COMPLEXES FORMED BETWEEN CALMODULIN AND THE ANTAGONISTS J-8 AND TFP IN SOLUTION

The binding of the antagonists N-(8-aminooctyl)-5-iodonaphthalene-1-su lfonamide (J-8) and trifluoperazine (TFP) to intact calcium-saturated bovine calmodulin (CaM) and also of J-8 to the C-terminal domain (tr2c ) has been investigated. Using a combination of NMR methods, including NOESY data, mobility measurements, and chemical shift and line-shape analysis, we show that the primary interaction between J-8 and tr2c is between the naphthalene ring of the antagonist and the hydrophobic po cket of the protein, similar to the binding of the hydrophobic side-ch ain residues of calmodulin target peptides. Comparison of the mobility of the drug, the intensity and pattern of intermolecular NOESY cross- peaks, and chemical shift changes shows that there is no significant c hange in the binding mode in J-8 CaM compared to J-8 tr2c, with one mo lecule binding to each domain. In particular, we find that the mobilit y of the aliphatic amino ''tail'' of J-8 remains highly mobile in both systems. This contrasts with the notion that the tail may bridge betw een the two domains to give a ''globular'' form of CaM. We also show t hat TFP induces very similar shift changes to J-8 and that the stoichi ometry of the major binding event in all three cases is one drug molec ule per domain. It also appears that secondary binding sites for the d rug molecules are present in all three systems.