Abstract
F-actin serves as a track for myosin's motor functions and activates its ATPase activity by several orders of magnitude, enabling actomyosin to produce effective force against load. Although actin activation is a ubiquitous property of all myosin isoforms, the molecular mechanism and physiological role of this activation are unclear. Here we describe a conserved actin-binding region of myosin named the 'activation loop', which interacts with the N-terminal segment of actin. We demonstrate by biochemical, biophysical and in vivo approaches using transgenic Caenorhabditis elegans strains that the interaction between the activation loop and actin accelerates the movement of the relay, stimulating myosin's ATPase activity. This interaction results in efficient force generation, but it is not essential for the unloaded motility. We conclude that the binding of actin to myosin's activation loop specifically increases the ratio of mechanically productive to futile myosin heads, leading to efficient muscle contraction.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Pollard, T.D. Reflections on a quarter century of research on contractile systems. Trends Biochem. Sci. 25, 607–611 (2000).
Sweeney, H.L. & Houdusse, A. Structural and functional insights into the Myosin motor mechanism. Annu. Rev. Biophys. 39, 539–557 (2010).
Odronitz, F. & Kollmar, M. Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species. Genome Biol. 8, R196 (2007).
Bauer, C.B., Holden, H.M., Thoden, J.B., Smith, R. & Rayment, I. X-ray structures of the apo and MgATP-bound states of Dictyostelium discoideum myosin motor domain. J. Biol. Chem. 275, 38494–38499 (2000).
Gyimesi, M. et al. The mechanism of the reverse recovery step, phosphate release, and actin activation of Dictyostelium myosin II. J. Biol. Chem. 283, 8153–8163 (2008).
Málnási-Csizmadia, A. & Kovacs, M. Emerging complex pathways of the actomyosin powerstroke. Trends Biochem. Sci. 35, 684–690 (2010).
Goody, R.S. & Hofmann-Goody, W. Exchange factors, effectors, GAPs and motor proteins: common thermodynamic and kinetic principles for different functions. Eur. Biophys. J. 31, 268–274 (2002).
Bhattacharyya, R.P. et al. The Ste5 scaffold allosterically modulates signaling output of the yeast mating pathway. Science 311, 822–826 (2006).
Holmes, K.C., Angert, I., Kull, F.J., Jahn, W. & Schroder, R.R. Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide. Nature 425, 423–427 (2003).
Oda, T., Iwasa, M., Aihara, T., Maeda, Y. & Narita, A. The nature of the globular- to fibrous-actin transition. Nature 457, 441–445 (2009).
Holmes, K.C., Schroder, R.R., Sweeney, H.L. & Houdusse, A. The structure of the rigor complex and its implications for the power stroke. Phil. Trans. R. Soc. Lond. B 359, 1819–1828 (2004).
Lorenz, M. & Holmes, K.C. The actin-myosin interface. Proc. Natl. Acad. Sci. USA 107, 12529–12534 (2010).
Liu, Y., Scolari, M., Im, W. & Woo, H.J. Protein-protein interactions in actin-myosin binding and structural effects of R405Q mutation: a molecular dynamics study. Proteins 64, 156–166 (2006).
Root, D.D. A computational comparison of the atomic models of the actomyosin interface. Cell Biochem. Biophys. 37, 97–110 (2002).
Kintses, B. et al. Reversible movement of switch 1 loop of myosin determines actin interaction. EMBO J. 26, 265–274 (2007).
Sasaki, N., Asukagawa, H., Yasuda, R., Hiratsuka, T. & Sutoh, K. Deletion of the myopathy loop of Dictyostelium myosin II and its impact on motor functions. J. Biol. Chem. 274, 37840–37844 (1999).
Gyimesi, M., Tsaturyan, A.K., Kellermayer, M.S. & Malnasi-Csizmadia, A. Kinetic characterization of the function of myosin loop 4 in the actin-myosin interaction. Biochemistry 47, 283–291 (2008).
Kojima, S. et al. Functional roles of ionic and hydrophobic surface loops in smooth muscle myosin: their interactions with actin. Biochemistry 40, 657–664 (2001).
Onishi, H., Mikhailenko, S.V. & Morales, M.F. Toward understanding actin activation of myosin ATPase: the role of myosin surface loops. Proc. Natl. Acad. Sci. USA 103, 6136–6141 (2006).
Furch, M., Remmel, B., Geeves, M.A. & Manstein, D.J. Stabilization of the actomyosin complex by negative charges on myosin. Biochemistry 39, 11602–11608 (2000).
Joel, P.B., Trybus, K.M. & Sweeney, H.L. Two conserved lysines at the 50/20-kDa junction of myosin are necessary for triggering actin activation. J. Biol. Chem. 276, 2998–3003 (2001).
