NMR studies of the interaction between (calcium ion)(4)-calmodulin and a peptide corresponding to the calmodulin-binding domain of the chicken smooth muscle myosin light chain kinase

The interaction between (Ca$\sp{2+}$)$\sb4$-chicken calmodulin and a peptide corresponding to the calmodulin-binding domain of the chicken smooth muscle myosin light chain kinase (smMLCK) has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. The study was facilitated by the use of $\sp{15}$N-labeled peptide in conjunction with $\sp{15}$N-edited and $\sp{15}$N-correlated $\sp1$H spectroscopy. The peptide and calcium-saturated calmodulin form a 1:1 complex (K$\sb{\rm d}$ = 1.0 nanomolar) which is in slow exchange with free peptide. The $\sp1$H and $\sp{15}$N resonances of the bound peptide have been assigned. The backbone conformation of the bound peptide has been determined by use of distance geometry and related computational methods using an extensive set of structural constraints for the bound peptide derived from the analysis of nuclear Overhauser effects and three bond coupling constants. The backbone conformation of the peptide has been determined to high precision and is generally indicative of helical secondary structure, although non-helical backbone conformations are seen in the middle and at the C-terminal end of the bound peptide. These studies provide the first direct confirmation of the amphiphilic helix model for the structure of peptides bound to calcium-saturated calmodulin. Extensive $\sp1$H and $\sp{15}$N resonance assignments of calmodulin in the complex have been obtained from the analysis of two and three dimensional nuclear magnetic resonance spectra. The assignment of calmodulin in the complex was facilitated by the use of selective labeling of the protein with $\alpha$-$\sp{15}$N labeled valine, alanine, lysine, leucine and glycine. These provided reference points during the main chain directed analysis of three dimensional spectra of complexes prepared with uniformly $\sp{15}$N-labeled calmodulin. The pattern of nuclear Overhauser effects seen among main chain amide NH, C$\sb\alpha$H and C$\sb\beta$H hydrogens indicate that the secondary structure of the globular domains of calmodulin in the complex closely corresponds to that observed in the calcium-saturated state of the protein in the absence of bound peptide. However, the backbone conformation of residues 76 through 84 adopts an extended chain conformation upon binding of the peptide in contrast to its helical conformation in the absence of peptide. The significance of these results are discussed in terms of recently proposed models for the structure of calmodulin-peptide complexes