Binding of levosimendan, a calcium sensitizer, to cardiac troponin C

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca²⁺-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca²⁺-loaded regulatory domain of recombinant cTnC_C35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnC_C35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnC_A-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnC_C35S and cTnC_A-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca²⁺-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN₃), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.