Mapping the domain of troponin T responsible for the activation of actomyosin ATPase activity - Identification of residues involved in binding to actin

The in vitro Ca2+ regulation of the actomyosin Mg2+-ATPase at physiological ratios of actin, tropomyosin, and troponin occurs only in the presence of troponin T. We have previously demonstrated that a polypeptide correspondin g to the first 191 amino acids of troponin T (TnT-(1-191)) activates the ac tomyosin Mg2+-ATPase in the presence of tropomyosin. In order to further ch aracterize this activation domain, we constructed troponin T fragments corr esponding to residues 1-157 (Tn-T(1-157)), 1-76 (TnT-(1-76)), 77-157 (TnT-( 77-157)), 77-191 (TnT-(77-191)), and 158-191 (TnT-(158-191)). Assays using these fragments demonstrated the following: (a) residues 1-76 do not bind t o tropomyosin or actin; (b) residues 158-191 bind to actin cooperatively bu t not to tropomyosin; (c) the sequence 77-157 is necessary for troponin int eraction with residue 263 of tropomyosin; (d) TnT-(77-191) on its own activ ates the actomyosin ATPase activity as described previously for TnT-(1-191) . TnT-(1-157), TnT-(1-76), TnT-(77-157), TnT-(158-191), and combinations of TnT-(158-191) with TnT-(1-157) or TnT-(77-157) showed no effect on the ATP ase activity. We conclude that the activation of actomyosin ATPase activity is mediated by a direct interaction between amino acids 77 and 191 of trop onin T, tropomyosin, and actin.