Isotope-Filtered 2D NMR of a Protein-Peptide Complex: Study of a Skeletal Muscle Myosin Light Chain Kinase Fragment Bound to Calmodulin

An NMR approach is demonstrated for elucidating the conformation of a peptide when tightly bound to a protein. A 26-residue peptide derived from rabbit skeletal muscle myosin light chain kinase, comprising the binding site for calmdulin, was complexed with uniformly (>95%) 15Nand %enriched calmodulin. Improved isotope-filtered two-dimensional NMR techiques were developed for suppressing NMR calmodulin signals. NOE patterns indicate that residues Arg-3 through Ser-21 of the bound peptide form an a-helix. NOE interactions between the peptide and the protein indicate that the N-terminal half of the peptide interacts with the C-terminal domain of calmodulin, and the C-terminal half interacts with the N-terminal calmodulin domain. Calmodulin (CaMI) is a ubiquitous intracellular protein of 148 residues (M, 16.7 kDa) that plays a key role in coupling Ca2+ transients, caused by a stimulus a t the cell surface, to events in the cytosol. It performs this role by calcium dependent binding to a host of intracellular enzymes. CaM contains four calcium binding sites of the “EF-hand” type. Its crystal structure2s indicates that the protein consists of two globular domains, each containing two calcium binding sites, connected by a continuous 26-residue a-helix that separates the two globular domains, giving CaM a dumbbell-type structure in the crystalline state. A recent NMR solution stud9 indicates, however, that the socalled ‘central helix” is disrupted from residue Asp-78 through Ser-8 1, with a high degree of flexibility in this region of the protein. Myosin light chain kinase (MLCK) is one of the enzymes that require CaZ+ and CaM for activity. It has been shown5 that CaM binds to residues 577-602 of rabbit skeletal MLCK. A 26-residue peptide comprising these residues, commonly referred to as M13, has a high affinity (lo4 M) for calcium-loaded CaM. The nature of the interaction between CaM and its targets has been the subject of many biophysical studies.616 However, because (1) Abbreviations used: CaM, calmodulin; MLCK, myosin light chain kinase; M13, a 26-residue peptide of the CaM-binding domain of rabbit skeletal muscle MLCK comprising residues 577-602; COSY, correlated spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; [F2-N]NOESY, NOESY with ”N-filtering in F2 dimension; [Fl-C,F2-C]-COSY, COSY with ”C-filtering in FI and F2 dimensions; [Fl-C,F2-C]-NOESY, NOESY with I3C-filtering in F, and F2 dimensions; [Fl-C/N,FZ-C/N]NOESY, NOESY with ”Cand IJN-filtering in FI and F2 dimensions. (2) Babu, Y. S.; Bugg, C. E.; Cook, W. J. J. Mol. Biol. 1988, 204, 191-204. (3) Kretsinger, R. H.; Rudnick, S. E.; Weissman, L. J. J . Inorg. Biochem. 1986. 28. 289-302. --. --. -~ (4) Ikura, M.; Spera, S.; Barbato, G.; Kay, L. E.; Krinks, M.; Bax, A. Biochemistry 1991, 30, 9216-9228. (5) Blumenthal, D. K.; Takio, K.; Edelman, A. M.; Charbonnean, H.; Titani, K.; Walsh, A.; Krebs, E. G. Proc. Narl. Acad. Sci. U.S.A. 1985,82, (6) For a review, see: Klee, C. B. In Molecular Aspects of Cellular Regulations; Cohen, P., Klee, C. B., Eds.; Elsevier: New York, 1988; Vol. (7) Jackson, A. E.; Carraway, K. L.; F’uett, D.; Brew, K. J. Biol. Chem. (8) Manalan, A. S.; Klee, C. B. Biochemistry 1987, 26, 1382-1390. (9) Klevit, R. E.; Blumenthal, D. K.; Wemmer, D. E.; Krebs, G. E. Bio(10) Seeholzer, S. H.; Cohen, M.; Putkey, J. A.; Means, A. R.; Crespi, H. (1 1) Linse, S.; Drakenberg, T.; Forsen, S. FEBS Lett. 1988, 119, 28-32. (12) Ikura, M.; Hasegawa, N.; Aimoto, S.; Yazawa, M.; Yagi, K.; Hikichi, (13) Seeholzer, S. H.; Wand, A. J. Biochemistry 1989, 28, 4011-4020. (14) Matsushima, N.; Izumi, Y.; Matsuo, T.; Yoshino, H.; Ueki, T.; Mi3 187-3 19 1.