Ca(2+) binding and energy coupling in the calmodulin-myosin light chain kinase complex.
We have previously shown that 3 Ca(2+) ions are released cooperatively and 1 independently from the complex between (Ca(2+))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(2+)-binding sites also function cooperatively in equilibrium Ca(2+) 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(2+)-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(2+) 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(2+) binding to one pair of sites is enhanced; the other pair is unaffected. In this complex reversal of enzyme activation occurs when Ca(2+) 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(2+)-independent interactions with calmodulin clearly play a major role in the enzyme complex, and appear to weaken Ca(2+)-dependent interactions with the core calmodulin-binding sequence.
Persechini
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Department of Pharmacology & Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA. ajp2o@crocus.medicine.rochester.edu
Yano
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Stemmer
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D020832:EF Hand Motifs; D004789:Enzyme Activation / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry*; D011485:Protein Binding; D011817:Rabbits; D016415:Sequence Alignment
D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase; D002118:Calcium