Intramolecular strain coordinates kinesin stepping behavior along microtubules

Kinesin advances 8 nm along a microtubule per ATP hydrolyzed, but the mechanism responsible for coordinating the enzymatic cycles of kinesin's two identical motor domains remains unresolved. Here, we have tested whether such coordination is mediated by intramolecular tension generated by the "neck linkers," mechanical elements that span between the motor domains. When tension is reduced by extending the neck linkers with artificial peptides, the coupling between ATP hydrolysis and forward stepping is impaired and motor's velocity decreases as a consequence. However, speed recovers to nearly normal levels when external tension is applied by an optical trap. Remarkably, external load also induces bidirectional stepping of an immotile kinesin that lacks its mechanical element (neck linker) and fuel (ATP). Our results indicate that the kinesin motor domain senses and responds to strain in a manner that facilitates its plus-end-directed stepping and communication between its two motor domains.     

PcrA helicase, a prototype ATP-driven molecular motor

Despite extensive studies, the mechanisms underlying molecular motor function are still poorly understood. Key to the mechanisms is the coupling of ATP hydrolysis to conformational changes of the motor protein. To investigate this coupling, we have conducted combined quantum mechanical/molecular mechanical simulations of PcrA helicase, a strikingly simple motor that translocates unidirectionally along single-stranded DNA (ssDNA). Our results reveal a close similarity in catalytic site structure and reaction pathway to those of F1-ATPase, and these similarities include a proton relay mechanism important for efficient ATP hydrolysis and an "arginine finger" residue that is key to the coupling of the chemical reaction to protein conformational changes. By means of in silico mutation studies, we identified the residue Q254 as being crucial for the coupling of ssDNA translocation to the actual catalytic event. Based on the present result for PcrA helicase and previous findings for F1-ATPase, we propose a general mechanism of ATP-driven molecular motor function.     

A simple theoretical model explains dynein's response to load

Recent experiment showed that cytoplasmic dynein 1, a molecular motor responsible for cargo transport in cells, functions as a gear in response to external load. In the presence of vanishing or small external load, dynein walks with 24- or 32-nm steps, whereas at high external load, its step size is reduced to 8 nm. A simple model is proposed to account for this property of dynein. The model assumes that the chemical energy of ATP hydrolysis is used through a loose coupling between the chemical reaction and the translocation of dynein along microtubule. This loose chemomechanical coupling is represented by the loosely coupled motions of dynein along two different reaction coordinates. The first reaction coordinate is tightly coupled to the chemical reaction and describes the protein conformational changes that control the chemical processes, including ATP binding and hydrolysis, and ADP-Pi release. The second coordinate describes the translocation of dynein along microtubule, which is directly subject to the influence of the external load. The model is used to explain the experimental data on the external force dependence of the dynein step size as well as the ATP concentration dependence of the stall force. A number of predictions, such as the external force dependence of speed of translocation, ATP hydrolysis rate, and dynein step sizes, are made based on this theoretical model. This model provides a simple understanding on how a variable chemomechanical coupling ratio can be achieved and used to optimize the biological function of dynein.     

Insights into the chemomechanical coupling of the myosin motor from simulation of its ATP hydrolysis mechanism

The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP x P(i) state of nucleotide-bound myosin.     

Single molecule processes on the stepwise movement of ATP-driven molecular motors

Movement is a fundamental characteristic of all living things. This biogenic function that is attributed to the molecular motors such as kinesin, dynein and myosin. Molecular motors generate forces by using chemical energy derived from the hydrolysis reaction of ATP molecules. Despite a large number of studies on this topic, the chemomechanical energy transduction mechanism is still unsolved. In this study, we have investigated the chemomechanical coupling of the ATPase cycle to the mechanical events of the molecular motor kinesin using single molecule detection (SMD) techniques. The SMD techniques allowed to detection of the movement of single kinesin molecules along a microtubule and showed that kinesin steps mainly in the forward direction, but occasionally in the backward. The stepping direction is determined by a certain load-dependent process, on which the stochastic behavior is well characterized by Feynman's thermal ratchet model. The driving force of the stepwise movement is essentially Brownian motion, but it is biased in the forward direction by using the free energy released from the hydrolysis of ATP.     

