Enhancement by Mg 2+ of domain specificity in Ca 2+ ‐dependent interactions of calmodulin with target sequences

Abstract

Mg 2+ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca 2+ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca 2+ ‐dependent target interactions. The binding of Mg 2+ does, however, produce significant spectroscopic changes in residues located in the Ca 2+ ‐binding loops, and the Mg‐calmodulin complex is significantly different from apo‐calmodulin in loop conformation. Direct measurement of Mg 2+ binding constants, and the effects of Mg 2+ on Ca 2+ binding to calmodulin, are consistent with specific binding of Mg 2+ , in competition with Ca 2+ . Mg 2+ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg 2+ has a direct role in conferring stability on both domains of apo‐calmodulin. Apo‐calmodulin binds typical target sequences from skeletal muscle myosin light chain kinase and neuromodulin with K d ∼ 70‐90 nM (at low ionic strength). These affinities are virtually unchanged by 5 mM Mg 2+ , in marked contrast to the strong enhancement of peptide affinity induced by Ca 2+ . Under conditions of stimulation and increased [Ca 2+ ], Mg 2+ has a role in directing the mode of initial target binding preferentially to the C‐domain of calmodulin, due to the opposite relative affinities for binding of Ca 2+ and Mg 2+ to the two domains. Mg 2+ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca 2+ threshold for enzyme activation and increases the importance of a partially Ca 2+ ‐saturated calmodulin‐target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca 2+ ‐dependent target interactions.