Miller, C.J., Wong, W.W., Bobkova, E., Rubenstein, P.A. & Reisler, E. Mutational analysis of the role of the N terminus of actin in actomyosin interactions. Comparison with other mutant actins and implications for the cross-bridge cycle. Biochemistry 35, 16557–16565 (1996).
Gu, J., Xu, S. & Yu, L.C. A model of cross-bridge attachment to actin in the A*M*ATP state based on X-ray diffraction from permeabilized rabbit psoas muscle. Biophys. J. 82, 2123–2133 (2002).
Sutoh, K. Mapping of actin-binding sites on the heavy chain of myosin subfragment 1. Biochemistry 22, 1579–1585 (1983).
Andreev, O.A. & Reshetnyak, Y.K. Mechanism of formation of actomyosin interface. J. Mol. Biol. 365, 551–554 (2007).
Van Dijk, J. et al. Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II. Eur. J. Biochem. 260, 672–683 (1999).
Jahn, W. The association of actin and myosin in the presence of γ-amido-ATP proceeds mainly via a complex with myosin in the closed conformation. Biochemistry 46, 9654–9664 (2007).
Wray, J. & Jahn, W. γ-amido-ATP stabilizes a high-fluorescence state of myosin subfragment 1. FEBS Lett. 518, 97–100 (2002).
Málnási-Csizmadia, A. et al. Kinetic resolution of a conformational transition and the ATP hydrolysis step using relaxation methods with a Dictyostelium myosin II mutant containing a single tryptophan residue. Biochemistry 40, 12727–12737 (2001).
Cooke, R., White, H. & Pate, E. A model of the release of myosin heads from actin in rapidly contracting muscle fibers. Biophys. J. 66, 778–788 (1994).
Nyitrai, M. et al. What limits the velocity of fast-skeletal muscle contraction in mammals? J. Mol. Biol. 355, 432–442 (2006).
Purcell, T.J. et al. Nucleotide pocket thermodynamics measured by EPR reveal how energy partitioning relates myosin speed to efficiency. J. Mol. Biol. 407, 79–91 (2011).
Forgacs, E. et al. Switch 1 mutation S217A converts myosin V into a low duty ratio motor. J. Biol. Chem. 284, 2138–2149 (2009).
Lin, T., Greenberg, M.J., Moore, J.R. & Ostap, E.M. A hearing loss-associated myo1c mutation (R156W) decreases the myosin duty ratio and force sensitivity. Biochemistry 50, 1831–1838 (2011).
Nagy, N.T. et al. Functional adaptation of the switch-2 nucleotide sensor enables rapid processive translocation by myosin-5. FASEB J. 24, 4480–4490 (2010).
Takács, B. et al. Myosin cleft closure determines the energetics of the actomyosin interaction. FASEB J. 25, 111–121 (2011).
Uyeda, T.Q., Abramson, P.D. & Spudich, J.A. The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc. Natl. Acad. Sci. USA 93, 4459–4464 (1996).
Waterston, R.H. The minor myosin heavy chain, mhcA, of Caenorhabditis elegans is necessary for the initiation of thick filament assembly. EMBO J. 8, 3429–3436 (1989).
Moerman, D.G., Plurad, S., Waterston, R.H. & Baillie, D.L. Mutations in the unc-54 myosin heavy chain gene of Caenorhabditis elegans that alter contractility but not muscle structure. Cell 29, 773–781 (1982).
Anderson, P. & Brenner, S. A selection for myosin heavy chain mutants in the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 81, 4470–4474 (1984).
Fire, A. & Waterston, R.H. Proper expression of myosin genes in transgenic nematodes. EMBO J. 8, 3419–3428 (1989).
Biro, N.A. & Szent-Gyorgyi, A.E. The effect of actin and physico-chemical changes on the myosin ATP-ase system, and on washed muscle. Hung. Acta Physiol. 2, 120–133 (1949).
Fischer, S., Windshugel, B., Horak, D., Holmes, K.C. & Smith, J.C. Structural mechanism of the recovery stroke in the myosin molecular motor. Proc. Natl. Acad. Sci. USA 102, 6873–6878 (2005).
Yu, H., Ma, L., Yang, Y. & Cui, Q. Mechanochemical coupling in the myosin motor domain. I. Insights from equilibrium active-site simulations. PLoS Comput. Biol. 3, e21 (2007).
Kintses, B., Yang, Z. & Malnasi-Csizmadia, A. Experimental investigation of the seesaw mechanism of the relay region that moves the myosin lever arm. J. Biol. Chem. 283, 34121–34128 (2008).
Johnson, K.A. Conformational coupling in DNA polymerase fidelity. Annu. Rev. Biochem. 62, 685–713 (1993).
Gromadski, K.B. & Rodnina, M.V. Kinetic determinants of high-fidelity tRNA discrimination on the ribosome. Mol. Cell 13, 191–200 (2004).
Egea, P.F. et al. Substrate twinning activates the signal recognition particle and its receptor. Nature 427, 215–221 (2004).
