A Modulatory Role for the Troponin T Tail Domain in Thin Filament Regulation

Abstract

In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnITnC complex with a tail anchoring it within the thin filament. We have examined the properties of TnT and the TnT 1 tail fragment (residues 1-158) upon reconstituted actin-tropomyosin filaments. Their regulatory effects have been characterized in both myosin S1 ATPase and S1 kinetic and equilibrium binding experiments. We show that both inhibit the actin-tropomyosin-activated S1 ATPase with TnT 1 producing a greater inhibitory effect. The S1 binding data show that this inhibition is not caused by the formation of the blocked B-state but by significant stabilization of the closed C-state with a 10-fold reduction in the C-to M-state equilibrium, K T , for TnT 1 . This suggests TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.

Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T

“…In block VII, Pro-333 of actin was occupied by TnT and myosin cannot bind strongly. Biochemical data also has suggested that TnT modulates the equilibrium between closed and open states (28). The proposed azimuthal shift of tropomyosin between blocks IV and V is supported by tropomyosin crystal structures (29), which showed that the coiled coil is disturbed around residues 151-162, located in the C-terminal half of block IV.…”

Differential Regulation of the Actomyosin Interaction by Skeletal and Cardiac Troponin Isoforms

“…Recent work has suggested that TnT may in fact have a significant role in modulating regulation, which may contribute to the differences between the two cardiac and skeletal complexes seen here (9,10). Although our study characterized skeletal TnT effects using similar assays to those presented here, the other used motility and ATPase measurements.…”

“…TnT also influences the Tm-Tm communication along the filament via its interaction at the Tm-Tm overlap (8) and has been shown recently to also influence regulation directly. Our own work has shown the TnT 1 fragment of skeletal TnT effects the open-closed equilibrium significantly reducing K T (9), whereas work on a similar cardiac TnT 1 fragment has shown strong inhibition of the actinactivated myosin ATPase (10).…”

The Role of Troponins in Muscle Contraction

“…In addition, actin-activated myosin ATPase assays on reconstituted thin filaments showed that these regions might also play a role in the inhibitory activity of Tn in the absence of Ca 2+ , with the HCTnT1 isoform demonstrating the greatest impairment (71). Other recent reports also suggest the importance of the N-terminal tail region of TnT in the inhibition of the myosin S1-actin interaction (72,73). These studies strongly suggest that TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.…”