Structural dynamics of troponin during activation of skeletal muscle

Abstract

Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s −1 ) matches the rate of Ca 2+ binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s −1 ) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s −1 ) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament-myosin head complex under physiological conditions. muscle regulation | excitation-contraction coupling | muscle signaling C ontraction of skeletal and cardiac muscle is initiated by a transient increase in the concentration of intracellular Ca 2+ ions, which bind to troponin in the thin filaments of the muscle sarcomere. This leads to azimuthal movement of tropomyosin around the thin filament, which uncovers the myosin binding sites on actin and allows the head domain of myosin from the thick filaments to bind to actin and generate force (1, 2). In vitro studies using isolated protein components showed that myosin head binding can produce a further motion of tropomyosin, at least in low [ATP] or rigor-like conditions (2-4), but the functional significance of this effect in physiological conditions and intact sarcomeres is not clear.To elucidate the molecular structural basis of muscle regulation and the role of myosin binding in situ, we introduced bifunctional fluorescent probes into the calcium-binding subunit of troponin, troponin C (TnC) (Fig. 1, yellow), in demembranated fibers from skeletal muscle (5-7). One probe cross-linked a pair of cysteines introduced into the C helix of TnC (Fig. 1, green), close to the regulatory Ca 2+ binding sites (Fig. 1, black spheres) in its N-terminal lobe, and reports the rotation and opening of this lobe on binding Ca 2+ (5). The N-lobe opening is associated with binding of the switch peptide of troponin I (TnI) (Fig. 1, blue) to a hydrophobic pocket on its surface, and this is a key step in the signaling pathway of calcium regulation (8, 9).A second probe was attached to the E helix of TnC (Fig. 1, magenta) in its C-terminal lobe, which contains a pair of divalent cation binding sites (Fig. 1, gray spheres) that can bind Mg 2+ as well as Ca 2+ . The C lobe of TnC is clasped between two long helices of TnI, one of which forms a coiled coil with part of the tropomyosin-binding component of troponin, troponin T (TnT) (Fig. 1, orange). The C lobe of TnC and these long TnI and TnT helices form a well-defined structural domain called the "IT arm" (9, 10). Although the C-lobe E helix of TnC is continuous with the N-lob...

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells

“…Strong binding of myosin heads to thin filaments is blocked by blebbistatin; and weak binding, which may not be blocked by blebbistatin, is almost independent of calcium and does not activate the thin filament (35). Moreover, the amplitude and kinetics of the calciuminduced changes in the orientation of the analogous C-and E-helix probes on TnC in skeletal muscle when active force is inhibited by N-benzyl-p-toluenesulphonamide, which has similar effects to blebbistatin, are the same as those produced when myosin binding to actin is completely abolished by removing overlap between thin and thick filaments (29). We conclude that the results of the blebbistatin experiments reported here exclude the possibility that changes in thin filament structure induced by C1mC2 are a secondary consequence of its effects on the thick filament.…”

“…A BR probe on the C-helix of TnC (BR-TnC-C) was used to monitor the orientation of its N-terminal domain containing the regulatory Ca 2+ site (29). A probe on the E-helix of TnC (BR-TnC-E) was used to monitor the orientation of the elongated "IT arm" domain of troponin that also contains segments of troponin I (TnI) and troponin T (TnT) (30).…”

“…A probe on the E-helix of TnC (BR-TnC-E) was used to monitor the orientation of the elongated "IT arm" domain of troponin that also contains segments of troponin I (TnI) and troponin T (TnT) (30). The E-helix probe monitors a step in the thin filament signaling pathway that is closely coupled to the azimuthal movement of tropomyosin that uncovers the myosinbinding sites on actin during Ca 2+ -mediated activation (29). BR-TnC-C or BR-TnC-E was introduced into skinned ventricular trabeculae to replace most of the native TnC, and the orientation of each probe was measured by polarized fluorescence (26) (SI Appendix, Table S1).…”

Order–Disorder Transitions in the Cardiac Troponin Complex

“…Kinetic studies of the troponin complex indicate that the movement of TnI C back into its blocked-state position likely precedes the movements of the switch peptide and IR. 21; 54 To confirm that a loss of Ca 2+ does not lead to dissociation of the switch peptide and IR from TnC N , we performed single-molecule Förster Resonant Energy Transfer (FRET) measurements on a solution-state troponin complex under high- and low- Ca 2+ conditions (methods and controls may be found in Metskas and Rhoades, 2015). 51 Donor and acceptor fluorophores were conjugated to TnI 152 (in the switch peptide) and TnC 35 (in TnC N ), and monitored for changes in FRET that would indicate dissociation of the interaction.…”

“…This concludes the pre-activation of the thin filament, and is consistent with findings that the principal components of thin filament activation are independent of myosin binding. 54 Activation then simply requires myosin binding, which dislodges TnI C . TnI C floats freely in solution, where its disordered conformation may allow low-affinity interaction with the thin filament to sample the binding state of myosin and determine whether contraction is still progressing.…”