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J. Biol. Chem., Vol. 265, Issue 17, 9764-9770, 06, 1990
LK MacLachlan, DG Reid, RC Mitchell, CJ Salter and SJ Smith
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.
Binding of a calcium sensitizer, bepridil, to cardiac troponin C. A fluorescence stopped-flow kinetic, circular dichroism, and proton nuclear magnetic resonance study
Department of Physical Organic Chemistry, Smith Kline & French Research Ltd., Welwyn, Hertfordshire, United Kingdom.
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