Ca2+ Binding and Energy Coupling in the Calmodulin-Myosin Light Chain Kinase Complex

• Published in: The Journal of biological chemistry Vol. 275; no. 6; pp. 4199 - 4204
• Main Authors: Persechini, Anthony, Yano, Kenji, Stemmer, Paul M.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 11-02-2000; American Society for Biochemistry and Molecular Biology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.6.4199

We have previously shown that 3 Ca2+ ions are released cooperatively and 1 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.