Enhancement by Mg²⁺ of domain specificity in Ca²⁺-dependent interactions of calmodulin with target sequences

Abstract

Mg²⁺ binds to calmodulin without inducing the changes in secondary structure that are characteristic of Ca²⁺ binding, or the exposure of hydrophobic surfaces that are involved in typical Ca²⁺-dependent target interactions. The binding of Mg²⁺ does, however, produce significant spectroscopic changes in residues located in the Ca²⁺-binding loops, and the Mg-calmodulin complex is significantly different from apo-calmodulin in loop conformation. Direct measurement of Mg²⁺ binding constants, and the effects of Mg²⁺ on Ca²⁺ binding to calmodulin, are consistent with specific binding of Mg²⁺, in competition with Ca²⁺. Mg²⁺ increases the thermodynamic stability of calmodulin, and we conclude that under resting, nonstimulated conditions, cellular Mg²⁺ 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 Mg²⁺, in marked contrast to the strong enhancement of peptide affinity induced by Ca²⁺. Under conditions of stimulation and increased [Ca²⁺], Mg²⁺ 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 Ca²⁺ and Mg²⁺ to the two domains. Mg²⁺ thus amplifies the intrinsic differences of the domains, in a target specific manner. It also contributes to setting the Ca²⁺ threshold for enzyme activation and increases the importance of a partially Ca²⁺-saturated calmodulin–target complex that can act as a regulatory kinetic and equilibrium intermediate in Ca²⁺-dependent target interactions.