TRIPLE-RESONANCE MULTIDIMENSIONAL NMR-STUDY OF CALMODULIN COMPLEXED WITH THE BINDING DOMAIN OF SKELETAL-MUSCLE MYOSIN LIGHT-CHAIN KINASE - INDICATION OF A CONFORMATIONAL CHANGE IN THE CENTRAL HELIX

Heteronuclear 3D and 4D NMR experiments have been used to obtain H-1, C-13, and N-15 backbone chemical shift assignments in Ca2+-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667] shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca2+-binding site I (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca2+-binding site IV (F141-M145). Analysis of backbone NOE connectivities indicates a change from alpha-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the C-alpha and carbonyl chemical shifts of these residues relative to M13-free calmodulin and by hydrogen-exchange experiments that indicate that the amide protons of residues 75-82 are in fast exchange (k(exch) > 10 s-1 at pH 7, 35-degrees-C) with the solvent. No changes in secondary structure are observed for the first helix of site I or the C-terminal helix of site IV. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site III.