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

Abstract

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.

Cryo-EM Unveils the Processivity Mechanism of Kinesin KIF1A and the Impact of its Pathogenic Variant P305L

“…We show that replacing KIF1A’s K-loop for loop-12 of the kinesin-3 motor, KIF14, which has only a single positively charged residue located close to the MT interface, results in a significant reduction of the run-length of the motor. This result is consistent with recent reports that show that KIF1A’s K-loop and its positively-charged residues are essential for KIF1A’s superprocessivity 74, 75 .…”

“…KIF1A, like other kinesin-3 family members, has an elongated Lys-rich loop-12 referred to as the K-loop. While it has been shown that the K-loop is important for KIF1A-MT binding [72][73][74][75] , how it interacts with MTs has not been clear as most of this loop is either disordered or not resolved in X-ray crystal structures of KIF1A and in lower resolution cryo-EM structures of KIF1A-MT complexes. Our high-resolution structures of KIF1A-MT complexes show the complete polypeptide path of the K-loop (Fig.…”

“…The specific role of KIF1A's K-loop in MT binding has been previously established 57,[80][81][82] , yet its interaction mechanism with MTs has remained unclear. Previous X-ray crystallography and lower-resolution cryo-EM studies have not captured the conformation of this loop and how it interacts with MTs [65][66][67][68]70,71 .…”

Manipulation of force geometry using optical tweezers reveals novel features of biomolecular interactions

“…It is worth mentioning that this type of behavior does not seem to be a generic feature of all kinesins. We recently studied the forces developed by KIF1A which is a member of the kinesin 3 family and we did not find such a variable type of behavior with microtubules in the three-bead assay [27]. However, in the single-bead assay the vertical force component Fz accelerated the detachment of KIF1A from the microtubule relative to the three-bead assay, which is in agreement with what we observed for KIF5B (kinesin 1) [27].…”

“…We recently studied the forces developed by KIF1A which is a member of the kinesin 3 family and we did not find such a variable type of behavior with microtubules in the three-bead assay [27]. However, in the single-bead assay the vertical force component Fz accelerated the detachment of KIF1A from the microtubule relative to the three-bead assay, which is in agreement with what we observed for KIF5B (kinesin 1) [27]. Since, as mentioned previously, the pair of opposing forces on the microtubule in the three-bead assay may apply in general along different protofilaments, the azimuthal angle  (cylindrical coordinates of the microtubule structure) between this pair of forces will be variable between different dumbbells (Fig.…”