Vol. 161, No. 3, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS June 30, 1989 Pages 1233-1238 l13Cd-NMR EVIDENCE FOR COOPERATIVE INTERACTION BETWEEN AMINO AND CARBOXYL-TERMINAL DOMAINS OF CALMODULIN Mitsuhiko Ikura#, Nobuko Ilasegawa, Saburo Aimotox, Michio Yazawa, Koichi Yagi, and Kunio Hikich? High-Resolution Nuclear Magnetic Resonance Laboratory and Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060, Japan *Institute for Protein Research, Osaka University, Suita 565, Japan Received May 17, 1989 SUMMARY:. 113Cd~NMR experiments were performed to characterize the nature of Cd2+ binding to calmodulin in the presence of a tetradecapeptide mastoparan or a 26-residue peptide Ml3 (calmodulin-binding region of skeletal muscle myosin light-chain kinase). The results indicate that binding of these peptides to calmodulin induces a positive cooperativity between Ca2+ binding to C- and N-terminal domains. The results imply that the activation of myosin light-chain kinase caused by the increase in Ca2+ concentration occurs as a result of cooperative interactions not only between two Ca2+ binding sites in each domain but also between the two domains. The inter- domain interaction manifests itself only in the presence of such peptides. D 1989 ACddCrnlC Press, 1°C. Calmodulin (C&I) is a ubiquitous intracellular Ca2+-binding protein, which activates a number of enzymes such as myosin light--chain kinase(MLCK) in a Ca2+-dependent manner(l,2). CaMhas four Ca2+ binding sites, two with a high affinity in the Cterminal half domain and the other two with a low affinity in the N-terminal half domain(3,4). Binding of two moles of Ca2+ to each domain occurs in a positively cooperative manner, while the two domains are independent of each other(3,4). The x-ray crystal structure analysis(5- 7) showed that CaM is in a dumbbell-like structure. Two lobes corresponding to the C- and N-terminal domains are connected by an m-helical rod, suggesting the independence of the two domains. The activation of enzymes by C&l is believed to be caused by formation of a CaMenzyme complex. Blumenthal et al. (8) reported that a 27-residue #Present Address: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892. +To whom correspondence should be addressed. 0006-291x/89 $1.50 1233 Copyright 0 1989 by Academic Press, Inc. All righfs of reproduction in any form reserved.