Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose.

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

Bidirectional cargo transport: moving beyond tug of war.

Vesicles, organelles and other intracellular cargo are transported by kinesin and dynein motors, which move in opposite directions along microtubules. This bidirectional cargo movement is frequently described as a 'tug of war' between oppositely directed molecular motors attached to the same cargo. However, although many experimental and modelling studies support the tug-of-war paradigm, numerous knockout and inhibition studies in various systems have found that inhibiting one motor leads to diminished motility in both directions, which is a 'paradox of co-dependence' that challenges the paradigm. In an effort to resolve this paradox, three classes of bidirectional transport models--microtubule tethering, mechanical activation and steric disinhibition--are proposed, and a general mathematical modelling framework for bidirectional cargo transport is put forward to guide future experiments.

KIF1A is kinetically tuned to be a superengaging motor under hindering loads.

KIF1A is a highly processive vesicle transport motor in the kinesin-3 family. Mutations in KIF1A lead to neurodegenerative diseases including hereditary spastic paraplegia. We applied optical tweezers to study the ability of KIF1A to generate and sustain force against hindering loads. We used both the three-bead assay, where force is oriented parallel to the microtubule, and the traditional single-bead assay, where force is directed along the radius of the bead, resulting in a vertical force component. The average force and attachment duration of KIF1A in the three-bead assay were substantially greater than those observed in the single-bead assay. Thus, vertical forces accelerate termination of force ramps of KIF1A. Average KIF1A termination forces were slightly lower than the kinesin-1 KIF5B, and the median attachment duration of KIF1A was >10-fold shorter than KIF5B under hindering loads. KIF1A rapidly reengages with microtubules after detachment, as observed previously. Strikingly, quantification enabled by the three-bead assay shows that reengagement largely occurs within 2 ms of detachment, indicating that KIF1A has a nearly 10-fold faster reengagement rate than KIF5B. We found that rapid microtubule reengagement is not due to KIF1A's positively charged loop-12; however, removal of charge from this loop diminished the unloaded run length at near physiological ionic strength. Both loop-12 and the microtubule nucleotide state have modulatory effects on reengagement under load, suggesting a role for the microtubule lattice in KIF1A reengagement. Our results reveal adaptations of KIF1A that lead to a model of superengaging transport under load.

Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-head-bound state.

KIF1A is an essential neuronal transport motor protein in the kinesin-3 family, known for its superprocessive motility. However, structural features underlying this function are unclear. Here, we determined that superprocessivity of KIF1A dimers originates from a unique structural domain, the lysine-rich insertion in loop-12 termed the 'K-loop', which enhances electrostatic interactions between the motor and the microtubule. In 80 mM PIPES buffer, replacing the native KIF1A loop-12 with that of kinesin-1 resulted in a 6-fold decrease in run length, whereas adding additional positive charge to loop-12 enhanced the run length. Interestingly, swapping the KIF1A loop-12 into kinesin-1 did not enhance its run length, consistent with the two motor families using different mechanochemical tuning to achieve persistent transport. To investigate the mechanism by which the KIF1A K-loop enhances processivity, we used microtubule pelleting and single-molecule dwell time assays in ATP and ADP. First, the microtubule affinity was similar in ATP and in ADP, consistent with the motor spending the majority of its cycle in a weakly bound state. Second, the microtubule affinity and single-molecule dwell time in ADP were 6-fold lower in the loop-swap mutant than WT. Thus, the positive charge in loop-12 of KIF1A enhances the run length by stabilizing binding of the motor in its vulnerable one-head-bound state. Finally, through a series of mutants with varying positive charge in the K-loop, we found that KIF1A processivity is linearly dependent on the charge of loop-12, further highlighting how loop-12 contributes to the function of this key motor protein.

Simulations suggest robust microtubule attachment of kinesin and dynein in antagonistic pairs.

Intracellular transport is propelled by kinesin and cytoplasmic dynein motors that carry membrane-bound vesicles and organelles bidirectionally along microtubule tracks. Much is known about these motors at the molecular scale, but many questions remain regarding how kinesin and dynein cooperate and compete during bidirectional cargo transport at the cellular level. The goal of the present study was to use a stochastic stepping model constructed by using published load-dependent properties of kinesin-1 and dynein-dynactin-BicD2 (DDB) to identify specific motor properties that determine the speed, directionality, and transport dynamics of a cargo carried by one kinesin and one dynein motor. Model performance was evaluated by comparing simulations to recently published experiments of kinesin-DDB pairs connected by complementary oligonucleotide linkers. Plotting the instantaneous velocity distributions from kinesin-DDB experiments revealed a single peak centered around zero velocity. In contrast, velocity distributions from simulations displayed a central peak around 100 nm/s, along with two side peaks corresponding to the unloaded kinesin and DDB velocities. We hypothesized that frequent motor detachment events and relatively slow motor reattachment rates resulted in periods in which only one motor is attached. To investigate this hypothesis, we varied specific model parameters and compared the resulting instantaneous velocity distributions, and we confirmed this systematic investigation using a machine-learning approach that minimized the residual sum of squares between the experimental and simulation velocity distributions. The experimental data were best recapitulated by a model in which the kinesin and dynein stall forces are matched, the motor detachment rates are independent of load, and the kinesin-1 reattachment rate is 50 s-1. These results provide new insights into motor dynamics during bidirectional transport and put forth hypotheses that can be tested by future experiments.

