Abstract
Heart muscle contraction is regulated by Ca(2+) binding to the thin filament protein troponin C. In cardiovascular disease, the myofilament response to Ca(2+) is often altered. Compounds that rectify this perturbation are of considerable interest as therapeutics. Plant flavonoids have been found to provide protection against a variety of human illnesses such as cancer, infection, and heart disease. (-)-Epigallocatechin gallate (EGCg), the prevalent flavonoid in green tea, modulates force generation in isolated guinea pig hearts (Hotta, Y., Huang, L., Muto, T., Yajima, M., Miyazeki, K., Ishikawa, N., Fukuzawa, Y., Wakida, Y., Tushima, H., Ando, H., and Nonogaki, T. (2006) Eur. J. Pharmacol. 552, 123-130) and in skinned cardiac muscle fibers (Liou, Y. M., Kuo, S. C., and Hsieh, S. R. (2008) Pflugers Arch. 456, 787-800; and Tadano, N., Yumoto, F., Tanokura, M., Ohtsuki, I., and Morimoto, S. (2005) Biophys. J. 88, 314a). In this study we describe the solution structure of the Ca(2+)-saturated C-terminal domain of troponin C in complex with EGCg. Moreover, we show that EGCg forms a ternary complex with the C-terminal domain of troponin C and the anchoring region of troponin I. The structural evidence indicates that the binding site of EGCg on the C-terminal domain of troponin C is in the hydrophobic pocket in the absence of troponin I, akin to EMD 57033. Based on chemical shift mapping, the binding of EGCg to the C-terminal domain of troponin C in the presence of troponin I may be to a new site formed by the troponin C.troponin I complex. This interaction of EGCg with the C-terminal domain of troponin C.troponin I complex has not been shown with other cardiotonic molecules and illustrates the potential mechanism by which EGCg modulates heart contraction.
Highlights
States, ϳ1 in 3 deaths in 2004 were caused by Cardiovascular disease (CVD) (1)
Two-dimensional 1H,15N HSQC and 1H,13C HSQC NMR experiments were used to monitor the binding of Epigallocatechin gallate (EGCg) to cTnC1⁄73Ca2ϩ, cCTnC1⁄72Ca2ϩ, and cCTnC1⁄72Ca2ϩ1⁄7cTnI-(34 – 71)
NMR has been utilized to show that W7 binds both the C- and N-terminal domains of cardiac TnC (cTnC) in the absence of cTnI (43); in the presence of cTnI-(34 –71) and cTnI-(128 –163), W7 associates exclusively in the N-terminal domain of cTnC (52)
Summary
Sample Preparation—The expression vectors for cCTnC(91–161) and cTnC were designed, and the uniformly labeled 13C,15N-cCTnC was isolated from Escherichia coli as described previously (31, 32). Where degradation of EGCg was witnessed, the solution first began to change from clear to a brownish hue, and eventually, precipitate started to form at the bottom of the NMR tube In this stage the 1H,15N HSQC spectrum revealed a slight recession of the amide correlation peaks toward the unbound chemical shifts of cCTnC. EGCg was titrated into a 80 M 15N-cCTnC NMR sample to final EGCg concentrations at each step of 8, 17, 25, 32, 41, 49, 57, 65, 83, 98, 113, 143, 218, 293, 378, 443, 578, and 713 M. The final refined ensemble has been deposited in the Protein Data Bank with the accession code of 2kdh
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