Interaction of cardiac troponin C with Ca2+ sensitizer EMD 57033 and cardiac troponin I inhibitory peptide

The binding of Ca2+ to cardiac troponin C (cTnC) triggers contraction in cardiac muscle. In diseased heart, the myocardium is often desensitized to Ca2+, leading to weak cardiac contractility. Compounds that can sensitize cardiac muscle to Ca2+ would have potential therapeutic value in treating heart failure. The thiadiazinone derivative EMD 57033 is an identified 'Ca2+ sensitizer', and cTnC is a potential target of the drug. In this work, we used 2D {H-1, N-15}-HSQC NMR spectroscopy to monitor the binding of EMD 57033 to cTnC in the Ca2+-saturated state. By mapping the chemical shift changes to the structure of cTnC, EMD 57033 is found to bind to the C-domain of cTnC, To test whether EMD 57033 competes with cardiac TnI (cTnI) for cTnC and interferes with the inhibitory function, we examined the interaction of cTnC with an inhibitory cTnI peptide (residues 128-147, cIp) in the absence and presence of EMD 57033, respectively. cTnC was also titrated with EMD 57033 in the presence of cIp, The results show that although both the drug and cIp interact with the C-domain of cTnC, they do not displace each other, suggesting noncompetitive binding sites for the two targets. Detailed chemical shift mapping of the binding sites reveals that the regions encompassing helix G-loop IV-helix I-I are more affected by EMD 57033, while residues located on helix E-loop III-helix F and the linker between sites III and IV are more affected by cIp, In both cases, the binding stoichiometry is 1:1, The binding affinities for the drug are 8.0 +/- 1.8 and 7.4 +/- 4.8 mu M in the absence and presence of cIp, respectively, while those for the peptide are 78.2 +/- 10.3 and 99.2 +/- 30.0 mu M in the absence and presence of EMD 57033, respectively. These findings suggest that EMD 57033 may exert its positive inotropic effect by not directly enhancing Ca2+ binding to the Ca2+ regulatory site of cTnC, but by binding to the structural domain of cTnC, modulating the interaction between cTnC and other thin filament proteins, and increasing the apparent Ca2+ sensitivity of the contractile system.