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The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca2+ on troponin to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D-EM, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca2+ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca2+ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca2+-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can then begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca2+, troponin operates as an inhibitor, while in activated muscles at high Ca2+, it acts as a promoter to initiate contraction.
Contraction in all muscles results from the relative sliding of thick and thin filaments. The process is driven by the myosin-crossbridge motors projecting from thick filaments and interacting cyclically with actin subunits on the thin filament molecular track. In skeletal and cardiac muscles, contraction is switched on and off by changes in sarcoplasmic free Ca2+ concentration and by the corresponding binding and dissociation of Ca2+ from the troponin complex, located on thin filaments. Crossbridge dynamics is a function of the myosin ATPase, which itself is activated by myosin-crossbridge binding to actin. Thus, any modulation of actin-activation would, in turn, serve to regulate contraction. Indeed, at low Ca2+, actin-myosin interaction is inhibited because myosin binding sites on actin become inaccessible. It is generally recognized that this inhibition occurs when Ca2+-free troponin impinges on elongated tropomyosin to then block myosin binding (reviewed in1,2). In contrast, the involvement of troponin in muscle activation is less well characterized. The crossbridge cycle may turn on simply because the troponin constraint is released at high Ca2+ and tropomyosin defaults to an unstrained, non-blocking position on actin. Alternatively, Ca2+-saturated troponin may play a more dynamic role and facilitate regulatory switching by actively promoting tropomyosin movement away from the blocking position. We have investigated these possibilities using electron microscopy and 3D reconstruction of thin filaments, and report here on troponin’s structural influence over tropomyosin at high Ca2+.
Tropomyosin is a ~40 nm long “coiled-coil” α-helical protein, which lies along the long-pitch double helical array of actin monomers on thin filaments3–5. Tropomyosin molecules associate together in an end-to-end fashion6 to form a continuous strand, with each tropomyosin spanning 7 successive actin molecules. This arrangement is possible because tropomyosin possesses a series of 7 quasi-repeating motifs designed to bind to neighboring actin monomers along filaments7–9. In turn, troponin, consisting of 3 subunits (TnT, TnI, and TnC), binds to tropomyosin at specific points along the tropomyosin molecule10–13; troponin complexes therefore assume the 40 nm periodicity of tropomyosin on thin filaments. Troponin is thought to have multiple, compartmentalized functions, with each subunit having a particular role in binding tropomyosin or Ca2+ or in inhibiting actomyosin ATPase14. TnC is well characterized and functions as the Ca2+ receptor. After binding Ca2+, it neutralizes the inhibition of actomyosin ATPase imposed by TnI (the inhibitory subunit). TnT, a fairly long asymmetric molecule15 (~19 nm), links the entire troponin complex to tropomyosin14,16,17. The N-terminal “tail” of TnT binds alongside tropomyosin on thin filaments, bridging the head-to-tail joint between adjacent tropomyosin molecules. However, the C-terminal part of TnT converges on TnC and TnI and interacts with them to form the core domain of troponin18,19. Other than providing a scaffold for TnI and TnC, its actions near or within the core domain are obscure. The view of TnT solely as a structural intermediary between troponin subunits and tropomyosin may be an oversimplification. In this regard, biochemical approaches have shown that in concert with tropomyosin and the rest of the troponin complex, TnT enhances actomyosin ATPase at high Ca2+(2,20,21), suggesting that it and the troponin core domain play more than just a permissive role during activation. Thus, troponin subunits may have a dual role in thin filament regulation, with TnI being linked to inhibition at low Ca2+ and TnT and the rest of the complex linked to activation at high Ca2+(22).