Myosin motor domain lever arm rotation is coupled to ATP hydrolysis

We have investigated coupling of lever arm rotation to the ATP binding and hydrolysis steps for the myosin motor domain. In several current hypotheses of the mechanism of force production by muscle, the primary mechanical feature is the rotation of a lever arm that is a subdomain of the myosin motor domain. In these models, the lever arm rotates while the myosin motor domain is free, and then reverses the rotation to produce force while it is bound to actin. These mechanical steps are coupled to steps in the ATP hydrolysis cycle. Our hypothesis is that ATP hydrolysis induces lever arm rotation to produce a more compact motor domain that has stored mechanical energy. Our approach is to use transient electric birefringence techniques to measure changes in hydrodynamic size that result from lever arm rotation when various ligands are bound to isolated skeletal muscle myosin motor domain in solution. Results for ATP and CTP, which do support force production by muscle fibers, are compared to those of ATPgammaS and GTP, which do not. Measurements are also made of conformational changes when the motor domain is bound to NDP's and PP(i) in the absence and presence of the phosphate analogue orthovanadate, to determine the roles the nucleoside moieties of the nucleotides have on lever arm rotation. The results indicate that for the substrates investigated, rotation does not occur upon substrate binding, but is coupled to the NTP hydrolysis step. The data are consistent with a model in which only substrates that produce a motor domain-NDP-P(i) complex as the steady-state intermediate make the motor domain more compact, and only those substrates support force production.     

Two distinct modes of processive kinesin movement in mixtures of ATP and AMP-PNP

An enzyme is frequently conceived of as having a single functional mechanism. This is particularly true for motor enzymes, where the necessity for tight coupling of mechanical and chemical cycles imposes rigid constraints on the reaction pathway. In mixtures of substrate (ATP) and an inhibitor (adenosine 5'-(beta,gamma-imido)triphosphate or AMP-PNP), single kinesin molecules move on microtubules in two distinct types of multiple-turnover "runs" that differ in their susceptibility to inhibition. Longer (less susceptible) runs are consistent with movement driven by the alternating-sites mechanism previously proposed for uninhibited kinesin. In contrast, kinesin molecules in shorter runs step with AMP-PNP continuously bound to one of the two active sites of the enzyme. Thus, in this mixture of substrate and inhibitor, kinesin can function as a motor enzyme using either of two distinct mechanisms. In one of these, the enzyme can accomplish high-duty-ratio processive movement without alternating-sites ATP hydrolysis.     

A deterministic mechanism producing the loose coupling phenomenon observed in an actomyosin system

Myosins are molecular motors that convert the chemical energy of ATP into mechanical work called a power stroke. Class II myosin engaged in muscle contraction is reported to show a "loose coupling phenomenon", in which the number of power strokes is greater than the number of ATP hydrolyses. This phenomenon cannot be explained by the lever-arm hypothesis, which is currently accepted as a standard theory for myosin motility. In this paper, a model is proposed to reproduce the loose coupling phenomenon. The model is based on a mechanochemical process called "Driven by Detachment (DbD)" mechanism, which assumes that the energy of the power strokes originates from the potential energy generated by the attractive force between myosin and actin. During the docking process, the potential energy is converted into an intramolecular strain in a myosin molecule, which drives the power stroke after the myosin is firmly attached to an actin filament. The energy of ATP is used to temporarily reduce the attractive force and to increase the potential energy. Therefore, it is not directly linked to the power strokes. When myosin molecules work as an aggregate, the sliding movement of a myosin filament driven by the power strokes of some myosin heads makes other myosin heads that have completed their power strokes detach from the actin without consuming ATP. Under the DbD mechanism, these passively detached myosins can be again engaged in power strokes after the next attachment to actin. As a result, the number of power strokes becomes greater than the number of ATP hydrolyses, and the loose coupling phenomenon will be observed. A theoretical analysis indicates that the efficiency of converting the potential energy into intramolecular elastic energy determines the number of power strokes per each ATP hydrolysis. Computer simulations showed that the DbD mechanism actually produced the loose coupling phenomenon. A critical requirement for this mechanism is that ATP must preferentially facilitate the detachment of myosins that have completed their power strokes, but are still strongly attached to the actin. This requirement may be fulfilled by ATP hydrolysis tightly depending on the conformation of a myosin molecule.     

Mechanochemical coupling in the myosin motor domain. I. Insights from equilibrium active-site simulations

Although the major structural transitions in molecular motors are often argued to couple to the binding of Adenosine triphosphate (ATP), the recovery stroke in the conventional myosin has been shown to be dependent on the hydrolysis of ATP. To obtain a clearer mechanistic picture for such "mechanochemical coupling" in myosin, equilibrium active-site simulations with explicit solvent have been carried out to probe the behavior of the motor domain as functions of the nucleotide chemical state and conformation of the converter/relay helix. In conjunction with previous studies of ATP hydrolysis with different active-site conformations and normal mode analysis of structural flexibility, the results help establish an energetics-based framework for understanding the mechanochemical coupling. It is proposed that the activation of hydrolysis does not require the rotation of the lever arm per se, but the two processes are tightly coordinated because both strongly couple to the open/close transition of the active site. The underlying picture involves shifts in the dominant population of different structural motifs as a consequence of changes elsewhere in the motor domain. The contribution of this work and the accompanying paper [] is to propose the actual mechanism behind these "population shifts" and residues that play important roles in the process. It is suggested that structural flexibilities at both the small and large scales inherent to the motor domain make it possible to implement tight couplings between different structural motifs while maintaining small free-energy drops for processes that occur in the detached states, which is likely a feature shared among many molecular motors. The significantly different flexibility of the active site in different X-ray structures with variable level arm orientations supports the notation that external force sensed by the lever arm may transmit into the active site and influence the chemical steps (nucleotide hydrolysis and/or binding).     

