MAPPING OF A 2ND ACTIN TROPOMYOSIN AND A 2ND TROPONIN-C BINDING-SITE WITHIN THE C-TERMINUS OF TROPONIN-I, AND THEIR IMPORTANCE IN THE CA2-DEPENDENT REGULATION OF MUSCLE-CONTRACTION()

To investigate the functional importance of the C-terminal residues 11 6 to 148 of troponin I (TnI) in the Ca2+-dependent regulation of verte brate skeletal muscle contraction, Mie have prepared several synthetic TnI peptide analogs corresponding to various regions within residues 96 to 148 of rabbit skeletal TnI, and analyzed each of these peptides in reconstituted thin filament assays. Our results show that the TnI p eptide 96 to 148 (TnI(96-148)) constitutes the minimal sequence of TnI capable of mediating an inhibitory activity similar to that of intact TnI protein. Truncation of residues 140 to 148 from this region (TnI( 96-139)) or substitution of residues K141, K142 and K144 with alanine (TnI(96-148A2)) completely abolishes the enhanced inhibitory effect of this region when compared with TnI(96-115). A synthetic peptide, resi dues 128 to 148 of TnI, containing residues 140 to 148, now termed the ''second actin-tropomyosin (actin-Tm) binding site'' is able to bind specifically to the actin-Tm filament and can induce a weak inhibitory activity on its own. Residues 116 to 131 of TnI do not appear to be i mportant for inhibition, but are critical for interacting with troponi n C (TnC). Specific investigations into this region have Shown that re sidues 116 to 126, located directly adjacent to the ''inhibitory regio n'' (residues 96 to 115), are critical for allowing TnC to neutralize fully and rapidly the acto-S1-Tm inhibition caused by the various TnI peptides. Furthermore, residues 116 to 131 of TnI, now termed the ''se cond TnC binding site'', can significantly enhance the binding affinit y of Me inhibitory region, residues 96 to 115, for TnC in a Ca2+-depen dent manner as determined by affinity chromatography analysis. The imp lication that TnI residues 116 to 131 bind to the N domain of TnC, and thus the inhibitory region (residues 96 to 115) binds to the C domain of TnC, has made us re-investigate the structural/functional role of the NH2-terminal region of TnI. Studies of competition between the N t erminus of TnI (Rp(1-40), residues 1 to 40) with the C-terminal peptid es TnI(96-115),TnI(96-131) and TnI(96-148) showed that only TnI(96-115 ) could be easily displaced from TnC. These results thus suggest that Ca2+ binding to the regulatory sites of TnC (N domain) alters the bind ing affinity between NH2 terminus and the C terminus of TnI for TnC, i .e. a Ca2+-dependent switch between these two sites of TnI for the C d omain of TnC. These results have been incorporated into a general mode l describing the Ca2+-dependent regulation of muscle contraction. (C) 1997 Academic Press Limited.