Triple-Resonance Multidimensional NMR Study of Calmodulin Complexed with a Conformational Change in the Central Helix? the Binding Domain of Skeletal Muscle Myosin Light-Chain Kinase: Indication of

ABSTRACT: Heteronuclear 3D and 4D NMR experiments have been used to obtain 'H, 13C, and 15N 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 29,4659-46671 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 (El 1-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 a-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the Car 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 (kexch> 10 s-I at pH 7, 35 "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 E l 14 at the end of the second helix of sit