Processivity of Kinesin-2 Results from Rear-Head Gating and Not Front-Head Gating

Abstract

The kinesin-2 family motor KIF3A/B coordinates with dynein to bidirectionally transport intraflagellar particles, melanosomes and neuronal vesicles. Compared to kinesin-1, kinesin-2 is less processive and its processivity is more sensitive to load, suggesting that the gating mechanisms that their control processivity may differ. To understand the motor roles that front-head gating and rear-head gating, we carried out stopped flow kinetics experiments using mant nucleotides, steady state assays, and single-molecule investigations to characterize the entire kinetic cycle a functional mouse KIF3A homodimer that exhibits similar motility to full-length KIF3A/B. Upon first encounter with the microtubule lattice, the motor exchanges mADP with an on-rate of 18 μM-1 s-1 and an off-rate of 27 s-1. When AMPPNP was used to entrap the motor in a two-head bound state, exchange kinetics were unchanged, indicating that rearward strain in the two-head-bound state does not alter nucleotide binding to the front head. Similar lack of front-head gating was found with mATP and when the neck linker domain was shortened from 17 to 14 residues to enhance intramolecular strain. In contrast, microtubule pelleting and single-molecule microscopy assays found that in ADP the motor dissociates with an off-rate of 2.1 s-1 and a KD of 0.5 μM, similar to its behavior in ATP. Hence, kinesin-2 processivity results from rear-head gating and not front-head gating. Based on the kinetics measurements, finally we propose a complete model of the kinesin-2 hydrolysis cycle that accounts for all of the kinetics and motility data. This study provides the direct evidence that rear-head gating does and not front-head gating does not play a role in kinesin-2 processivity and suggests that kinesin-2 mechanochemistry is specifically adapted for bidirectional transport.