Concept: Molecular motor
We have observed by NMR spectroscopy that the diffusive movement of a ruthenium-based Grubbs' catalyst increases during ring-closing metathesis as a function of the substrate concentration. This is one of the smallest single molecule motors to exhibit catalytically driven motion.
DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism.
Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 1 year ago
The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5'-3' through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.
Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds actin in a controlled and manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an angstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head-head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps.
There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes.
Molecular machines are among the most complex of all functional molecules and lie at the heart of nearly every biological process. A number of synthetic small-molecule machines have been developed, including molecular muscles, synthesizers, pumps, walkers, transporters and light-driven and electrically driven rotary motors. However, although biological molecular motors are powered by chemical gradients or the hydrolysis of adenosine triphosphate (ATP), so far there are no synthetic small-molecule motors that can operate autonomously using chemical energy (that is, the components move with net directionality as long as a chemical fuel is present). Here we describe a system in which a small molecular ring (macrocycle) is continuously transported directionally around a cyclic molecular track when powered by irreversible reactions of a chemical fuel, 9-fluorenylmethoxycarbonyl chloride. Key to the design is that the rate of reaction of this fuel with reactive sites on the cyclic track is faster when the macrocycle is far from the reactive site than when it is near to it. We find that a bulky pyridine-based catalyst promotes carbonate-forming reactions that ratchet the displacement of the macrocycle away from the reactive sites on the track. Under reaction conditions where both attachment and cleavage of the 9-fluorenylmethoxycarbonyl groups occur through different processes, and the cleavage reaction occurs at a rate independent of macrocycle location, net directional rotation of the molecular motor continues for as long as unreacted fuel remains. We anticipate that autonomous chemically fuelled molecular motors will find application as engines in molecular nanotechnology.
In contrast to processive molecular motors, skeletal myosins form a large motor ensemble for contraction of muscles against high loads. Despite numerous information on the molecular properties of skeletal myosin, its ensemble effects on collective force generation have not been rigorously clarified. Here we show 4 nm stepwise actin displacements generated by synthetic myofilaments beyond a load of 30 pN, implying that steps cannot be driven exclusively by single myosins, but potentially by coordinated force generations among multiple myosins. The simulation model shows that stepwise actin displacements are primarily caused by coordinated force generation among myosin molecules. Moreover, the probability of coordinated force generation can be enhanced against high loads by utilizing three factors: strain-dependent kinetics between force-generating states; multiple power stroke steps; and high ATP concentrations. Compared with other molecular motors, our findings reveal how the properties of skeletal myosin are tuned to perform cooperative force generation for efficient muscle contraction.
Intracellular cargo transport relies on myosin Va molecular motor ensembles to travel along the cell’s three-dimensional (3D) highway of actin filaments. At actin filament intersections, the intersecting filament is a structural barrier to and an alternate track for directed cargo transport. Here we use 3D super-resolution fluorescence imaging to determine the directional outcome (that is, continues straight, turns or terminates) for an ∼10 motor ensemble transporting a 350 nm lipid-bound cargo that encounters a suspended 3D actin filament intersection in vitro. Motor-cargo complexes that interact with the intersecting filament go straight through the intersection 62% of the time, nearly twice that for turning. To explain this, we develop an in silico model, supported by optical trapping data, suggesting that the motors' diffusive movements on the vesicle surface and the extent of their engagement with the two intersecting actin tracks biases the motor-cargo complex on average to go straight through the intersection.
Cell migration, which is central to many biological processes including wound healing and cancer progression, is sensitive to environmental stiffness, and many cell types exhibit a stiffness optimum, at which migration is maximal. Here we present a cell migration simulator that predicts a stiffness optimum that can be shifted by altering the number of active molecular motors and clutches. This prediction is verified experimentally by comparing cell traction and F-actin retrograde flow for two cell types with differing amounts of active motors and clutches: embryonic chick forebrain neurons (ECFNs; optimum ∼1 kPa) and U251 glioma cells (optimum ∼100 kPa). In addition, the model predicts, and experiments confirm, that the stiffness optimum of U251 glioma cell migration, morphology and F-actin retrograde flow rate can be shifted to lower stiffness by simultaneous drug inhibition of myosin II motors and integrin-mediated adhesions.
Photodriven molecular motors are able to convert light energy into directional motion and hold great promise as miniaturized powering units for future nanomachines. In the current state of the art, considerable efforts have still to be made to increase the efficiency of energy transduction and devise systems that allow operation in ambient and non-damaging conditions with high rates of directional motions. The need for ultraviolet light to induce the motion of virtually all available light-driven motors especially hampers the broad applicability of these systems. We describe here a hemithioindigo-based molecular motor, which is powered exclusively by nondestructive visible light (up to 500 nm) and rotates completely directionally with kHz frequency at 20 °C. This is the fastest directional motion of a synthetic system driven by visible light to date permitting materials and biocompatible irradiation conditions to establish similarly high speeds as natural molecular motors.