Protein Science, Vol 6, Issue 4 794-807, Copyright © 1997 by Cold Spring Harbor Laboratory Press

ARTICLE

Intermolecular tuning of calmodulin by target peptides and proteins: Differential effects on Ca(2+) binding and implications for kinase activation

O. B. PEERSEN, T. S. MADSEN and J. J. FALKE
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca(2+) binding and Ca(2+) dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase, CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca(2+) binding affinities and kinetics of the two CaM domains over a wide range of Ca(2+) concentrations and time scales. The five peptides increase the Ca(2+) affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca(2+) dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca(2+) affinity 8- to 100-fold and slow dissociation kinetics 13- to 32-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca(2+) affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca(2+) dissociation from the protein complex follows monoexponential kinetics in which all four Ca(2+) ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca(2+) ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca(2)(2+)-CaM or Ca(4)(2+)-CaM after a Ca(2+) signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca(2+) ions from the bound C-terminal domain or the dissociation of Ca(2+) loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca(2+) affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.
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