conformation very similar to t he calcium-loaded state. These results are similar to those observed f or the (Cd2+)(1) state of the wild-type protein, a model for the compl ementary half-saturated state with an ion bound in the C-terminal site (II). Thus, with respect to cooperativity in either of the binding pa thways, binding of the first ion drives the conformation and dynamics of the protein far toward the (Ca2+)(2) state, thereby facilitating bi nding of the second ion. Comparison with the half-saturated state of t he analogous E65Q mutant confirms that mutation of this critical biden tate calcium ligand at position 12 of the consensus EF-hand binding lo op causes very significant structural perturbations. This result has i mportant implications regarding numerous studies that have utilized mu tation of this critical residue for site deactivation.