A proton nuclear magnetic resonance and molecular modeling study of calmidazolium (R24571) binding to calmodulin and skeletal muscle troponin C.
1H NMR spectroscopy at 360 MHz has been used to study the interactions between the calmodulin function inhibitor calmidazolium (R24571) and (i) calmodulin (CaM) and (ii) skeletal muscle troponin C (sTnC). One equivalent of racemic calmidazolium binds tightly to CaM and perturbs a number of protein signals, corresponding to residues in both dicalcium-binding domains, in a manner characteristic of slow exchange. Calmidazolium binds with lower affinity to sTnC but still induces widespread perturbations in both domains. Extensive spectral overlap precludes definite assignment of intermolecular nuclear Overhauser effect (NOEs) although intraprotein NOEs do indicate the nature of some drug-induced conformational changes. Relaxation enhancements induced by two spin-labeled calmidazolium analogues demonstrate that several methionine residues of CaM, significantly immobilized by calmidazolium binding, are in fact located at or near its binding sites. These and other residue-specific broadening effects have enabled low resolution models to be constructed of the predominantly hydrophobic drug-binding sites on each domain of CaM. The hydrophobic portions of calmidazolium itself, and its analogues, contact side chains of Ala-15, Leu-18, Phe-19, Val-35, Met-36, Leu-37, Leu-39, Met-51, Met-71, Met-72, and Met-76 in the N-terminal domain of calmodulin, and Ala-88, Val-91, Phe-92, Val-108, Met-109, Leu-112, Phe-141, and Met-145 in its C-terminal domain. The model, and an analogous one of sTnC, can be used to rationalize drug-induced changes in intraprotein NOEs. Issues pertaining to the possible simultaneous binding of calmidazolium to both globular domains of the proteins are discussed in terms of the experimental results and the overall structures of each protein.
Reid
D G
DG
Department of Physical Organic Chemistry, Smith Kline & French Research Ltd., Weiwyn, Herts, United Kingdom.
MacLachlan
L K
LK
Gajjar
K
K
Voyle
M
M
King
R J
RJ
England
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D002147:Calmodulin; D004791:Enzyme Inhibitors; D007093:Imidazoles; D014336:Troponin; D019209:Troponin C; C031938:calmidazolium