Engineering controllable bidirectional molecular motors based on myosin

Abstract

Cytoskeletal motors drive the transport of organelles and molecular cargoes within cells1, and have potential applications in molecular detection and diagnostic devices2,3. Engineering molecular motors with dynamically controllable properties will allow selective perturbation of mechanical processes in living cells, and yield optimized device components for complex tasks such as molecular sorting and directed assembly3. Biological motors have previously been modified by introducing activation/deactivation switches that respond to metal ions4,5 and other signals6. Here we show that myosin motors can be engineered to reversibly change their direction of motion in response to a calcium signal. Building on previous protein engineering studies7–11 and guided by a structural model12 for the redirected power stroke of myosin VI, we constructed bidirectional myosins through the rigid recombination of structural modules. The performance of the motors was confirmed using gliding filament assays and single fluorophore tracking. Our general strategy, in which external signals trigger changes in the geometry and mechanics of myosin lever arms, should enable spatiotemporal control over a range of motor properties including processivity, stride size13, and branchpoint turning14.

Controllable molecular motors engineered from myosin and RNA

“…Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport, and can be dynamically controlled using programmed transitions in lever arm structure 7,9 . We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy, and structural probing 16 .…”

“…Protein engineering has previously been used to replace the myosin lever arm with alternative structures that can reproduce or modify wild-type behavior 6,7,9,19 , including modules that function as gearshifts that respond to optical or chemical signals 7,9 . Since nucleic acid engineering offers a complementary approach for precise design of programmable molecular structures and devices 20,21 , we asked whether a functional myosin lever arm could also be constructed from RNA.…”

“…M6-RB (for Myosin VI - RNA-Binding) was generated by fusing myosin VI to the L7Ae kink-turn binding domain 22 . Following a design principle established in previous work on engineered myosin lever arms 6,7,9,19 , we made use of a helix-sharing junction in which the C-terminal helix of the truncated myosin VI lever arm was fused to the N-terminal helix of the L7Ae domain. Guided by crystal structures of L7Ae-RNA 22 and of myosin VI 23,24 , we aligned the terminal helices and optimized the phasing of the junction to orient a bound kink-turn motif as a structural foundation from which to build extended RNA lever arms.…”

“…We tested the M6-RB:ktL complex for its designed function using dual-labeled gliding filament assays 7,9 (Fig. 1c).…”

“…Changing the directionality of myosins by altering the structure of the lever arm has provided rigorous tests of the swinging crossbridge model and represents an additional demonstration that an attached structure functions as a lever 6,7,9,19 . Following a similar strategy used in protein engineering studies of myosins and kinesin-14 9,31 , we aimed to reverse the direction of the motor by designing an RNA lever arm that inverted the projection of the power stroke, yielding plus-end directed motility.…”