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Intermolecular tuning of calmodulin by target peptides and proteins: Differential effects on Ca 2+ binding and implications for kinase activation
Author(s) -
Peersen Olve B.,
Madsen Travis S.,
Falke Joseph J.
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060406
Subject(s) - calmodulin , chemistry , kinetics , peptide , receptor–ligand kinetics , biophysics , myosin light chain kinase , kinase , enzyme kinetics , biochemistry , stereochemistry , enzyme , myosin , active site , biology , receptor , physics , quantum mechanics
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 11, 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 CaZ2+‐CaM or Cq2+‐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.