Processive Steps in the Reverse Direction Require Uncoupling of the Lead Head Lever Arm of Myosin VI

Menetrey, J.; Isabet, T.; Ropars, Virginie; Mukherjea, Monalisa; Pylypenko, Olena; Liu, Xiaoyan; Pérez, Javier; Vachette, Patrice; Sweeney, H. Lee; Houdusse, Anne

doi:10.1016/j.molcel.2012.07.034

Cited by 23 publications

(29 citation statements)

References 35 publications

(64 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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. The interaction of L7Ae with kink-turn motifs has been previously exploited in nanotechnology and synthetic biology applications 25–27 , and yields stable complexes with reported K d values of ~1 nM and dissociation rates of ~2–7 × 10 −4 s −1 25,27,28 .…”

mentioning

confidence: 99%

Controllable molecular motors engineered from myosin and RNA

et al. 2017

View full text Add to dashboard Cite

Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems1 or in living cells2. Previously, synthetic nucleic acid motors3–5 and modified natural protein motors6–10 have been developed in separate complementary strategies for achieving tunable and controllable motor function. Integrating protein and nucleic acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number, and composition of tethered protein motors11–15. 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 structure7,9. We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy, and structural probing16. Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement17 reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10–20 nm s−1. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.

show abstract

mentioning

confidence: 99%

Controllable molecular motors engineered from myosin and RNA

et al. 2017

View full text Add to dashboard Cite

show abstract

“…We suggest that the location of the hinge around which the lever arm rotates is close to residue N785 (located in ins2), at the beginning of a region identified as pliant in the PrePS state (41). Within a few microseconds from the start of the PrePS→R transition N785 binds the converter and then fluctuates around an average position (SI Appendix, Figs.…”

Section: Discussionmentioning

confidence: 99%

“…It has been suggested that the power stroke of MVI occurs with a two-step mechanism, in which first the converter rotates, and then, once the backward load from the actin-bound trailing head is relieved, the lever arm rotates toward the R-state configuration (41). In this picture, it was argued that at the end of the first step the converter domain is in a PrePS state-like configuration, but it has moved toward the R-state position on the motor domain.…”

Section: Discussionmentioning

confidence: 99%

“…Although models of the dimer incorporating the flexibility of the MVI lever arm are capable of reproducing known features of MVI motility (36), experiments using a chimeric MVI with the MV lever arm showed that the stepsize distribution was similar to the wild type (32,37), suggesting that the elusive structure of the MVI lever arm might not be the key ingredient needed to explain the peculiar stepping mechanism of MVI. Following experimental studies, structural modeling, and simulations, it is tempting to conclude that the uncoupling of the MVI leading-head lever arm from the movement of the converter, or the pliancy of the lever arm, and the conformational transition in the converter may contribute to the large step-size distribution (30,(38)(39)(40)(41). Furthermore, according to a recently proposed model (31, 37), short steps occur if the free, trailing head of a myosin dimer binds F-actin while the lever arm of the actin-bound, leading head remains in the prestroke orientation.…”

Section: Prepsmentioning

confidence: 99%

“…Although experimental evidence supports the model of the uncoupling or pliancy of the lever arm (30,(38)(39)(40)(41), direct evidence requires a structural model capable of describing the dynamics of the lever arm swing. We use a combination of coarse-grained (CG) simulations and theory to probe the dynamics of the swing of the lever arm in MVI.…”

Section: Prepsmentioning

confidence: 99%

See 2 more Smart Citations

Kinematics of the Lever Arm Swing in Myosin VI

Mugnai

Thirumalai

2017

Biophysical Journal

View full text Add to dashboard Cite

Myosin VI (MVI) is the only known member of the myosin superfamily that, upon dimerization, walks processively toward the pointed end of the actin filament. The leading head of the dimer directs the trailing head forward with a power stroke, a conformational change of the motor domain exaggerated by the lever arm. Using a unique coarse-grained model for the power stroke of a single MVI, we provide the molecular basis for its motility. We show that the power stroke occurs in two major steps. First, the motor domain attains the poststroke conformation without directing the lever arm forward; and second, the lever arm reaches the poststroke orientation by undergoing a rotational diffusion. From the analysis of the trajectories, we discover that the potential that directs the rotating lever arm toward the poststroke conformation is almost flat, implying that the lever arm rotation is mostly uncoupled from the motor domain. Because a backward load comparable to the largest interhead tension in a MVI dimer prevents the rotation of the lever arm, our model suggests that the leading-head lever arm of a MVI dimer is uncoupled, in accord with the inference drawn from polarized total internal reflection fluorescence (polTIRF) experiments. Without any adjustable parameter, our simulations lead to quantitative agreement with polTIRF experiments, which validates the structural insights. Finally, in addition to making testable predictions, we also discuss the implications of our model in explaining the broad step-size distribution of the MVI stepping pattern.myosin VI | power stroke | coarse-grained simulations | uncoupled lever arm swing | quantitative experimental predictions L ike their counterparts dyneins and kinesins, myosins are molecular motors that transform chemical energy harvested in the hydrolysis of ATP into mechanical work. They do so by undergoing a reaction cycle ( Fig. 1) involving ATP hydrolysis coupled with binding to and unbinding from the filamentous actin (F-actin) (1, 2). Myosins loaded with a hydrolyzed ATP bind to F-actin, release the products of ATP hydrolysis, and undergo a structural change known as a power stroke. The structural change of the N-terminal part of the motor domain, where the actin and nucleotide binding sites reside, is communicated to the converter domain that moves from the prepower stroke (PrePS) state to the postpower stroke (or rigor, R) state conformation. The movement of the converter is exaggerated by the large swing of the lever arm, an oblong domain bound to light chains or calmodulins (CaMs). When a new ATP molecule binds the nucleotide-free myosin (in the R state), the motor detaches from actin and it is ready to begin a new cycle.Much of the work on nonmuscle myosins has focused on myosin V (MV). However, since the discovery that myosin VI (MVI) has an unusual structure, there has been an increasing interest in the motility of MVI. In addition to its biophysical importance, MVI has been implicated in a wide variety of cellular functions in different organisms (3, 4). For...

show abstract