Málnási-Csizmadia, A., Woolley, R.J. & Bagshaw, C.R. Resolution of conformational states of Dictyostelium myosin II motor domain using tryptophan (W501) mutants: implications for the open-closed transition identified by crystallography. Biochemistry 39, 16135–16146 (2000).
Yang, Y., Kovacs, M., Xu, Q., Anderson, J.B. & Sellers, J.R. Myosin VIIB from Drosophila is a high duty ratio motor. J. Biol. Chem. 280, 32061–32068 (2005).
Pardee, J.D. & Spudich, J.A. Purification of muscle actin. Methods Cell Biol. 24, 271–289 (1982).
Cooper, J.A., Walker, S.B. & Pollard, T.D. Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J. Muscle Res. Cell Motil. 4, 253–262 (1983).
Hutter, J.L. & Bechhoefer, J. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64, 1868–1873 (1993).
Acknowledgements
We thank A.G. Szent-Györgyi, C.R. Bagshaw and M. Kovács for insightful discussions and helpful comments. We thank E. Málnási-Csizmadia for the processing of videos. This work was supported by the European Research Council (European Community's Seventh Framework Programme (FP7/2007-2013)/European Research Council grant agreement no. 208319), the European Union in collaboration with the European Social Fund (grant agreement no. TAMOP-4.2.1/B-09/1/KMR), the National Office for Research and Technology and the European Union (European Regional Development Fund), under the sponsorship of the National Technology Programme (NTP TECH_08_A1/2-2008-0106). T.V. is a grantee of the János Bolyai scholarship.
Author information
Authors and Affiliations
Contributions
B.H.V. and A.M.-C. designed, conducted and analyzed all experiments and wrote the paper. Z.Y. conducted the in silico experiments. B.K. designed experiments and contributed to the writing of the paper. T.V. and P.E. contributed to the C. elegans experiments. I.B.-N. contributed to the in vitro motility experiments. A.L.K. carried out electron microscopy. M.K. designed and conducted the AFM experiment. P.H. designed the analysis of the movement of C. elegans.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1 and 2, Supplementary Table 1, Supplementary Discussion and Supplementary Methods (PDF 388 kb)
Supplementary Movie 1
Fluorescent videos of transgenic animals. Fluorescent video recordings of transgenic animals from strains Δunc-54;unc-54::gfp and Δunc-54;unc-54K525E::gfp expressing UNC-54::GFP or UNC-54K525E::GFP, respectively. Recordings were taken with a Zeiss Lumar.V12 microscope combined with Zeiss AxioCam using AxioVision 4.8 software. GFP was excited with a UV lamp and a combination of 470nm-510nm and 525nm-575nm transmission filters were used on the excitation and emission side, respectively. (MOV 1043 kb)
Supplementary Movie 1a. Fluorescent video recording of a Δunc-54;unc-54K525E::gfp animal.
Supplementary Movie 1b. Fluorescent video recording of a Δunc-54;unc-54::gfp animal.
Supplementary Movie 2
Videos of the four C. elegans strains during AFM experiments. Video recordings of the four C. elegans strains (Δunc-54, Δunc-54;unc-54::gfp, Δunc-54;unc-54K525E::gfp and wild type) during the force measurement experiments with AFM. One typical behaviour from each strain is presented here. In the field of view the animal, the base of the cantilever and the cantilever can be seen from above. Recordings were taken from the moment of catching the animal with the tip of the cantilever until ~40 seconds or until their escape. During this period, several push-and-pull phases took place on one animal. The animal's mechanical activity was measured between a push and a pull phase. Animals from different strains were of similar size. The spring constant of the cantilever was kept around 50pN/μm2 within all experiments. (MOV 10386 kb)
Supplementary Movie 2a. Video recording of a Δunc-54 animal.
Supplementary Movie 2b. Video recording of a Δunc-54;unc-54K525E::gfp animal.
Supplementary Movie 2c. Video recording of a Δunc-54;unc-54::gfp animal.
Supplementary Movie 2d. Video recording of a wild type animal.
Rights and permissions
About this article
Cite this article
Várkuti, B., Yang, Z., Kintses, B. et al. A novel actin binding site of myosin required for effective muscle contraction. Nat Struct Mol Biol 19, 299–306 (2012). https://doi.org/10.1038/nsmb.2216
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.2216
This article is cited by
-
Cytosolic actin isoforms form networks with different rheological properties that indicate specific biological function
Nature Communications (2023)
-
Filamentous tangles with nemaline rods in MYH2 myopathy: a novel phenotype
Acta Neuropathologica Communications (2021)
-
Investigating the correlation of muscle function tests and sarcomere organization in C. elegans
Skeletal Muscle (2021)
-
Coherent Raman Imaging of Live Muscle Sarcomeres Assisted by SFG Microscopy
Scientific Reports (2017)
-
Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution
Nature (2016)