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3.15: Troponin C

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    The contraction of striated muscle is triggered by Ca2+ ions. Muscle cells are highly specialized, and contain two types of filaments that may slide past each other in an energy-consuming process. One of the filaments, the thin filament, is built up by actin molecules (Mr ≈ 42 kDa) polymerized end-to-end in a double helix. In the grooves of this helix runs a long rod-like molecule, tropomyosin; and located on this molecule at every seventh actin, is a complex of three proteins, troponin. The three proteins in the troponin complex are troponin I (TnI), troponin T (TnT), and troponin C (TnC). A schematic picture of the organization of the thin filament is shown in Figure 3.20.

    Figure 3.20 - Schematic diagram of the organization of skeletal muscle thin filament, showing the position of tropo-myosin and the troponin complex on the actin filament. The binding of Ca2+ to TnC, the calcium-binding subunit of the troponin complex, removes TnI, the inhibitory subunit, from actin and thus permits an interaction with a specialized protein, myosin, on neighboring thick muscle filaments (not shown). An ATP-driven conformation change in the myosin head group makes the thick and thin filaments move relative to one another, so that muscle contraction occurs.

    Troponin C is the Ca2 -binding subunit of troponin, and it is structurally highly homologous to calmodulin. Skeletal-muscle troponin C (sTnC; Mr ≈ 18 kDa) can bind four Ca2+ ions, but cardiac-muscle troponin C (cTnC) has one of the four calcium sites modified, so that it binds only three Ca2+ ions. The x-ray structures of sTnC from turkey and chicken skeletal muscle have been determined to resolutions of 2.8 and 3.0 Å, respectively.102,103 The structure of turkey sTnC is shown in Figure 3.21.

    Figure 3.17).

    The similarity between the structures of CaM (Figure 3.17) and sTnC is obvious. In sTnC we again find two domains, each with two potential Ca2+ sites, separated by a 9-turn \(\alpha\)-helix. The crystals were grown in the presence of Ca2+ at a low pH (pH = 5), and only two Ca2+ ions are found in the C-terminal domain. The two Ca2+-binding sites in this domain have the same helix-loop-helix motif that is found in CaM, and they both conform to the archetypal EF-hand structure. The interhelix angles between helices E and F and between G and H are close to 110°. By contrast, the helices in the N-terminal domain, where no Ca2+ ions are bound, are closer to being antiparallel, with interhelix angles of 133° (helices A and B) and 151° (helices C and D).

    Both sTnC and cTnC have two high-affinity Ca2+-binding sites (see Table 3.2) that also bind Mg2+ ions competitively, although with a much lower affinity. These two sites are usually called "the Ca2+-Mg2+ sites." 76,104 In sTnC there are also two (in cTnC, only one) Ca2+-binding sites of lower affinity (KBCa2+ ≈ 105 M-1) that bind Mg2+ weakly and therefore have been called "the Ca2+-specific sites." Since Ca2+ binding to the latter sites is assumed to be the crucial step in the contractile event, they are often referred to as "the regulatory sites" (see below). The existence of additional weak Mg2+ sites (KB ≈ 300 M-1) on sTnC, not in direct competition with Ca2+, has also been inferred.76,104,105 Spectroscopic studies have shown that the two strong Ca2+- Mg2+ sites are located in the C-terminal domain, and the weaker Ca2+-specific sites in the N-terminal domain of sTnC.106 This pattern is similar to that observed with CaM. NMR spectroscopic studies strongly suggest that binding of Ca2+ to both sTnC and cTnC is cooperative.107 In sTnC the C-terminal domain binds Mg2+ much more strongly than the N-terminal domain, by contrast to CaM, where the reverse is true.

    The rates of dissociation of Ca2+ and Mg2+ from sTnC have been measured by both stopped-flow and 43Ca NMR techniques.76,108 As with CaM, the actual numbers depend on the solution conditions, ionic strength, presence of Mg2+, etc. (see Table 3.4). On the rate of Mg2+ dissociation from the Ca2+- Mg2+ sites, quite different results have been obtained by stopped-flow studies76 of fluorescence-labeled sTnC (koffMg2+ ≈ 8 s-1) and by 25Mg NMR (koffMg2+ \(\simeq\) 800-1000 s-1).109 This apparent discrepancy seems to have been resolved by the observation that both binding and release of Mg2+ ions to the Ca2+-Mg2+ sites occur stepwise, with koffMg2+ < 20 s-1 for one of the ions, and koffMg2+ ≥ 800 s-1 for the other.110 The rates of dissociation of the Mg2+ ions are important, since under physiological conditions the Ca2+-Mg2+ sites of sTnC are likely to be predominantly occupied by Mg2+ ions, release of which determines the rate at which Ca2+ can enter into these sites.

    Spectroscopic and biochemical data111 indicate that upon binding Ca2+, sTnC and cTnC undergo significant conformation changes. Comparisons of NMR spectroscopic changes on Ca2+ binding to intact sTnC, as well as to the two fragments produced by tryptic cleavage (essentially the N-terminal and C-terminal halves of the molecule, just as was the case with CaM), have shown that the conformation changes induced are mainly localized within the domain that is binding added ions.110,112 Thus the central \(\alpha\)-helix connecting the domains seems unable to propagate structural changes from one domain to the other. It has been suggested that the structural differences found in the x-ray structure of turkey sTnC between the C-terminal domain, which in the crystal contains two bound Ca2+ ions, and the N-terminal domain, in which no Ca2+ ions were found, may represent these conformational changes.113 This rather substantial conformational change is schematically depicted in Figure 3.22.

    Figure kindly provided by N. C. J. Strynadka and M. N. G. James.

    However, preliminary structure calculations114 of the calcium-saturated and calcium-free forms of calbindin D9k indicate that much more subtle conformational changes take place upon binding Ca2+ in calbindin D9k. Interestingly, 1H NMR spectroscopy has provided evidence for the concept that the structural change induced by Mg2+ binding to the C-terminal domain of sTnC must be very similar to that induced by Ca2+ ions. Another result obtained by 113Cd NMR studies108 is that the cadmium-loaded N-terminal domain of sTnC in solution undergoes a rapid interchange between two or more conformations, with an exchange rate on the order of 103-104 s-1.

    Just as CaM exerts its biological function in complexes with other proteins, TnC participates in the three-protein troponin complex. It presently appears that TnC and TnI form a primary complex that is anchored by TnT to a binding site on tropomyosin.115 In the troponin complex the Ca2+ affinity is increased by a factor of about ten over that in isolated sTnC, both at the Ca2+-Mg2+ sites and at the Ca2+-specific sites. A similar increase in affinity is found for Mg2+. Given the amounts of "free" Mg2+ inside muscle cells (1 to 3 mM), it seems likely that the Ca2+-Mg2+ sites in the resting state of troponin are filled with Mg2+, so that a transitory release of Ca2+ leads primarily to rapid Ca2+ binding to the Ca2+-specific sites, and subsequently to conformation change and contraction.

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