Kinesin walks processively on microtubules (MTs) in an asymmetric hand-over-hand manner consuming one ATP molecule per 16-nm step. The individual contributions due to docking of the approximately 13-residue neck linker to the leading head (deemed to be the power stroke) and diffusion of the trailing head (TH) that contributes in propelling the motor by 16 nm have not been quantified. We use molecular simulations by creating a coarse-grained model of the MT-kinesin complex, which reproduces the measured stall force as well as the force required to dislodge the motor head from the MT, to show that nearly three-quarters of the step occurs by bidirectional stochastic motion of the TH. However, docking of the neck linker to the leading head constrains the extent of diffusion and minimizes the probability that kinesin takes side steps, implying that both the events are necessary in the motility of kinesin and for the maintenance of processivity. Surprisingly, we find that during a single step, the TH stochastically hops multiple times between the geometrically accessible neighboring sites on the MT before forming a stable interaction with the target binding site with correct orientation between the motor head and the [Formula: see text] tubulin dimer.

Conventional Kinesin (Kin-1), which is responsible for directional transport of cellular vesicles, takes multiple nearly uniform 8.2 nm steps by consuming one ATP molecule per step as it walks towards the plus end of the microtubule (MT). Despite decades of intensive experimental and theoretical studies there are gaps in the elucidation of key steps in the catalytic cycle of kinesin. For example, how the motor waits for ATP to bind to the leading head has become controversial. Two experiments using a similar protocol, which follow the movement of a large gold nanoparticle attached to one of the motor heads, have arrived at different conclusions. One of them (1) asserts that kinesin waits for ATP in a state with both heads bound to the MT, whereas the other (2) shows that ATP binds to the leading head after the trailing head is detached. In order to discriminate between these two scenarios, we developed a minimal model, which analytically predicts the outcomes of a number of experimental observables quantities, such as the distribution of run length [P (n)], the distribution of velocity [P (v)], and the randomness parameter as a function of an external resistive force (F ) and ATP concentration ([T]). We find that P (n) is insensitive to the waiting state of kinesin. The bimodal velocity distribution P (v) depends on the ATP waiting states of kinesin. The differences in P (v) as a function of F between the two models may be amenable to experimental testing. Most importantly, we predict that the F and [T] dependence of the randomness parameters differ qualitatively depending on whether ATP waits with both heads bound to the MT or with detached tethered head. The randomness parameters as a function of F and [T] can be quantitatively measured from stepping trajectories with very little prejudice in data analysis. Therefore, an accurate measurement of the randomness parameter and the velocity distribution as a function of load and nucleotide concentration could resolve the apparent controversy, thus providing insights into the waiting state of kinesin for ATP. A. Chemical randomness parameter, r C 8 B. Mechanical randomness parameter, r M 9 C. Derivation of mechanical randomness parameter with backward steps 11 IV. Two models for how kinesin waits for ATP 17 V. Variant of 1HB model: k − is independent of [T] 20 VI. Fitting theory to experimental data 21 References 23 2 I. DERIVATION OF THE RUN LENGTH DISTRIBUTION The summation in Eq.(4) in the main text, P (n) = ∞ m,l=0(m + l)! m!l! can be carried out for n > 0, leading to, P (n > 0) =

Conventional kinesin walks by a hand-over-hand mechanism on the microtubule (MT) by taking ∼8 nm discrete steps and consumes one ATP molecule per step. The time needed to complete a single step is on the order of 20 μs. We show, using simulations of a coarse-grained model of the complex containing the two motor heads, the MT and the coiled coil, that to obtain quantitative agreement with experiments for the stepping kinetics hydrodynamic interactions (HIs) have to be included. In simulations without hydrodynamic interactions, spanning nearly 20 μs, not a single step was completed in one hundred trajectories. In sharp contrast, nearly 14% of the steps reached the target binding site within 6 μs when HIs were included. Somewhat surprisingly, there are qualitative differences in the diffusion pathways in simulations with and without HI. The extent of movement of the trailing head of kinesin on the MT during the diffusion stage of stepping is considerably greater in simulations with HI than in those without HI. It is likely that inclusion of HI is crucial in the accurate description of motility of other motors as well.

SummaryCytoplasmic Dynein, a motor with an unusual architecture made up of a motor domain belonging to the AAA+ family, walks on microtubule towards the minus end. Prompted by the availability of structures in different nucleotide states, we performed simulations based on a new coarse-grained model to illustrate the molecular details of the dynamics of allosteric transitions in the motor. The simulations show that binding of ATP results in the closure of the cleft between the AAA1 and AAA2, which in turn triggers conformational changes in the rest of the motor domain, thus poising dynein in the pre-power stroke state. Interactions with the microtubule, which are modeled implicitly, substantially enhances the rate of ADP release, and formation of the post-power stroke state.The dynamics associated with the key mechanical element, the linker (LN) domain, which changes from a straight to a bent state and vice versa, are highly heterogeneous suggestive of multiple routes in the pre power stroke to post power stroke transition. We show that persistent interactions between the LN and the insert loops in the AAA2 domain prevent the formation of pre-power stroke state when ATP is bound to AAA3, thus locking dynein in a non-functional repressed state. Motility in such a state may be rescued by applying mechanical force to the LN domain. Taken together, these results show how the intricate signaling dynamics within the motor domain facilitate the stepping of dynein. IntroductionMolecular motors are spectacular nanomachines that transport cargos by moving processively along filamentous actin and microtubules (MT). 1-3 Of the three motor families, kinesins and dyneins walk along MTs whereas myosins step on filamentous actin. Although a number of questions still remain unanswered, it is fair to say that the mechanism of processive motion of kinesin-1 and myosin V is fairly well understood, thanks to remarkable experimental studies conducted over the last twenty five years. 2,[4][5][6][7][8][9][10][11][12][13][14][15][16] In contrast, the characterization of the motility of cytoplasmic dynein has lagged behind.Although discovered over fifty years ago, 17 it is only in the last decade the complexity 2 and variations in the architecture of dyneins have been revealed, raising various questions that challenge our views of information processing in nanoscale biological machines. Even though this issue, which relates to to allosteric communication in protein complexes, is most interesting, our focus here is to provide quantitative insights into the molecular basis of the dynamics of allosteric transitions between distinct functional states in the motility of dynein, a most intriguing molecular motor.Cytoplasmic dynein transports cargo along MTs, and is a minus end-directed motor. Among dyne's different cargoes are endosomes, lysosomes, and mitochondria. [18][19][20] Additionally, cytoplasmic dynein (or dynein from now on) is involved in the process of positioning of the spindle during cell division. 21 In almost all respects, dynein...