Trim9 and Klp61F promote polymerization of new dendritic microtubules along parallel microtubules.

Axons and dendrites are distinguished by microtubule polarity. In Drosophila, dendrites are dominated by minus-end-out microtubules, whereas axons contain plus-end-out microtubules. Local nucleation in dendrites generates microtubules in both orientations. To understand why dendritic nucleation does not disrupt polarity, we used live imaging to analyze the fate of microtubules generated at branch points. We found that they had different rates of success exiting the branch based on orientation: correctly oriented minus-end-out microtubules succeeded in leaving about twice as often as incorrectly oriented microtubules. Increased success relied on other microtubules in a parallel orientation. From a candidate screen, we identified Trim9 and kinesin-5 (Klp61F) as machinery that promoted growth of new microtubules. In S2 cells, Eb1 recruited Trim9 to microtubules. Klp61F promoted microtubule growth in vitro and in vivo, and could recruit Trim9 in S2 cells. In summary, the data argue that Trim9 and kinesin-5 act together at microtubule plus ends to help polymerizing microtubules parallel to pre-existing ones resist catastrophe.

Measurement of the persistence length of cytoskeletal filaments using curvature distributions.

Cytoskeletal filaments, such as microtubules and actin filaments, play important roles in the mechanical integrity of cells and the ability of cells to respond to their environment. Measuring the mechanical properties of cytoskeletal structures is crucial for gaining insight into intracellular mechanical stresses and their role in regulating cellular processes. One of the ways to characterize these mechanical properties is by measuring their persistence length, the average length over which filaments stay straight. There are several approaches in the literature for measuring filament deformations, such as Fourier analysis of images obtained using fluorescence microscopy. Here, we show how curvature distributions can be used as an alternative tool to quantify biofilament deformations, and investigate how the apparent stiffness of filaments depends on the resolution and noise of the imaging system. We present analytical calculations of the scaling curvature distributions as a function of filament discretization, and test our predictions by comparing Monte Carlo simulations with results from existing techniques. We also apply our approach to microtubules and actin filaments obtained from in vitro gliding assay experiments with high densities of nonfunctional motors, and calculate the persistence length of these filaments. The presented curvature analysis is significantly more accurate compared with existing approaches for small data sets, and can be readily applied to both in vitro and in vivo filament data through the use of the open-source codes we provide.

Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A.

Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency.

Microtubule binding kinetics of membrane-bound kinesin-1 predicts high motor copy numbers on intracellular cargo.

Bidirectional vesicle transport along microtubules is necessary for cell viability and function, particularly in neurons. When multiple motors are attached to a vesicle, the distance a vesicle travels before dissociating is determined by the race between detachment of the bound motors and attachment of the unbound motors. Motor detachment rate constants (koff) can be measured via single-molecule experiments, but motor reattachment rate constants (kon) are generally unknown, as they involve diffusion through the bilayer, geometrical considerations of the motor tether length, and the intrinsic microtubule binding rate of the motor. To understand the attachment dynamics of motors bound to fluid lipid bilayers, we quantified the microtubule accumulation rate of fluorescently labeled kinesin-1 motors in a 2-dimensional (2D) system where motors were linked to a supported lipid bilayer. From the first-order accumulation rate at varying motor densities, we extrapolated a koff that matched single-molecule measurements and measured a 2D kon for membrane-bound kinesin-1 motors binding to the microtubule. This kon is consistent with kinesin-1 being able to reach roughly 20 tubulin subunits when attaching to a microtubule. By incorporating cholesterol to reduce membrane diffusivity, we demonstrate that this kon is not limited by the motor diffusion rate, but instead is determined by the intrinsic motor binding rate. For intracellular vesicle trafficking, this 2D kon predicts that long-range transport of 100-nm-diameter vesicles requires 35 kinesin-1 motors, suggesting that teamwork between different motor classes and motor clustering may play significant roles in long-range vesicle transport.

Direct observation of individual tubulin dimers binding to growing microtubules.

