INTERACTION OF CALMODULIN WITH ITS BINDING DOMAIN OF RAT CEREBELLAR NITRIC-OXIDE SYNTHASE - A MULTINUCLEAR NMR-STUDY

The intercellular messenger nitric oxide is produced through the actio n of nitric oxide synthases, a class of enzymes that is regulated by c alcium-calmodulin (CaM). In this work, the interaction of CaM with a 2 3-amino-acid residue synthetic peptide, encompassing the CaM-binding d omain of constitutive rat cerebellar nitric oxide synthase (cNOS), was investigated by various NMR methods, Cadmium-113 NMR studies showed t hat binding of the cNOS peptide increased the affinity of CaM for meta l ions and induced interdomain cooperativity in metal ion binding as e arlier observed for complexes of CaM with myosin light chain kinase (M LCK) peptides. By using specific isotopically labeled [C-13]methyl-Met and selenomethionine-substituted CaM in two dimensional proton-detect ed C-13 and Se-77 NMR studies, we obtained evidence for the involvemen t of the Met residues of CaM in the binding of the cNOS peptide. These residues form two hydrophobia: surface areas on CaM, and they are als o involved in the binding of other target proteins. A nitroxide spin-l abeled version of the cNOS peptide caused broadening only for NMR reso nances in the N-terminal. half of CaM, showing that the peptide binds with a C to N orientation to the N- and C-terminal domains of CaM, pH titration experiments of CaM dimethylated with [C-13]formaldehyde show that Lys-75 (and Lys-148) experience a large increase in pK(a) upon p eptide binding; this indicates an unraveling of part of the helical li nker region of CaM upon cNOS peptide binding. Taken together, our data show that the cNOS and MLCK peptides bind in a closely analogous fash ion to CaM.