In the complete absence of other factors, tropomyosin is thought to be able to oscillate laterally over a narrow region of the flat surface of actin23–28. It is generally assumed, although not explicitly established, that this intrinsic ability to shift azimuthally around actin filaments at low energy cost is inherent to and necessary for the thin filament regulatory mechanism23–25. Presumably, tropomyosin location becomes biased towards specific regulatory positions on actin25,29 in the presence of troponin and/or myosin, and depending on levels of Ca2+ binding to troponin and myosin binding to actin. At low Ca2+, tropomyosin localizes over the outer domain of actin (on actin subdomains 1 and 2, covering myosin binding sites; the B-state position). At high Ca2+, tropomyosin moves to actin’s inner domain (to the edge of subdomains 3 and 4, exposing most but not all of the myosin binding site; the C-state position), and after myosin binding it moves further onto the inner domain (exposing the entire myosin binding site; the M-state position)29. Thus the Ca2+-induced movement of tropomyosin is thought to increase the probability of myosin binding, and the resulting myosin interaction leads to a further tropomyosin shift and full activation of the thin filament29.
We recently showed that the C-terminal end of TnI (cTerm-TnI) drives tropomyosin to the B-state blocking position on the actin outer domain at low Ca2+(30,31). Here we have extended these studies to characterize how troponin influences tropomyosin in the presence of Ca2+. Our results demonstrate that a troponin extension, likely to involve TnT and/or parts of the troponin core domain complex, promotes tropomyosin movement away from the blocking position and stabilizes it in the C-state position. Our work therefore supports the view that troponin exhibits dual function, inhibiting actin-myosin interaction at low Ca2+ and facilitating interaction at high Ca2+. Our results suggest further that troponin transforms tropomyosin’s fundamentally ambiguous position on striated muscle actin by dampening tropomyosin’s oscillatory behavior, while actively promoting its movement to B- or C-state configurations.
Thin filaments were reconstituted from F-actin, cardiac troponin and tropomyosin under conditions known to saturate the filaments with the regulatory proteins25,26. Filaments were then negatively stained25,26. EM images of the reconstituted thin filaments showed characteristic double-helical arrays of actin monomers, tropomyosin strands, and troponin densities repeating with a 40 nm periodicity (Fig. 1). The visually apparent, in-register binding of troponin on each helical strand of F-actin at 40 nm intervals has been directly quantified by analysis of class averaged 2D projections of these same filaments32, corroborating that troponin-tropomyosin binds with the same structural arrangement and molar stoichiometry displayed by native thin filaments isolated directly from muscle. Troponin densities were absent in actin-tropomyosin controls (Fig. 1).
Helical reconstructions of Ca2+-treated filaments showed actin subunits and densities that were attributable to tropomyosin, as previously observed (Fig. 2c, d). As expected, the longitudinally continuous tropomyosin strands were well defined and localized over the outer part of the inner domain of actin. This position is distinctly different from that assumed by tropomyosin on the actin outer domain in reconstructions of EGTA-treated filaments (Fig. 2e, f, cf.25,26). In an effort to identify material derived from troponin, the threshold density cutoff was reduced to 1.5 to 3.0 sigma standard deviation units above the mean density (normally >5.0 σ is used). Additional densities, not previously detected, were then evident in the maps of high Ca2+ filaments, but were absent at low Ca2+ at the same or considerably lower threshold density levels. The densities emerged from the center of actin subdomain 1 and traversed the face of the domain in a path that approached and then abutted the C-state tropomyosin (Fig. 2d); i.e. they bridged sites on actin normally occupied by B-state tropomyosin at low Ca2+. As these densities were not seen in actin-tropomyosin controls (Fig. 2b), we attribute them to troponin. As they were also not seen in reconstructions of low Ca2+-treated troponin-tropomyosin filaments (Fig. 2e), we ascribe them to a part of troponin that is visualized only in the Ca2+-saturated conformation.
Helical reconstruction is an averaging technique that treats each successive actin along thin filaments with its associated proteins as equivalent units. Thus well-ordered troponin domains present only on every seventh actin along thin filaments will distribute as if they derived from every single actin and would be expected to appear so in reconstructions. The distribution process dilutes the troponin signal which becomes comparatively low in amplitude relative to actin and tropomyosin. In this case, the amplitude of each extra density observed on every actin in the reconstructions of Ca2+-treated filaments at best may represent one seventh that of the original troponin mass. Nonetheless, the troponin densities observed in the reconstructions were quite robust and displayed high statistical significance at greater than the 99% confidence level (Fig. 3).