The biochemical basis of microtubule growth has remained elusive for over 30 years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make direct, single-molecule measurements of tubulin association and dissociation rates. We detect two populations of transient dwell times and determine via binding-interface mutants that they are distinguished by the formation of one interprotofilament bond. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data and, furthermore, predicts plus-end tapering. Overall, we provide the most direct and complete experimental quantification of how microtubules grow to date.

Load-dependent detachment kinetics plays a key role in bidirectional cargo transport by kinesin and dynein.

Bidirectional cargo transport along microtubules is carried out by opposing teams of kinesin and dynein motors. Despite considerable study, the factors that determine whether these competing teams achieve net anterograde or retrograde transport in cells remain unclear. The goal of this work is to use stochastic simulations of bidirectional transport to determine the motor properties that most strongly determine overall cargo velocity and directionality. Simulations were carried out based on published optical tweezer characterization of kinesin-1 and kinesin-2, and for available data for cytoplasmic dynein and the dynein-dynactin-BicD2 (DDB) complex. By varying dynein parameters and analyzing cargo trajectories, we find that net cargo transport is predicted to depend minimally on the dynein stall force, but strongly on dynein load-dependent detachment kinetics. In simulations, dynein is dominated by kinesin-1, but DDB and kinesin-1 are evenly matched, recapitulating recent experimental work. Kinesin-2 competes less well against dynein and DDB, and overall, load-dependent motor detachment is the property that most determines a motor's ability to compete in bidirectional transport. It follows that the most effective intracellular regulators of bidirectional transport are predicted to be those that alter motor detachment kinetics rather than motor velocity or stall force.

A kinetic dissection of the fast and superprocessive kinesin-3 KIF1A reveals a predominant one-head-bound state during its chemomechanical cycle.

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.

Insights into Kinesin-1 Stepping from Simulations and Tracking of Gold Nanoparticle-Labeled Motors.

High-resolution tracking of gold nanoparticle-labeled proteins has emerged as a powerful technique for measuring the structural kinetics of processive enzymes and other biomacromolecules. These techniques use point spread function (PSF) fitting methods borrowed from single-molecule fluorescence imaging to determine molecular positions below the diffraction limit. However, compared to fluorescence, gold nanoparticle tracking experiments are performed at significantly higher frame rates and utilize much larger probes. In the current work, we use Brownian dynamics simulations of nanoparticle-labeled proteins to investigate the regimes in which the fundamental assumptions of PSF fitting hold and where they begin to break down. We find that because gold nanoparticles undergo tethered diffusion around their anchor point, PSF fitting cannot be extended to arbitrarily fast frame rates. Instead, camera exposure times that allow the nanoparticle to fully populate its stationary positional distribution achieve a spatial averaging that increases fitting precision. We furthermore find that changes in the rotational freedom of the tagged protein can lead to artifactual translations in the fitted particle position. Finally, we apply these lessons to dissect a standing controversy in the kinesin field over the structure of a dimer in the ATP waiting state. Combining new experiments with simulations, we determine that the rear kinesin head in the ATP waiting state is unbound but not displaced from its previous microtubule binding site and that apparent differences in separately published reports were simply due to differences in the gold nanoparticle attachment position. Our results highlight the importance of gold conjugation decisions and imaging parameters to high-resolution tracking results and will serve as a useful guide for the design of future gold nanoparticle tracking experiments.

The Orphan Kinesin PAKRP2 Achieves Processive Motility via a Noncanonical Stepping Mechanism.

Phragmoplast-associated kinesin-related protein 2 (PAKRP2) is an orphan kinesin in Arabidopsis thaliana that is thought to transport vesicles along phragmoplast microtubules for cell plate formation. Here, using single-molecule fluorescence microscopy, we show that PAKRP2 is the first orphan kinesin to exhibit processive plus-end-directed motility on single microtubules as individual homodimers. Our results show that PAKRP2 processivity is achieved despite having an exceptionally long (32 residues) neck linker. Furthermore, using high-resolution nanoparticle tracking, we find that PAKRP2 steps via a hand-over-hand mechanism that includes frequent side steps, a prolonged diffusional search of the tethered head, and tight coupling of the ATP hydrolysis cycle to the forward-stepping cycle. Interestingly, truncating the PAKRP2 neck linker to 14 residues decreases the run length of PAKRP2; thus, the long neck linker enhances the processive behavior. Based on the canonical model of kinesin stepping, such a long neck linker is expected to decrease the processivity and disrupt the coupling of ATP hydrolysis to forward stepping. Therefore, we conclude that PAKRP2 employs a noncanonical strategy for processive motility, wherein a long neck linker is coupled with a slow ATP hydrolysis rate to allow for an extended diffusional search during each step without sacrificing processivity or efficiency.

