Regulatory Properties of the NH2- and COOH-terminal Domains of Troponin T

Abstract

The contraction of skeletal muscle is regulated by Ca 2؉ binding to troponin C, which results in an internal reorganization of the interactions within the troponin-tropomyosin complex. Troponin T is necessary for Ca 2؉ -dependent inhibition and activation of actomyosin. Tro-ponin T consists of an extended NH 2 -terminal domain that interacts with tropomyosin and a globular COOH-terminal domain that interacts with tropomyosin, tro-ponin I, and troponin C. In this study we used recombi-nant troponin T and troponin I fragments to delimit further the structural and regulatory interactions with the thin filament. Our results show the following: (i) the NH 2 -terminal region of troponin T activates the actomy-osin ATPase in the presence of tropomyosin; (ii) the interaction of the globular domain of troponin T with the thin filament blocks ATPase activation in the ab-sence of Ca 2؉ ; and (iii) the COOH-terminal region of the globular domain anchors the troponin C-troponin I bi-nary complex to troponin T through a direct Ca 2؉ -inde-pendent interaction with the NH 2 -terminal region of tro-ponin I. This interaction is required for Ca 2؉ -dependent activation of the actomyosin ATPase activity. Based on these results we propose a refined model for the tropo-nin complex and its interaction with the thin filament. Troponin (Tn) 1 and tropomyosin (Tm) mediate the Ca 2ϩ -de-pendent regulation of skeletal muscle contraction through con-trol of conformational features of the actin-based thin filament (1–5). Actin, Tm, and Tn are present in the thin filaments in a molar ratio of 7:1:1 (6 – 8). The binary troponin C-troponin I (TnC⅐TnI) complex confers little Ca 2ϩ sensitivity to the acto-myosin ATPase activity at physiological ratios of actin to Tn, is not stably assembled on the actin filament, and is not capable of activating the ATPase at high Ca 2ϩ levels. Troponin T (TnT) is necessary for full Ca 2ϩ sensitivity of the actomyosin ATPase (9 –13) and for restoration of full velocity in sliding filament assays (14). The mechanism through which TnT exerts its role in the regulatory function of the troponin complex is not fully under-stood. TnT interacts with TnC, TnI, and Tm and holds the TnC/TnI dimer in the thin filament irrespective of the Ca