Measurement system for simultaneous observation of myosin V chemical and mechanical events

Myosin V is an actin-based processive molecular motor driven by the chemical energy of ATP hydrolysis. Although the chemo-mechanical coupling in processive movement has been postulated by separate structural, mechanical and biochemical studies, no experiment has been able to directly test these conclusions. Therefore the relationship between ATP-turnover and force generation remains unclear. Currently, the most direct method to measure the chemo-mechanical coupling in processive motors is to simultaneously observe ATP-turnover cycles and displacement at the single molecule level. In this study, we developed a simultaneous measurement system suitable for mechanical and chemical assays of myosin V in order to directly elucidate its chemo-mechanical coupling.     

A physical model for motor proteins

A general stochastic theory is outlined for chemical to mechanical energy transduction by motor enzymes. In addition to ATP hydrolysis and fiber binding phenomena, thermal noise effects are taken into account. A minimal, 4-state model is identified that gives the hydrolysis rate as well as mechanical quantities such as sliding velocity and generated force, as functions of ATP concentration and the number of motors. It explains in a unified way many results of recent in vitro assays, both in myosins/actin and kinesins or dyneins/microtubule systems.     

Mechanical properties of BiP protein determined by nano‐rheology

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N‐terminal Nucleotide‐Binding Domain (NBD), and the C‐terminal Substrate‐Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano‐rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides‐ ATP, ADP, and AMP‐PNP‐, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (K_D) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.     

Coupled chemical and mechanical reaction steps in a processive Neurospora kinesin.

We show using single molecule optical trapping and transient kinetics that the unusually fast Neurospora kinesin is mechanically processive, and we investigate the coupling between ATP turnover and the mechanical actions of the motor. Beads carrying single two-headed Neurospora kinesin molecules move in discrete 8 nm steps, and stall at approximately 5 pN of retroactive force. Using microtubule-activated release of the fluorescent analogue 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mantADP) to report microtubule binding, we found that initially only one of the two motor heads binds, and that the binding of the other requires a nucleotide 'chase'. mantADP was released from the second head at 4 s(-1) by an ADP chase, 5 s(-1) by 5'-adenylylimidodiphosphate (AMPPNP), 27 s(-1) by ATPgammaS and 60 s(-1) by ATP. We infer a coordination mechanism for molecular walking, in which ATP hydrolysis on the trailing head accelerates leading head binding at least 15-fold, and leading head binding then accelerates trailing head unbinding at least 6-fold.     

Altered Nucleotide-Microtubule Coupling and Increased Mechanical Output by a Kinesin Mutant

Kinesin motors hydrolyze ATP to produce force and do work in the cell – how the motors do this is not fully understood, but is thought to depend on the coupling of ATP hydrolysis to microtubule binding by the motor. Transmittal of conformational changes from the microtubule- to the nucleotide-binding site has been proposed to involve the central β-sheet, which could undergo large structural changes important for force production. We show here that mutation of an invariant residue in loop L7 of the central β-sheet of the Drosophila kinesin-14 Ncd motor alters both nucleotide and microtubule binding, although the mutated residue is not present in either site. Mutants show weak-ADP/tight-microtubule binding, instead of tight-ADP/weak-microtubule binding like wild type – they hydrolyze ATP faster than wild type, move faster in motility assays, and assemble long spindles with greatly elongated poles, which are also produced by simulations of assembly with tighter microtubule binding and faster sliding. The mutated residue acts like a mechanochemical coupling element – it transmits changes between the microtubule-binding and active sites, and can switch the state of the motor, increasing mechanical output by the motor. One possibility, based on our findings, is that movements by the residue and the loop that contains it could bend or distort the central β-sheet, mediating free energy changes that lead to force production.     

