Interactions of structural C and regulatory N domains of troponin C with repeated sequence motifs in troponin I.

Abstract

The actomyosin ATPase inhibitory protein troponin I (TnI) plays a central regulatory role in skeletal and cardiac muscle contraction and relaxation through its calcium-dependent interactions with troponin C (TnC) and actin. Previously we have demonstrated the utility of F29W and F105W mutants of TnC for measurement of binding affinities of inhibitory peptide TnI(96-116) to its regulatory N and structural C domains, both in isolation and in the intact TnC molecule [Pearlstone, J. R. & Smillie, L. B. (1995) Biochemistry 34, 6932-6940]. This approach is now extended to fragment TnI(96-148). Curve-fitting analyses of fluorescence changes induced in the intact TnC mutants and the isolated N and C domains by increasing [TnI(96-148)] have permitted the assignments of K(D) values (designated K(D,N) and K(D,C)) to the interaction of TnI(96-148) with the N and C domains, respectively, of intact TnC. Taken together with the previous data for TnI(96-116) binding, it can be concluded that, within TnI(96-148), residues 96-116 are primarily responsible for binding to C domain of intact TnC and residues 117-148 to its N domain. Inspection of the available mammalian and avian skeletal muscle TnI amino acid sequences reveals a previously unrecognized conserved motif repeated 3-fold, once in the inhibitory peptide region (approximately residues 101-114; designated alpha) and twice more in the region of residues approximately 121-132 (beta) and approximately 135-146 (gamma). The number and distribution of these motifs have important structural implications for the TnI x C complex. In the beta motif of cardiac TnI, as compared with skeletal, several changes in charged amino acids are suggested as candidates responsible for the greater sensitivity of cardiac Ca2+-regulated actomyosin to acidic pH as in ischemia.