Motor Dynamics Underlying Cargo Transport by Pairs of Kinesin-1 and Kinesin-3 Motors.

Intracellular cargo transport by kinesin family motor proteins is crucial for many cellular processes, particularly vesicle transport in axons and dendrites. In a number of cases, the transport of specific cargo is carried out by two classes of kinesins that move at different speeds and thus compete during transport. Despite advances in single-molecule characterization and modeling approaches, many questions remain regarding the effect of intermotor tension on motor attachment/reattachment rates during cooperative multimotor transport. To understand the motor dynamics underlying multimotor transport, we analyzed the complexes of kinesin-1 and kinesin-3 motors attached through protein scaffolds moving on immobilized microtubules in vitro. To interpret the observed behavior, simulations were carried out using a model that incorporated motor stepping, attachment/detachment rates, and intermotor force generation. In single-molecule experiments, isolated kinesin-3 motors moved twofold faster and had threefold higher landing rates than kinesin-1. When the positively charged loop 12 of kinesin-3 was swapped with that of kinesin-1, the landing rates reversed, indicating that this "K-loop" is a key determinant of the motor reattachment rate. In contrast, swapping loop 12 had negligible effects on motor velocities. Two-motor complexes containing one kinesin-1 and one kinesin-3 moved at different speeds depending on the identity of their loop 12, indicating the importance of the motor reattachment rate on the cotransport speed. Simulations of these loop-swapped motors using experimentally derived motor parameters were able to reproduce the experimental results and identify best fit parameters for the motor reattachment rates for this geometry. Simulation results also supported previous work, suggesting that kinesin-3 microtubule detachment is very sensitive to load. Overall, the simulations demonstrate that the transport behavior of cargo carried by pairs of kinesin-1 and -3 motors are determined by three properties that differ between these two families: the unloaded velocity, the load dependence of detachment, and the motor reattachment rate.

The axonal transport motor kinesin-2 navigates microtubule obstacles via protofilament switching.

Axonal transport involves kinesin motors trafficking cargo along microtubules that are rich in microtubule-associated proteins (MAPs). Much attention has focused on the behavior of kinesin-1 in the presence of MAPs, which has overshadowed understanding the contribution of other kinesins such as kinesin-2 in axonal transport. We have previously shown that, unlike kinesin-1, kinesin-2 in vitro motility is insensitive to the neuronal MAP Tau. However, the mechanism by which kinesin-2 efficiently navigates Tau on the microtubule surface is unknown. We hypothesized that mammalian kinesin-2 side-steps to adjacent protofilaments to maneuver around MAPs. To test this, we used single-molecule imaging to track the characteristic run length and protofilament switching behavior of kinesin-1 and kinesin-2 motors in the absence and presence of 2 different microtubule obstacles. Under all conditions tested, kinesin-2 switched protofilaments more frequently than kinesin-1. Using computational modeling that recapitulates run length and switching frequencies in the presence of varying roadblock densities, we conclude that kinesin-2 switches protofilaments to navigate around microtubule obstacles. Elucidating the kinesin-2 mechanism of navigation on the crowded microtubule surface provides a refined view of its contribution in facilitating axonal transport.

Motor Reattachment Kinetics Play a Dominant Role in Multimotor-Driven Cargo Transport.

Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multimotor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be fourfold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of a gold-nanoparticle-labeled motor with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than does kinesin-1. Finally, compared to cargo transported by two kinesin-1, cargo transported by two kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multimotor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads whereas kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle.

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.

Kinesin Processivity Is Determined by a Kinetic Race from a Vulnerable One-Head-Bound State.

Kinesin processivity, defined as the average number of steps that occur per interaction with a microtubule, is an important biophysical determinant of the motor's intracellular capabilities. Despite its fundamental importance to the diversity of tasks that kinesins carry out in cells, no existing quantitative model fully explains how structural differences between kinesins alter kinetic rates in the ATPase cycle to produce functional changes in processivity. Here we use high-resolution single-molecule microscopy to directly observe the stepping behavior of kinesin-1 and -2 family motors with different length neck-linker domains. We characterize a one-head-bound posthydrolysis vulnerable state where a kinetic race occurs between attachment of the tethered head to its next binding site and detachment of the bound head from the microtubule. We find that greater processivity is correlated with faster attachment of the tethered head from this vulnerable state. In compliment, we show that slowing detachment from this vulnerable state by strengthening motor-microtubule electrostatic interactions also increases processivity. Furthermore, we provide evidence that attachment of the tethered head is irreversible, suggesting a first passage model for exit from the vulnerable state. Overall, our results provide a kinetic framework for explaining kinesin processivity and for mapping structural differences to functional differences in diverse kinesin isoforms.