In order to assess the impact of troponin on the structure of thin filaments further, we examined the structural interactions of tropomyosin on troponin-free actin, particularly since biochemical studies suggest that cardiac tropomyosin binding is weakened when troponin is absent20. Helical reconstruction of 500 to 1000 nm stretches of actin-tropomyosin (free of troponin) revealed that the mean location of tropomyosin was midway between B- and C-state configurations, possibly reflecting an assemblage of filaments with variable tropomyosin position (Fig. 4a, b). Inspection of reconstructions of the individual filaments contributing to the average map confirmed this possibility. About half the filament reconstructions analyzed could be clearly identified by eye (as well as by fitting protocols) as either being distinctly B- or C-state examples of tropomyosin association. Dividing these two filament classes into separate data sets and averaging each set independently yielded two distinctly different reconstructions where C- and B-state modes were each indistinguishable from those displayed by troponin-tropomyosin filaments at corresponding high or low Ca2+ (Fig. 4c, d; cf.25–27). This demonstrates that approximately half the filaments possessed long stretches that on average were in one or another well-defined regulatory state. Hence, a mixed population of B- and C-state conformations does coexist in these cardiac filament preparations, in part explaining the diffuse localization of cardiac muscle tropomyosin in averaged data26. The more ambiguous positioning of tropomyosin, seemingly in between B- and C-state positions on remainder of the filaments, may reflect a disconnect between the tropomyosin position on the two opposing actin helical strands and/or a disordering of tropomyosin position resulting from a dynamic fluctuation between states.
Previous single particle reconstructions of 40 nm long segments of actin – cardiac tropomyosin filaments suggested a similar positional ambiguity for tropomyosin26. Attempts were made here to classify segments from the above set of filaments showing poorly localized tropomyosin. Cross-correlation against B-, C- or intermediate state models did not succeed in sorting the data into demonstrably separate categories and reconstructions generated showed no uniquely distinct tropomyosin positional states. Thus, single particle methods and helical analysis both identified a set of filaments characterized by an apparent local disordering of cardiac tropomyosin on actin in the absence of troponin.
By contrast, marked variability in tropomyosin position has not been observed for preparations of filaments composed of cardiac troponin-tropomyosin; without exception, helical reconstruction showed that on average tropomyosin along all single filaments examined at low Ca2+ could be readily classified as belonging to the B-state mode and analogously tropomyosin along all filaments in high Ca2+ to the C-state configuration. Similarly, single particle analysis of short segments of these filaments indicated that while localized oscillation between positional modes appears to occur, tropomyosin occupied characteristic regulatory state positions along roughly 80 percent of filament lengths26.
The precision with which tropomyosin is positioned is not simply a matter of troponin being present or absent, but rather also depends on the isoform of tropomyosin examined. For example, polymorphic positioning of tropomyosin was not observed here or previously25,33 on thin filaments containing smooth muscle tropomyosin (without troponin), where a single mode of binding interaction was detected. In fact, the variance associated with densities connecting cardiac tropomyosin to actin is higher than that for the smooth muscle tropomyosin connection, particularly near subdomain 1 (Fig. 5).
