Pathogenic mutations in the kinesin-3 motor KIF1A diminish force generation and movement through allosteric mechanisms.

The kinesin-3 motor KIF1A functions in neurons, where its fast and superprocessive motility facilitates long-distance transport, but little is known about its force-generating properties. Using optical tweezers, we demonstrate that KIF1A stalls at an opposing load of ~3 pN but more frequently detaches at lower forces. KIF1A rapidly reattaches to the microtubule to resume motion due to its class-specific K-loop, resulting in a unique clustering of force generation events. To test the importance of neck linker docking in KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders. Molecular dynamics simulations predict that V8M and Y89D mutations impair neck linker docking. Indeed, both mutations dramatically reduce the force generation of KIF1A but not the motor's ability to rapidly reattach to the microtubule. Although both mutations relieve autoinhibition of the full-length motor, the mutant motors display decreased velocities, run lengths, and landing rates and delayed cargo transport in cells. These results advance our understanding of how mutations in KIF1A can manifest in disease.