Myosin cleft movement and its coupling to actomyosin dissociation


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 options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Models to show the location of Dd residues 416 (cyan) and 537 (yellow) that are labeled with pyrene.
Figure 2: Fluorescence emission spectra of pyr-Md (365 nm excitation).
Figure 3: Time-resolved pyrene fluorescence measurements.
Figure 4: Stopped-flow records of pyrene excimer fluorescence on interaction of pyr-Md with actin and nucleotides.

Accession codes


Protein Data Bank


  1. 1

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  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).

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  12. 12

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  PubMed  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  28. 28

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

    CAS  PubMed  Google Scholar 

Download references


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



Corresponding author

Correspondence to Clive R Bagshaw.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Conibear, P., Bagshaw, C., Fajer, P. et al. Myosin cleft movement and its coupling to actomyosin dissociation. Nat Struct Mol Biol 10, 831–835 (2003).

Download citation

Further reading


Nature Briefing

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing