Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Molecular engineering of a backwards-moving myosin motor


All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin- and nucleotide-binding sites and produces force and movement1,2. The light-chain-binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm3,4. Myosins move on polarized actin filaments either forwards to the barbed (+ ) or backwards to the pointed (- ) end5,6. Here, we describe the engineering of an artificial backwards-moving myosin from three pre-existing molecular building blocks. These blocks are: a forward-moving class I myosin motor domain, a directional inverter formed by a four-helix bundle segment of human guanylate-binding protein-1 and an artificial lever arm formed by two α-actinin repeats. Our results prove that reverse-direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180°.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Mechanical models for myosin-based forwards and backwards movement.
Figure 2: Molecular models of MD-2R and E698-Ω2R attached to F-actin.
Figure 3: Direction of movement of myosin constructs.


  1. Toyoshima, Y. Y. et al. Myosin subfragment-1 is sufficient to move actin filaments in vitro. Nature 328, 536–539 (1987)

    ADS  CAS  Article  Google Scholar 

  2. Manstein, D. J., Ruppel, K. M. & Spudich, J. A. Expression and characterization of a functional myosin head fragment in Dictyostelium discoideum. Science 246, 656–658 (1989)

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  4. Berg, J. S., Powell, B. C. & Cheney, R. E. A millennial myosin census. Mol. Biol. Cell 12, 780–794 (2001)

    CAS  Article  Google Scholar 

  5. Wells, A. L. et al. Myosin VI is an actin-based motor that moves backwards. Nature 401, 505–508 (1999)

    ADS  CAS  Article  Google Scholar 

  6. Homma, K., Yoshimura, M., Saito, J., Ikebe, R. & Ikebe, M. The core of the motor domain determines the direction of myosin movement. Nature 412, 831–834 (2001)

    ADS  CAS  Article  Google Scholar 

  7. Kollmar, M., Dürrwang, U., Kliche, W., Manstein, D. J. & Kull, F. J. Crystal structure of the motor domain of a class-I myosin. EMBO J. 21, 2517–2525 (2002)

    CAS  Article  Google Scholar 

  8. Veigel, C. et al. The motor protein myosin-I produces its working stroke in two steps. Nature 398, 530–533 (1999)

    ADS  CAS  Article  Google Scholar 

  9. Prakash, B., Renault, L., Praefcke, G. J., Herrmann, C. & Wittinghofer, A. Triphosphate structure of guanylate-binding protein 1 and implications for nucleotide binding and GTPase mechanism. EMBO J. 19, 4555–4564 (2000)

    CAS  Article  Google Scholar 

  10. 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)

    ADS  CAS  Article  Google Scholar 

  11. Anson, M., Geeves, M. A., Kurzawa, S. E. & Manstein, D. J. Myosin motors with artificial lever arms. EMBO J. 15, 6069–6074 (1996)

    CAS  Article  Google Scholar 

  12. Ruff, C., Furch, M., Brenner, B., Manstein, D. J. & Meyhöfer, E. Single-molecule tracking of myosins with genetically engineered amplifier domains. Nature Struct. Biol. 8, 226–229 (2001)

    CAS  Article  Google Scholar 

  13. Kliche, W., Fujita-Becker, S., Kollmar, M., Manstein, D. J. & Kull, F. J. Structure of a genetically engineered molecular motor. EMBO J. 20, 40–46 (2001)

    CAS  Article  Google Scholar 

  14. Kull, F. J. & Endow, S. A. Kinesin: switch I & II and the motor mechanism. J. Cell Sci. 115, 15–23 (2002)

    CAS  PubMed  Google Scholar 

  15. Vale, R. D. & Milligan, R. A. The way things move: looking under the hood of molecular motor proteins. Science 288, 88–95 (2000)

    ADS  CAS  Article  Google Scholar 

  16. Furch, M., Geeves, M. A. & Manstein, D. J. Modulation of actin affinity and actomyosin adenosine triphosphatase by charge changes in the myosin motor domain. Biochemistry 37, 6317–6326 (1998)