A simulation model of the conventional kinesin based on the Driven-by-Detachment mechanism

Kinesins are molecular motors that unidirectionally move along microtubules using the chemical energy of ATP. Although the core structure of kinesins is similar to that of myosins, the lever-arm hypothesis, which is widely accepted as a plausible mechanism to explain the behaviors of myosins, cannot be directly applied to kinesins. Masuda has proposed a mechanochemical process called the ‘Driven-by-Detachment (DbD)’ mechanism to explain the characteristic behaviors of myosins, including the backward movement of myosin VI and the loose coupling phenomenon of myosin II. The DbD mechanism assumes that the energy of ATP is mainly used to detach a myosin head from an actin filament by temporarily reducing the affinity of the myosin against the actin. After the affinity is recovered, the detached head has potential energy originating from the attractive force between the myosin and the actin. During the docking process, the potential energy is converted into elastic energy within the myosin molecule, and the intramolecular elastic energy is finally used to produce the power strokes. In the present paper, the DbD mechanism was used to explain the hand-over-hand motion of the conventional kinesin. The neck linker of the kinesin is known to determine the directionality of the motility but, in this paper, it was assumed that the neck linker was not directly engaged in the power strokes, which were driven by the attractive force between the kinesin head and the microtubule. Based on this assumption, simple mechanical simulations showed that the model of a kinesin dimer processively moved along a microtubule protofilament, if the affinity of the kinesin against the microtubule is appropriately controlled. Moreover, if an external force was applied to the center of the kinesin dimer, the dimer moved backward along a microtubule, as observed in experimental motility assays.     

Complex cooperativity of ATP hydrolysis in the F(1)-ATPase molecular motor

F(1)-ATPase catalyses ATP hydrolysis and converts the cellular chemical energy into mechanical rotation. The hydrolysis reaction in F(1)-ATPase does not follow the widely believed Michaelis-Menten mechanism. Instead, the hydrolysis mechanism behaves in an ATP-dependent manner. We develop a model for enzyme kinetics and hydrolysis cooperativity of F(1)-ATPase which involves the binding-state changes to the coupling catalytic reactions. The quantitative analysis and modeling suggest the existence of complex cooperative hydrolysis between three different catalysis sites of F(1)-ATPase. This complexity may be taken into account to resolve the arguments on the binding change mechanism in F(1)-ATPase.     

Coupling between ATP hydrolysis and protein conformational change in maltose transporter

As the intracellular part of maltose transporter, MalK dimer utilizes the energy of ATP hydrolysis to drive protein conformational change, which then facilitates substrate transport. Free energy evaluation of the complete conformational change before and after ATP hydrolysis is helpful to elucidate the mechanism of chemical‐to‐mechanical energy conversion in MalK dimer, but is lacking in previous studies. In this work, we used molecular dynamics simulations to investigate the structural transition of MalK dimer among closed, semi‐open and open states. We observed spontaneous structural transition from closed to open state in the ADP‐bound system and partial closure of MalK dimer from the semi‐open state in the ATP‐bound system. Subsequently, we calculated the reaction pathways connecting the closed and open states for the ATP‐ and ADP‐bound systems and evaluated the free energy profiles along the paths. Our results suggested that the closed state is stable in the presence of ATP but is markedly destabilized when ATP is hydrolyzed to ADP, which thus explains the coupling between ATP hydrolysis and protein conformational change of MalK dimer in thermodynamics. Proteins 2017; 85:207–220. © 2016 Wiley Periodicals, Inc.     

Rapid double 8-nm steps by a kinesin mutant

The mechanism by which conventional kinesin walks along microtubules is poorly understood, but may involve alternate binding to the microtubule and hydrolysis of ATP by the two heads. Here we report a single amino-acid change that affects stepping by the motor. Under low force or low ATP concentration, the motor moves by successive 8-nm steps in single-motor laser-trap assays, indicating that the mutation does not alter the basic mechanism of kinesin walking. Remarkably, under high force, the mutant motor takes successive 16-nm displacements that can be resolved into rapid double 8-nm steps with a short dwell between steps, followed by a longer dwell. The alternating short and long dwells under high force demonstrate that the motor stepping mechanism is inherently asymmetric, revealing an asymmetric phase in the kinesin walking cycle. Our findings support an asymmetric two-headed walking model for kinesin, with cooperative interactions between the two heads. The sensitivity of the 16-nm displacements to nucleotide and load raises the possibility that ADP release is a force-producing event of the kinesin cycle.     

An electromechanical model of myosin molecular motors

There is a long-running debate on the working mechanism of myosin molecular motors, which, by interacting with actin filaments, convert the chemical energy of ATP into a variety of mechanical work. After the development of technologies for observing and manipulating individual working molecules, experimental results negating the widely accepted 'lever-arm hypothesis' have been reported. In this paper, based on the experimental results so far accumulated, an alternative hypothesis is proposed, in which motor molecules are modelled as electromechanical components that interact with each other through electrostatic force. Electrostatic attractive force between myosin and actin is assumed to cause a conformational change in the myosin head during the attachment process. An elastic energy resulting from the conformational change then produces the power stroke. The energy released at the ATP hydrolysis is mainly used to detach the myosin head from actin filaments. The mechanism presented in this paper is compatible with the experimental results contradictory to the previous theories. It also explains the behavior of myosins V and VI, which are engaged in cellular transport and move processively along actin filaments.