Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 May 15;388(4):673-81.
doi: 10.1016/j.jmb.2009.03.060. Epub 2009 Mar 31.

Structural basis for the activation of muscle contraction by troponin and tropomyosin

William Lehman  1 , Agnieszka Galińska-Rakoczy, Victoria Hatch, Larry S Tobacman, Roger Craig
Affiliations

Structural basis for the activation of muscle contraction by troponin and tropomyosin

William Lehman et al. J Mol Biol. .

Abstract

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca(2+) on troponin (Tn) 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 electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca(2+) 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 Ca(2+) allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca(2+)-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can 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 Ca(2+), troponin operates as an inhibitor, while in activated muscles at high Ca(2+), it acts as a promoter to initiate contraction.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Electron micrographs of negatively stained filaments. (a) F-actin-troponin-tropomyosin, (b) F-actin-tropomyosin (b). Note that the presence of troponin (arrowheads), distributed with a 40 nm periodicity in (a) increases the maximum width of thin filaments; also note obliquely oriented tropomyosin strands in (a) and (b) (arrows). Filaments are shown with their pointed ends facing up; polarity was determined by alignment tools in reference. Scale bar = 50 nm. Preparation of proteins: F-actin, bovine cardiac troponin and tropomyosin were purified as previously. SDS-PAGE indicated that the cardiac tropomyosin consisted of greater than 90% aa-isoform and the remainder αβ-isoform; it was presumed to be 20 to 30 percent phosphorylated, as is the tropomyosin in adult cardiac tissue of other mammals,. The cardiac troponin as isolated was monophosphorylated and not biphosphorylated as occurs after adrenergic stimulation. EM and image processing: We have previously shown that a moderate excess of troponin-tropomyosin relative to actin is needed to saturate filaments at the low protein actin concentrations required for successful EM work,. Filaments were thus prepared by mixing a two-fold molar excess of tropomyosin or troponin–tropomyosin (40 μM) with F-actin (20 μM) to optimize binding, in 100mM NaCl, 3mM MgCl2, 1mM NaN3, 0.2mM EGTA, 1mM dithiothreitol, 5mM sodium phosphate/5mM Pipes buffer (pH 7.0) at 25°C,. The mixture was diluted 20-fold, applied to carbon-coated grids and negatively stained with 1% uranyl acetate,. Filaments from two preparations of F-actin-troponin-tropomyosin and three preparations of F-actin-tropomyosin were recorded and processed in these studies. EM was done on a Philips CM120 EM at a magnification of X60,000 under low dose conditions (~12 e−/Å). Helical reconstruction was performed by standard methods as previously,. Helical reconstruction was particularly well suited to identify the relatively low density troponin signal observed, as the method takes advantage of layer line indexing information to filter out noise, which otherwise would have obscured the troponin density observed. In addition, robust statistical programs accompany the helical reconstruction package, allowing the significance of weak densities to be assessed by Student’s t-test methodology,. Real space reconstruction methods, treating filament segments as single particles, were also used to confirm results obtained by helical reconstruction and to attempt to sort filament segments according to their tropomyosin positional modes, as previously.
Fig. 2
Fig. 2
Surface views of thin filament reconstructions showing the position of the high Ca2+ troponin extension. Reconstructions of: (a) F-actin (cyan, subdomains numbered on one actin subunit) and (b) F-actin-tropomyosin (green) controls, (c, d, e) F-actin-troponin-tropomyosin: (c, d) high Ca2+ (gold), (e) low Ca2+ (maroon). Note tropomyosin positions indicated by arrows. High Ca2+ reconstructions displayed at two density cutoff thresholds ((c) 5 σ, (d) 2.5 σ greater than the mean density). Note the presence of extra densities in (d) emerging from the centers of subdomain 1, which traverse the face of the domain to abut C-state tropomyosin (open arrowheads). Note also that these densities are absent from F-actin-tropomyosin controls (b) and low Ca2+ F-actin-troponin-tropomyosin (e) (each displayed at 2.5 σ). (f) Reconstructions in (d) and (e) superimposed for comparison. Note that the respective tropomyosin strands occupy different positions, with tropomyosin from the high and low Ca2+ maps in C- and B-states. Also note that Ca2+ specific extra density attributed to troponin (open arrows) crosses the B-state tropomyosin position. All of the reconstructions of thin filaments obtained were aligned to each other and are shown with filament pointed ends facing up. Each map shows densities with amplitudes that are at a threshold of 2.5 sigma over the mean density (with the exception of the map in (c)).