    CAS  Article  Google Scholar 

  17. 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)

    CAS  Article  Google Scholar 

  18. 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)

    CAS  Article  Google Scholar 

  19. Knetsch, M. L. W., Uyeda, T. Q. & Manstein, D. J. Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction. J. Biol. Chem. 274, 20133–20138 (1999)

    CAS  Article  Google Scholar 

  20. Tsiavaliaris, G. et al. Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium. EMBO Rep. 3, 1099–1105 (2002)

    CAS  Article  Google Scholar 

  21. Ponomarev, M. A., Furch, M., Levitsky, D. I. & Manstein, D. J. Charge changes in loop 2 affect the thermal unfolding of the myosin motor domain bound to F-actin. Biochemistry 39, 4527–4532 (2000)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  23. Ringler, P. & Schulz, G. E. Self-assembly of proteins into designed networks. Science 302, 106–109 (2003)

    ADS  CAS  Article  Google Scholar 

  24. Kron, S. J. & Spudich, J. A. Fluorescent actin filaments move on myosin fixed to a glass surface. Proc. Natl Acad. Sci. USA 83, 6272–6276 (1986)

    ADS  CAS  Article  Google Scholar 

  25. Lehrer, S. S. & Kerwar, G. Intrinsic fluorescence of actin. Biochemistry 11, 1211–1217 (1972)

    CAS  Article  Google Scholar 

  26. Lorenz, M., Poole, K. J., Popp, D., Rosenbaum, G. & Holmes, K. C. An atomic model of the unregulated thin filament obtained by X-ray fiber diffraction on oriented actin-tropomyosin gels. J. Mol. Biol. 246, 108–119 (1995)

    CAS  Article  Google Scholar 

  27. 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 

  28. Schröder, R. R. et al. Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1. Nature 364, 171–174 (1993)

    ADS  Article  Google Scholar 

  29. Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997)

    CAS  Article  Google Scholar 

  30. Herm-Götz, A. et al. Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor. EMBO J. 21, 2149–2158 (2002)

    Article  Google Scholar 

Download references


We thank S. Zimmermann for excellent technical assistance, R. Fedorov for providing Fig. 1, H. Faulstich for providing rhodaminephalloidin, C. Herrmann for the hGBP-1 cDNA, R. S. Goody, K. C. Holmes, M. A. Geeves, F. J. Kull, R. Maytum and D. P. Mulvihill for comments and discussions, and K. C. Holmes for continuous support. The work was supported by grants from the Deutsche Forschungsgemeinschaft (to D.J.M.).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Dietmar J. Manstein.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information


Supplementary Figure: Graphical representation of the back and forth movement of four actin filaments on a lawn of non-specifically adsorbed E698-WΩ2R. (GIF 17 kb)

Supplementary Movie 1: Molecular model of an artificial pointed (-) end directed myosin motor that is attached in the ‘pre-power-stroke’ state to an actin protofilament consisting of five actin monomers (green and blue). The motor is created by fusing three pre-existing molecular building blocks: a class-I myosin motor domain (grey), a directional inverter formed by a segment of human guanylate binding protein-1 (red), and an artificial lever arm formed by two α-actinin repeats (orange). (MOV 1426 kb)

Supplementary Movie 2: Actin-filaments move with their pointed (-) ends leading, on surfaces that are coated with HMM. (MOV 776 kb)

Supplementary Movie 3: Actin-filaments move with their pointed (-) ends leading, on surfaces that are coated with E698-2R. This result is in agreement with myosin II-derived motors moving towards the barbed (+) end. (MOV 236 kb)

Supplementary Movie 4: E698-Ω2R attached to an anti-His-tag antibody-coated surface moves filaments with their pointed (-) ends trailing, indicating that E698-Ω2R is a pointed (-) end directed motor. (MOV 615 kb)

Supplementary Figure and Movie Legends. (DOC 20 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tsiavaliaris, G., Fujita-Becker, S. & Manstein, D. Molecular engineering of a backwards-moving myosin motor. Nature 427, 558–561 (2004).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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