The location of tropomyosin on actin is defined by weak electrostatic contacts8. In turn, both the collective strength of these weak interactions and the stiffness of the tropomyosin strand on actin will influence tropomyosin’s responsiveness to mechanical or chemical perturbations. Maytum et al.28 (2008) likened the behavior of different tropomyosin isoforms to variably vibrating guitar strings being held taut or not, where the additional displacement of the guitar strings by a pick, would be the counterpart of troponin or other proteins disturbing preset average equilibrium positions. Thus in a case in which tropomyosin is well localized on actin, for example in smooth muscle filaments25,, the tropomyosin in effect is taut and well positioned by electrostatics. Non-muscle tropomyosins, also well localized on actin, could be similarly described25,28,34. A high degree of chemomechanical specificity may be required in these cases of troponin-free filaments, in order to maximally stabilize tropomyosin on actin. In contrast, we have shown here that the position of cardiac striated muscle tropomyosin on actin is less well defined, as if the isoform were held more loosely on actin, at least in the absence of troponin (cf.21). Given this extra degree of plasticity, limited as it might be to yielding azimuthal oscillations amounting to between 15 Å and 25 Å(27), the behavior of tropomyosin on actin filaments may be more easily fine-tuned by troponin.
We have previously demonstrated that the mechanochemical equilibrium balance of tropomyosin that determines its positional state is easily perturbed by small changes in electrostatic interactions between actin and tropomyosin25. We also showed that the content of αα-, ββ-, or αβ-tropomyosin isoforms may strongly influence the equilibrium balance of tropomyosin on actin25. Less well characterized variation in the end-to-end contacts between successive tropomyosins along tropomyosin and their degree of phosphorylation may also influence tropomyosin interactions. Whether such subtleties affect tropomyosin positioning in vivo and are physiologically meaningful is not known.
We have recently indicated that at low Ca2+, a C-terminal domain of TnI (cTerm-TnI) binds to actin, displaces tropomyosin from the C-state and stabilizes it in the B-state position31. This would account for the unambiguous position of tropomyosin in low Ca2+-treated thin filaments. We suggest here that at high Ca2+, C-terminal domains of TnT or parts of the troponin core domain complex move over the B-state binding sites, promote tropomyosin movement and then stabilize tropomyosin in the C-state, thus accounting for tropomyosin’s positional fidelity in Ca2+-treated filaments. Hence the presence of troponin appears to diminish the positional promiscuity of cardiac tropomyosin at both low and high Ca2+. We also previously indicated that the N-terminal TnT tail domain stabilizes tropomyosin in a B-state position35. Thus the TnT tail alone cannot promote the transition of tropomyosin to the C-state at high Ca2+. Clearly additional parts of troponin, whose domain structures are yet to be identified, are required for inducing tropomyosin movement to the C-state.
We have suggested that the blocked, B-state is brought about and muscle relaxation ensues at low Ca2+ because tropomyosin is wedged in an inhibitory position between the cTerm-TnI regulatory domain on one side and TnT and the troponin core domain on the other31. This configuration may compress the C-terminal end of TnT against the troponin core domain complex. At low Ca2+, the core domain may also become constricted and thus more limited in its binding to targets on actin. It follows that once cTerm-TnI dissociates from actin at high Ca2+, such strain will be released, thrusting tropomyosin toward the C-state position (Fig. 6). The extra density seen in high Ca2+ reconstructions is likely to represent an obliquely oriented segment of troponin that associates closely with actin and drives this process. Comparable densities are not resolved from tropomyosin in low Ca2+-reconstructions, possibly because they may be compressed linearly against the elongated protein or become disordered by a radial displacement from actin.
Our results support the hypothesis that troponin is intimately involved in both the inhibition and the activation of muscle contraction. We propose that the counterpunching action of the mobile parts of troponin determines the positions of tropomyosin at low and high Ca2+, thereby regulating the thin filament. We argue further that the inherent positional ambiguity of striated muscle tropomyosin on actin is an adaptation attuned to troponin function. While we have not defined how the entire troponin complex reconfigures during muscle activation and inhibition, our results indicate that key structural domains of troponin bring about striking steric effects that control actin-activation of myosin ATPase and consequently contraction.
We thank Ms. Karen Moore (Moore Design) for artwork in Figure 6. This research was supported by grants from the National Institutes of Health to W.L. (HL36153, HL86655), L.S.T. (HL38834, HL63774) and R.C (AR34711, RR08426).
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