Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca 2+ -ligated calmodulin

Abstract

Significance Complex formation between calmodulin and target proteins underlies numerous calcium signaling processes in biology, yet structural and mechanistic details, which entail major conformational changes in both calmodulin and its substrates, have been unclear. We show that a combination of time-resolved electron paramagnetic and NMR measurements can elucidate the molecular mechanism, at the quantitative kinetic and structural levels, of the binding pathway of a peptide substrate from skeletal muscle myosin light-chain kinase to calcium-loaded calmodulin. The mechanism involves coupled folding and binding and comprises a bifurcated process, with rapid, direct complex formation when the peptide interacts first with the C-terminal domain of calmodulin or a slower, two-step complex formation when the peptide interacts initially with the N-terminal domain.