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 wild-type 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.

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 tenfold 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 novel model of super-engaging transport under load.

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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.

KIF1A is an essential neuronal transport motor protein known for its superprocessive motility. 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 loop-12 of KIF1A with that of kinesin-1, resulted in a 6-fold decrease in run length, and 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 times 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 ~4-fold lower in the loop-swap mutant. Thus, the positive charge in loop-12 of KIF1A enhances the run length by stabilizing the motor binding in its vulnerable one-head-bound state. Finally, through a series of mutants with varying positive charge in the K-loop, we found that the KIF1A processivity is linearly dependent on the charge of loop-12.