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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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.).
The authors declare that they have no competing financial interests.
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)
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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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