Enhancement by Mg2+ of domain specificity in Ca2+-dependent interactions of calmodulin with target sequences.
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 Kd approximately 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.
Martin
S R
SR
Division of Physical Biochemistry, National Institute for Medical Research, London, United Kingdom.
Masino
L
L
Bayley
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D004330:Drosophila / Q000737:chemistry; D019922:GAP-43 Protein / Q000737:chemistry; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis
D002147:Calmodulin; D019922:GAP-43 Protein; D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D008274:Magnesium; D002118:Calcium