Ca2+ binding and energy coupling in the calmodulin-myosin light chain kinase complex

Abstract

We have previously shown that 3 Ca2+ ions are released cooperatively and I independently from the complex between (Ca2+)4-calmodulin and skeletal muscle myosin light chain kinase or a peptide containing its core calmodulin-binding sequence. We now have found that three Ca2+-binding sites also function cooperatively in equilibrium Ca2+ binding to these complexes. Replacement of sites I and II in calmodulin by a copy of sites III and IV abolishes these cooperative effects. Energy coupling-dependent increases in Ca2+-binding affinity in the mutant and native calmodulin complexes with enzyme are considerably less than in the peptide complexes, although the complexes have similar affinities. Ca2+ binding to three sites in the native calmodulin-enzyme complex is enhanced; the affinity of the remaining site is slightly reduced. In the mutant enzyme complex Ca2+ binding to one pair of sites is enhanced; the other pair is unaffected. In this complex reversal of enzyme activation occurs when Ca2+ dissociates from the pair of sites with enhanced affinity; more rapid dissociation from the other pair has no effect, although both pairs participate in activation. Ca2+-independent interactions with calmodulin clearly play a major role in the enzyme complex, and appear to weaken Ca2+-dependent interactions with the core calmodulin-binding sequence.