Mapping of a second actin-tropomyosin and a second 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 116 to 148 of troponin I (TnI) in the Ca2+-dependent regulation of vertebrate skeletal muscle contraction, we 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 peptide 96 to 148 (TnI96-148) 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 (TnI96-139) or substitution of residues K141, K142 and K144 with alanine (TnI96-148A2) completely abolishes the enhanced inhibitory effect of this region when compared with TnI96-115. A synthetic peptide, residues 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 important for inhibition, but are critical for interacting with troponin C (TnC). Specific investigations into this region have shown that residues 116 to 126, located directly adjacent to the "inhibitory region" (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 "second TnC binding site", can significantly enhance the binding affinity of the inhibitory region, residues 96 to 115, for TnC in a Ca2+-dependent manner as determined by affinity chromatography analysis. The implication 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 terminus of TnI (Rp1-40, residues 1 to 40) with the C-terminal peptides TnI96-115, TnI96-131 and TnI96-148 showed that only TnI96-115 could be easily displaced from TnC. These results thus suggest that Ca2+ binding to the regulatory sites of TnC (N domain) alters the binding affinity between the 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 domain of TnC. These results have been incorporated into a general model describing the Ca2+-dependent regulation of muscle contraction.
Tripet
B
B
Department of Biochemistry and the MRC Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Van Eyk
J E
JE
Hodges
R S
RS
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D004791:Enzyme Inhibitors; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000502:physiology; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011817:Rabbits; D019209:Troponin C / Q000378:metabolism*; D019210:Troponin I / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D004791:Enzyme Inhibitors; D010455:Peptides; D019209:Troponin C; D019210:Troponin I; D000251:Adenosine Triphosphatases; D002118:Calcium