Fig. 3
Fig. 3
Statistical significance of densities contributing to reconstructions of Ca2+-treated F-actin-troponin-tropomyosin. (a) z-section of reconstruction shown in figure 2d (gold). Note actin subdomains (numbered) and tropomyosin positions (indicated by arrows and labeled Tm) on the inner domain of actin over subdomains 3 and 4. Note also the extra density emerging from subdomain 1 and connecting to tropomyosin (indicated by open arrowhead; the outer edge of the extra density is highlighted in cyan). (b) Densities associated with the z-section in (a) that have a confidence level greater than 99 % (pink). (c) The z-section in (a) superimposed on the statistical map in (b), showing that all densities in (a), including the extra density attributable to troponin, have a high level of statistical significance.
Fig. 4
Fig. 4
Reconstructions of troponin-free actin – tropomyosin. (a) z-section of reconstruction shown in figure 2b of cardiac tropomyosin bound to F-actin (no troponin). Note that tropomyosin (arrows) occupies a position mid-way between the inner and outer domains of actin, i.e. between actin subdomains 1 and 3 on one side of the filament and between subdomains 2 and 4 on the other side. (b) Tropomyosin densities determined by subtracting densities in the F-actin reconstruction from those of the map in (a) and then superimposing them on the corresponding z-section of F-actin in order to highlight the position of tropomyosin. (c, d) the filaments contributing to the average density map in (a) were sorted according to their best alignment to B- or C-state models. (c) Reconstruction of the sorted filaments that were in the C-state mode; note tropomyosin densities on the actin inner domain (i.e. associated with actin subdomains 3 and 4). (d) Reconstruction of the sorted filaments that were in the B-state mode, with tropomyosin densities on the outer domain (i.e. associated with subdomains 1 and 2). Comparable results were obtained when the skeletal muscle isoform of tropomyosin was examined on troponin-free F-actin, viz. an average position midway between B- and C-state, but here individual filament reconstructions showed a slightly greater bias for the C-state mode than with a found will cardiac tropomyosin.
Fig. 5
Fig. 5
Comparison of cardiac and smooth muscle tropomyosin positions on actin. z-sections of reconstructions of F-actin combined with cardiac muscle tropomyosin (a) and aortic smooth muscle tropomyosin (b). Note the closer association of the smooth muscle tropomyosin density with the actin outer domain (compare white arrows in (a) and (b)). (c) Maps plotting the variance associated with the contributing density points, in the reconstruction in (a) show that a high variance (red) occurs over the site where tropomyosin normally makes contact with actin in the B-state. (d) In contrast, corresponding variance maps of actin – smooth muscle tropomyosin reconstructions show low variance at this site and only comparably high variance (red) at the very edges of the tropomyosin density.
Fig. 6
Fig. 6
Cartoon representation of the organization of the thin filament at high Ca2+. As previously, the troponin core domain complexes on either side of F-actin are depicted as W-shaped TnIT structures supporting dumbbell-shaped TnC,; actin, grey; tropomyosin, salmon; TnI, cyan; TnC, red; TnT, yellow. At high Ca2+, cTerm-TnI dissociates from actin and binds to the N-terminal lobe of TnC. We argue that this releases chemomechanical constraints on troponin and tropomyosin. Troponin then unfurls, here depicted for simplicity as TnT propelling tropomyosin to the C-state position. Other related depictions of troponin on thin filaments can be found in references,,.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Gordon AM, Homsher E, Regnier M. Regulation of contraction in striated muscle. Physiol Rev. 2000;80:853–924. - PubMed
    1. Tobacman LS. Thin filament-mediated regulation of cardiac contraction. Annu Rev Physiol. 1996;58:447–481. - PubMed
    1. Moore PB, Huxley HE, DeRosier DJ. Three-dimensional reconstruction of F-actin, thin filaments and decorated thin filaments. J Mol Biol. 1970;50:279–292. - PubMed
    1. O’Brien EJ, Bennett PM, Hanson J. Optical diffraction studies of myofibrillar structure. Philos Trans Roy Soc Lond B Biol Sci. 1971;261:201–208. - PubMed
    1. Spudich JA, Huxley HE, Finch JT. The regulation of skeletal muscle contraction. II Structural studies of the interaction of the tropomyosin-troponin complex with actin. J Mol Biol. 1972;72:619–632. - PubMed

Publication types

LinkOut - more resources