The net charge of the K-loop regulates KIF1A superprocessivity by enhancing microtubule affinity in the one-head-bound state

Abstract

KIF1A is an essential neuronal transport motor protein in the kinesin-3 family, 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 6-fold lower in the loop-swap mutant compared to wild type. 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.