Drug binding to cardiac troponin C.

Compounds that sensitize cardiac muscle to Ca(2+) by intervening at the level of regulatory thin filament proteins would have potential therapeutic benefit in the treatment of myocardial infarctions. Two putative Ca(2+) sensitizers, EMD 57033 and levosimendan, are reported to bind to cardiac troponin C (cTnC). In this study, we use heteronuclear NMR techniques to study drug binding to [methyl-(13)C]methionine-labeled cTnC when free or when complexed with cardiac troponin I (cTnI). In the absence of Ca(2+), neither drug interacted with cTnC. In the presence of Ca(2+), one molecule of EMD 57033 bound specifically to the C-terminal domain of free cTnC. NMR and equilibrium dialysis failed to demonstrate binding of levosimendan to free cTnC, and the presence of levosimendan had no apparent effect on the Ca(2+) binding affinity of cTnC. Changes in the N-terminal methionine methyl chemical shifts in cTnC upon association with cTnI suggest that cTnI associates with the A-B helical interface and the N terminus of the central helix in cTnC. NMR experiments failed to show evidence of binding of levosimendan to the cTnC.cTnI complex. However, levosimendan covalently bound to a small percentage of free cTnC after prolonged incubation with the protein. These findings suggest that levosimendan exerts its positive inotropic effect by mechanisms that do not involve binding to cTnC.

Identification of binding sites for bepridil and trifluoperazine on cardiac troponin C.

The solution structure of cardiac troponin C (cTnC) (Sia, S., Li, M. X., Spyracopoulos, L., Gagne, S. M., Liu, W., Putkey, J. A. & Sykes, B. D. (1997) J. Biol. Chem. 272, 18216-18221) challenges existing structure/function models for this critical regulatory protein. For example, it is clear that the closed conformation of the regulatory N-terminal domain in Ca2+-bound cardiac troponin C (cTnC) presents a much different binding surface for Ca2+-sensitizing compounds than previously thought. We report here the use of Met methyl groups as site-specific structural markers to identify drug binding sites for trifluoperazine and bepridil on cTnC. Drug dependent changes in the NMR heteronuclear single-quantum coherence spectra of [methyl-13C]Met-labeled cTnC indicate that bepridil and trifluoperazine bind to similar sites but only in the presence of Ca2+. There are 3-4 drug binding sites in the N- and C-terminal domains of intact cTnC that exhibit fast exchange on the NMR time scale. Use of a novel spin-labeled phenothiazine and detection of isotope-filtered nuclear Overhauser effects allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain and on two hydrophobic surfaces on the N-terminal regulatory domain. The data presented here, coupled with our previous study using covalent blocking groups, support a model in which the Ca2+-sensitizing binding site includes Met-45 in helix B of site I, and Met-60 and -80 in helices B and C of the regulatory site II. This subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds.

Cardiac troponin I induced conformational changes in cardiac troponin C as monitored by NMR using site-directed spin and isotope labeling.

Conformational changes in both free cardiac troponin C (cTnC) and in complex with a recombinant troponin I protein [cTnI(33-211), cTnI(33-80), or cTnI (86-211)] were observed by means of a combination of selective carbon-13 and spin labeling. The paramagnetic effect from the nitroxide spin label, MTSSL, attached to cTnC(C35S) at Cys 84 allowed measurement of the relative distances to the 13C-methyl groups of the 10 methionines of cTnC in the monomer or complex. All 10 1H-13C correlations in the heteronuclear single- and multiple-quantum coherence (HSMQC) spectrum of [13C-methyl] Met cTnC in the complex with cTnI(33-211) were previously assigned [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., & Rosevear, P. R. (1994) J. Biol. Chem. 269, 23731-23735]. In the presence of oxidized spin label, nine of the 10 Met methyl 1H-13C correlations of cTnC were significantly broadened in the cTnC(C35S) monomer. This suggests flexibility within the central helix, or interdomain D/E helical linker, bringing the N- and C-terminal domains in closer proximity than predicted from the crystallographic structure of TnC. In the spin-labeled cTnC(C35S). cTnI(33-211) complex only N-terminal Met methyl 1H-13C correlations of cTnC(C35S) were paramagnetically broadened beyond detection, whereas correlations for Met residues (103, 120, 137, and 157) in the C-terminal domain were not. Thus, complex formation with cTnI decreases interdomain flexibility and maintains cTnC in an extended conformation. This agrees with the recently published study suggesting that sTnC is extended when bound to sTnI [Olah, G. A., & Trewhella, J. (1994) Biochemistry 33, 12800-12806]. The recombinant N-terminal domain of cTnI, cTnI(33-80), gave similar results as observed with cTnI(33-211) when complexed with spin-labeled cTnC(C35S). However, complex formation with the C-terminal fragment, cTnI(86-211), which contains the inhibitory sequence, is insufficient to maintain cTnC extended to the amount observed with either cTnI(33-211) or cTnI(33-80); although compared to that observed in free cTnC, it does cause decreased flexibility in the interdomain linker. In the absence of the N-terminal domain of cTnI, there is a decrease in flexibility within the N-terminal domain of cTnC. Interestingly, the N-terminal domain of cTnC in the reduced spin-labeled complex with cTnI(86-211), in the presence of ascorbate, showed two distinct conformations which were not seen in the complex with cTnI(33-211).(ABSTRACT TRUNCATED AT 400 WORDS)

NMR studies delineating spatial relationships within the cardiac troponin I-troponin C complex.

NMR spectroscopy and selective isotope labeling of both recombinant cardiac troponin C (cTnC3) and a truncated cardiac troponin I (cTnI/NH2) lacking the N-terminal 32-amino acid cardiac-specific sequence have been used to probe protein-protein interactions central to muscle contraction. Using [methyl-13C]Met-labeled cTnC3, all 10 cTnC Met residues of Ca(2+)-saturated cTnC3 could be resolved in the two-dimensional heteronuclear single- and multiple-quantum coherence spectrum of the cTnI.cTnC complex. Based on the known Met assignments in cTnC3, the largest chemical shift changes were observed for Met81, Met120, Met137, and Met157. Methionines 120, 137, and 157 are all located in the C-terminal domain of cTnC. Methionine 81 is located at the N terminus of the central helix. Minimal chemical shift changes were observed for Met45, Met47, and Met103 of cTnC3 in the cTnI.cTnC complex. All 6 Met residues in [methyl13C]Met-labeled cTnI/NH2 could be resolved in the cTnI.cTnC complex, suggesting that both cTnI and cTnC form a stable homogeneous binary complex under the conditions of the NMR experiment. In the absence of added protease inhibitors in the cTnI.cTnC complex, cTnI/NH2 was found to undergo selective proteolysis to yield a 5.5-kDa N-terminal fragment corresponding to residues 33-80. Judging from the NMR spectra of [methyl13C]Met-labeled cTnC3, cTnI-(33-80) was sufficient for interaction with the C-terminal domain of cTnC in a manner identical to that observed for native cTnI/NH2. However, in the presence of the proteolytic fragment cTnI-(33-80), the chemical shift of Met81 was not perturbed from its position in free cTnC3. Thus, residues located C-terminal to Arg80 in cTnI appear to be responsible for interaction with the N-terminal half of cTnC. Taken together, these results provide strong evidence for an antiparallel arrangement for the two proteins in the troponin complex such that the N-terminal portion of cTnI interacts with the C-terminal domain of cTnC. This interaction likely plays a role in maintaining the stability of the TnI.TnC complex.