Binding of cardiac troponin-I147-163 induces a structural opening in humancardiac tropoin-C

The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central r ole in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an "opening" of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1 997) J. Biol. Chem. 272, 18216-18221; Spyracopoulos et al. (1997) Biochemis try 36, 12138-12146], which is in contrast to the regulatory N-domain of sk eletal TnC (sTnC). This implies that the mode of interaction between cTnC a nd cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115-131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol, Chem. 272, 28494-28500]. The present study demonstr ates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 co mplex, with an affinity approximately six times weaker than that between th e skeletal counterparts. Thus, while Ca2+ does not cause opening, it is req uired for muscle regulation. The solution structure of the cNTnC . Ca2+. cT nI(147-163) complex has been determined by multinuclear multidimensional NM R spectroscopy. The structure reveals an open conformation for cNTnC, simil ar to that of Ca2+-saturated sNTnC. The bound peptide adopts a alpha-helica l conformation spanning residues 150-157. The C-terminus of the peptide is unstructured. The open conformation for Ca2+-saturated cNTnC in the presenc e of cTnI (residues 147-163) accommodates hydrophobic interactions between side chains of the peptide and side chains at the interface of A and B heli ces of cNTnC. Thus the mechanistic differences between the regulation of ca rdiac and skeletal muscle contraction can be understood in terms of differe nt thermodynamics and kinetics equilibria between essentially the same stru cture states.