Pathogenic Mutations in the Kinesin-3 Motor KIF1A Diminish Force Generation and Movement through Allosteric Mechanisms

ABSTRACT The kinesin-3 motor KIF1A functions in neurons where its fast and superprocessive motility is thought to be critical for long-distance transport. However, little is known about the force-generating properties of kinesin-3 motors. Using optical tweezers, we demonstrate that KIF1A and its C. elegans homolog UNC-104 undergo force-dependent detachments at ~3 pN and then rapidly reattach to the microtubule to resume motion, resulting in a sawtooth pattern of clustered force generation events that is unique among the kinesin superfamily. Whereas UNC-104 motors stall before detaching, KIF1A motors do not. To examine the mechanism of KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders, V8M and Y89D, based on their location in structural elements required for force generation in kinesin-1. Molecular dynamics simulations predict that the V8M and Y89D mutations impair docking of the N-terminal (β9) or C-terminal (β10) portions of the neck linker, respectively, to the KIF1A motor domain. Indeed, both mutations dramatically impair force generation of KIF1A but not the motor’s ability to rapidly reattach to the microtubule track. Homodimeric and heterodimeric mutant motors also display decreased velocities, run lengths, and landing rates and homodimeric Y89D motors exhibit a higher frequency of non-productive, diffusive events along the microtubule. In cells, cargo transport by the mutant motors is delayed. Our work demonstrates the importance of the neck linker in the force generation of kinesin-3 motors and advances our understanding of how mutations in the kinesin motor domain can manifest in disease.