Binding of a calcium sensitizer, bepridil, to cardiac troponin C. A fluorescence stopped-flow kinetic, circular dichroism, and proton nuclear magnetic resonance study.
Stopped-flow fluorescence kinetic measurements, circular dichroism (CD), and 1H nuclear magnetic resonance (NMR) spectroscopy at 360 MHz have been used to study the interaction of the calcium-channel blocker and calmodulin antagonist bepridil with cardiac troponin C (cTnC) in the presence of calcium. The kinetic data show that bepridil reduces the rate of calcium release only from the low affinity, calcium-specific site and not from the two high affinity calcium/magnesium sites. CD measurements indicate that drug binding leads to a small increase in the alpha-helical content of the complex. 1H NMR shows that the protein binds one equivalent of bepridil, with a dissociation constant of approximately 20 microM, only when the low affinity calcium site is occupied. Exchange is fast or intermediate on the chemical shift time scale. Drug binding is shown to be largely localized in the N-terminal domain, containing the low affinity calcium site, by observing the shifting and broadening of several resonances associated with that domain. These include assigned aromatic signals together with methionyl and other methyl signals. Observation of intermolecular nuclear Overhauser effects was precluded by extensive spectral overlap. Consideration of the data from the three techniques permitted a model of the bepridil-cTnC complex to be constructed, using the model of cTnC derived from the x-ray structure of calmodulin (MacLachlan L. K., Reid, D. G., and Carter, N. (1990) J. Biol. Chem. 265, 9754-9763). Binding of bepridil to a prominent hydrophobic depression in the N-terminal domain can be invoked to explain many of the induced changes in the spectral and kinetic properties of the protein. The implications of the model for the calcium sensitizing action of bepridil are discussed.
MacLachlan
L K
LK
Department of Physical Organic Chemistry, Smith Kline & French Research Ltd., Welwyn, Hertfordshire, United Kingdom.
Reid
D G
DG
Mitchell
R C
RC
Salter
C J
CJ
Smith
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D015764:Bepridil / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D015764:Bepridil