Ca Binding and Energy Coupling in the Calmodulin-Myosin Light Chain Kinase Complex*

We have previously shown that 3 Ca ions are released cooperatively and 1 independently from the complex between (Ca)4-calmodulin and skeletal muscle myosin light chain kinase or a peptide containing its core calmodulin-binding sequence. We now have found that three Ca-binding sites also function cooperatively in equilibrium Ca 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 Ca-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. Ca 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 Ca binding to one pair of sites is enhanced; the other pair is unaffected. In this complex reversal of enzyme activation occurs when Ca 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. Ca-independent interactions with calmodulin clearly play a major role in the enzyme complex, and appear to weaken Ca-dependent interactions with the core calmodulin-binding sequence.