Molecular engineering of a backwards-moving myosin motor

Abstract

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°.

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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.

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Acknowledgements

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.).

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Correspondence to Dietmar J. Manstein.

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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)

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Tsiavaliaris, G., Fujita-Becker, S. & Manstein, D. Molecular engineering of a backwards-moving myosin motor. Nature 427, 558–561 (2004). https://doi.org/10.1038/nature02303

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