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 ca rdiac muscle. In diseased heart, the myocardium is often desensitized to Ca 2+, leading to weak cardiac contractility. Compounds that can sensitize car diac muscle to Ca2+ would have potential therapeutic value in treating hear t failure. The thiadiazinone derivative EMD 57033 is an identified 'Ca2+ se nsitizer', and cTnC is a potential target of the drug. In this work, we use d 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 cTn C, To test whether EMD 57033 competes with cardiac TnI (cTnI) for cTnC and interferes with the inhibitory function, we examined the interaction of cTn C with an inhibitory cTnI peptide (residues 128-147, cIp) in the absence an d presence of EMD 57033, respectively. cTnC was also titrated with EMD 5703 3 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, su ggesting noncompetitive binding sites for the two targets. Detailed chemica l 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 lo cated on helix E-loop III-helix F and the linker between sites III and IV a re more affected by cIp, In both cases, the binding stoichiometry is 1:1, T he 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 p ositive 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, a nd increasing the apparent Ca2+ sensitivity of the contractile system.