Structural dynamics of troponin during activation of skeletal muscle

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 Ca²⁺ 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.Contraction of skeletal and cardiac muscle is initiated by a transient increase in the concentration of intracellular Ca²⁺ 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 Ca²⁺ binding sites (Fig. 1, black spheres) in its N-terminal lobe, and reports the rotation and opening of this lobe on binding Ca²⁺ (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).Open in a separate windowFig. 1.Troponin core complex with bifunctional probes on the muscle thin filament. On the left, the structure of the core complex of troponin from skeletal muscle in the Ca²⁺-saturated form (10) is shown, containing TnC (yellow) and parts of TnI (blue) and TnT (orange). BR probes cross-linked cysteines 56 and 63 (red spheres) along the C helix (green) in the TnC N lobe or cysteines 96 and 103 along the E helix (magenta) in the TnC C lobe. Black spheres indicate the Ca²⁺ regulatory sites and gray spheres the Ca²⁺/Mg²⁺ sites. On the right is the schematic representation of the troponin core complex oriented on the actin filament (light gray) according to the model of Knowles et al. (7). Each complex is anchored to one tropomyosin strand (dark gray) through the N terminus of TnT (orange arrow). Orange and blue sticks form the IT arm. The C terminus of TnI contains the two actin-binding regions (small blue rectangles), the switch peptide (blue triangle) and the remainder of the mobile domain (large blue rectangle). The position of the probes labeling C and E helices of TnC is marked by green and magenta rectangles respectively.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 Mg²⁺ as well as Ca²⁺. 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-lobe D helix in the Ca²⁺-bound crystal structure shown in Fig. 1, the D/E helix is broken in situ, as it is in the crystal structures of the Ca²⁺-bound cardiac isoform and the apo state of the skeletal isoform (9, 10). Thus, the C- and E-helix probes give independent information about the orientations of the TnC N lobe and the IT arm, respectively, in a muscle fiber.We separated the structural effects of Ca²⁺ and myosin binding during activation of demembranated muscle fibers in physiological conditions kinetically, using rapid jumps in intracellular [Ca²⁺] produced by photolysis of nitrophenyl-EGTA (NP-EGTA or caged Ca). Binding of Ca²⁺ to the regulatory sites of troponin is at least 10–20 times faster than myosin binding in the conditions used here, so we were able to resolve the kinetics of intermediate structural changes in the troponin signaling pathway, and relate them to those of calcium binding to troponin, myosin binding to actin, and force generation. We used three additional protocols to assess the role of myosin binding in muscle regulation in physiological conditions: we (i) imposed rapid ramp shortening on active muscle fibers to drive myosin detachment, (ii) abolished active force generation with a small molecule inhibitor, and (iii) stretched the muscle fibers to remove the overlap between the thick and thin filaments. The results lead to a four-state model that describes the sequence of structural changes in troponin and the thin filament during muscle activation.