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

To follow Mg2+ binding to the N-terminal of calmodulin (CaM), we substituted Phe in position 19, which immediately precedes the first Ca2+/Mg2+ binding loop, with Trp, thus making F19WCaM (W-Z). W-Z has four acidic residues in chelating positions, two of which form a native Z-acid pair. We then generated seven additional N-terminal CaM mutants to examine the role of chelating 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 exhibited 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-fold 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 an X- or Y-acid pair could not restore the high Mg2+ binding lost with removal of the Z-acid pair. Therefore, the Z-acid pair in the first EF-hand of CaM supports high Mg2+ binding primarily by increasing the rate of Mg2+ association.