Calmodulin binding to myosin light chain kinase begins at substoichiometric Ca2+ concentrations: A small-angle scattering study of binding and conformational transitions

We have used small-angle scattering to study the calcium dependence of the interactions between calmodulin (CaM) and skeletal muscle myosin light chai n kinase (MLCK), as well as the conformations of the complexes that form. S cattering data were measured from equimolar mixtures of a functional MLCK a nd CaM or a mutated CaM (B12QCaM) incompetent to bind Ca2+ in its N-termina l domain, with increasing Ca2+ concentrations. To evaluate differences betw een CaM-enzyme versus CaM-peptide interactions, similar Ca2+ titration expe riments were performed using synthetic peptides based on the CaM-binding se quence from MLCK (MLCK-I), Our data show there are different determinants f or CaM binding the isolated peptide sequence compared to CaM binding to the same sequences within the enzyme. For example, binding of either CaM or B1 2QCaM to the MLCK-I peptide is observed even in the presence of EGTA, where as binding of CaM to the enzyme requires Ca2+. The peptide studies also sho w that the conformational collapse of CaM requires both the N and C domains of CaM to be competent for Ca2+ binding as well as interactions with each end of MLCK-I, and it occurs at similar to 2 mol of Ca2+/mol of CaM. We sho w that CaM binding to the MLCK enzyme begins at substoichiometric concentra tions of Ca2+ (less than or equal to 2 mol of Ca2+/mol of CaM), but that th e final compact structure of CaM with the enzyme requires saturating Ca2+. in addition, MLCK enzyme does bind to 2Ca(2+). B12QCaM, although this compl ex is more extended than the complex with native CaM, Our results support t he hypothesis that CaM regulation of MLCK involves an initial binding step at less than saturating Ca2+ concentrations and a subsequent activation ste p at higher Ca2+ concentrations.