Modifying Mg2+ binding and exchange with the N-terminal of calmodulin

TO follow Mg2+ binding to the N-terminal of calmodulin (CaM), we substitute d Phe in position 19, which immediately precedes the first Ca2+/Mg2+ bindin g loop, with Trp, thus making F19WCaM (W-Z). W-Z has four acidic residues i n chelating positions, two of which form a native Z-acid pair. We then gene rated seven additional N-terminal CaM mutants to examine the role of chelat ing acidic residues in Mg2+ binding and exchange with the first EF-hand of CaM. A CaM mutant with acidic residues in all of the chelating positions ex hibited Mg2+ affinity similar to that of W-Z. Only CaM mutants that had a Z -acid pair were able to bind Mg2+ With physiologically relevant affinities. Removal of the Z-acid pair from the first EF-hand produced a dramatic 58-f old decrease in its Mg2+ affinity. Additionally, removal of the Z-acid pair led to a 1.8-fold increase in the rate of Mg2+ dissociation. Addition of a n X- or Y-acid pair could not restore the high Mg2+ binding lost with remov al of the Z-acid pair. Therefore, the Z-acid pair in the first EF-hand of C aM supports high Mg2+ binding primarily by increasing the rate of Mg2+ asso ciation.