Article | Published:

Myosin cleft movement and its coupling to actomyosin dissociation

Nature Structural & Molecular Biologyvolume 10pages831835 (2003) | Download Citation

Subjects

Abstract

It has long been known that binding of actin and binding of nucleotides to myosin are antagonistic, an observation that led to the biochemical basis for the crossbridge cycle of muscle contraction. Thus ATP binding to actomyosin causes actin dissociation, whereas actin binding to the myosin accelerates ADP and phosphate release. Structural studies have indicated that communication between the actin- and nucleotide-binding sites involves the opening and closing of the cleft between the upper and lower 50K domains of the myosin head. Here we test the proposal that the cleft responds to actin and nucleotide binding in a reciprocal manner and show that cleft movement is coupled to actin binding and dissociation. We monitored cleft movement using pyrene excimer fluorescence from probes engineered across the cleft.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accession codes

Accessions

Protein Data Bank

References

  1. 1

    Lymn, R.W. & Taylor, E.W. Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry 10, 4617–4624 (1971).

  2. 2

    Fisher, A.J. et al. X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4. Biochemistry 34, 8960–8972 (1995).

  3. 3

    Dominguez, R., Freyzon, Y., Trybus, K.M. & Cohen, C. Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state. Cell 94, 559–571 (1998).

  4. 4

    Geeves, M.A. & Holmes, K.C. Structural mechanism of muscle contraction. Annu. Rev. Biochem. 68, 687–728 (1999).

  5. 5

    Volkmann, N. et al. Evidence for cleft closure in actomyosin upon ADP release. Nat. Struct. Biol. 7, 1147–1155 (2000).

  6. 6

    Himmel, D.M. et al. Crystallographic findings on the internally uncoupled and near-rigor states of myosin: further insights into the mechanics of the motor. Proc. Natl. Acad. Sci. USA 24, 12645–12650 (2002).

  7. 7

    Holmes, K.C., Angert, I., Kull, F.J., Jahn, W. & Schroeder, R.R. Electron cryo-microscopy reveals how myosin strong binding to actin releases nucleotide. Nature in the press (2003).

  8. 8

    Reubold, T.F., Eschenburg, S., Becker, A., Kull, F.J. & Manstein, D.J. A structural model for actin-induced nucleotide release in myosin. Nat. Struct. Biol. 10, 826–830 (2003).

  9. 9

    Coureux, P.D. et al. The myosin V motor visualized at 2.0 Å without bound nucleotide reveals a new structural state of myosin. Nature in the press (2003).

  10. 10

    Rayment, I. et al. Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 261, 50–58 (1993).

  11. 11

    Houdusse, A., Szent-Gyorgyi, A.G. & Cohen, C. Three conformational states of scallop myosin S1. Proc. Natl. Acad. Sci. USA 97, 11238–11243 (2000).

  12. 12

    Rayment, I. et al. Structure of the actin-myosin complex and its implications for muscle contraction. Science 261, 58–65 (1993).

  13. 13

    Shih, W.M., Gryczynski, Z., Lakowicz, J.R. & Spudich, J.A. A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin. Cell 102, 683–694 (2000).

  14. 14

    Yengo, C.M., De La Cruz, E.M., Chrin, L.R., Gaffney, D.P. 2nd & Berger, C.L. Actin-induced closure of the actin-binding cleft of smooth muscle myosin. J. Biol. Chem. 277, 24114–24119 (2002).

  15. 15

    Wakelin, S. et al. Engineering Dictyostelium discoideum myosin II for the introduction of site-specific probes. J. Muscle Res. Cell Motil. 23, 673–683 (2002).

  16. 16

    Birks, J.B., Kazzaz, A.A. & King, T.A. 'Excimer' fluorescence IX. Lifetime studies of pyrene crystals. Proc. R. Soc. Lond. A 291, 556–569 (1966).

  17. 17

    Lehrer, S.S. Intramolecular pyrene excimer fluorescence: a probe of proximity and protein conformational change. Methods Enzymol. 278, 286–295 (1997).

  18. 18

    Kuhlman, P.A. & Bagshaw, C.R. ATPase kinetics of the Dictyostelium discoideum myosin II motor domain. J. Muscle Res. Cell Motil. 19, 491–504 (1998).

  19. 19

    Malnasi-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).

  20. 20

    Kovacs, M., Malnasi-Csizmadia, A., Woolley, R.J. & Bagshaw, C.R. Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site. J. Biol. Chem. 277, 28459–28467 (2002).

  21. 21

    Geeves, M.A., Jeffries, T.E. & Millar, N.C. ATP-induced dissociation of rabbit skeletal actomyosin subfragment 1. Characterization of an isomerization of the ternary acto-S1-ATP complex. Biochemistry 25, 8454–8458 (1986).

  22. 22

    Taylor, E.W. Kinetic studies on the association and dissociation of myosin subfragment 1 and actin. J. Biol. Chem. 266, 294–302 (1991).

  23. 23

    Cremo, C.R. & Geeves, M.A. Interaction of actin and ADP with the head domain of smooth muscle myosin: implications for strain-dependent ADP release in smooth muscle. Biochemistry 37, 1969–1978 (1998).

  24. 24

    Geeves, M.A. Dynamic interaction between actin and myosin subfragment 1 in the presence of ADP. Biochemistry 28, 5864–5871 (1989).

  25. 25

    Manstein, D.J., Schuster, H.P., Morandini, P. & Hunt, D.M. Cloning vectors for the production of proteins in Dictyostelium discoideum. Gene 162, 129–134 (1995).

  26. 26

    Manstein, D.J. & Hunt, D.M. Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein. J Muscle Res. Cell Motil. 16, 325–232 (1995).

  27. 27

    Malnasi-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).

  28. 28

    Sarkar, G. & Sommer, S.S. The 'megaprimer' method of site-directed mutagenesis. Biotechniques 8, 404–407 (1990).

Download references

Acknowledgements

We thank W. Shih and J. Spudich for the cysteine-deficient construct and K. Holmes and R. Schroeder for the coordinates of their actomyosin model. We are grateful to the Wellcome Trust, the UK Biotechnology and Biological Sciences Research Council, the US National Science Foundation and the Magyary Zoltán Foundation for financial support.

Author information

Author notes

    • Mihály Kovács

    Present address: Laboratory of Molecular Cardiology, National Heart, Lung and Blood Institute, Bethesda, Maryland, 20892-1762, USA

Affiliations

  1. Department of Biochemistry, University of Leicester, Leicester, LE1 7RH, UK

    • Paul B Conibear
    • , Clive R Bagshaw
    •  & András Málnási-Csizmadia
  2. Institute of Molecular Biophysics, Florida State University, Tallahassee, 32310, Florida, USA

    • Piotr G Fajer
  3. Department of Biochemistry, Eötvös University, Budapest, H-1117, Hungary

    • Mihály Kovács
    •  & András Málnási-Csizmadia

Authors

  1. Search for Paul B Conibear in:

  2. Search for Clive R Bagshaw in:

  3. Search for Piotr G Fajer in:

  4. Search for Mihály Kovács in:

  5. Search for András Málnási-Csizmadia in:

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Clive R Bagshaw.

About this article

Publication history

Received

Accepted

Published

Issue Date

DOI

https://doi.org/10.1038/nsb986

Further reading