Processivity of the Kinesin-2 KIF3A Results From Rear-Head Gating and Not Front-Head Gating [Molecular Biophysics]

February 5th, 2015 by Chen, G.-Y., Arginteanu, D. F. J., Hancock, W. O.

The kinesin-2 family motor KIF3A/B works together 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 processivity may be controlled by different gating mechanisms. We used stopped flow and steady-state kinetics experiments, along with single-molecule and multi-motor assays to characterize the entire kinetic cycle of a KIF3A homodimer that exhibits similar motility to full-length KIF3A/B. Upon first encounter with a microtubule, the motor rapidly exchanges both mADP and mATP. 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. A similar lack of front-head gating was found when intramolecular strain was enhanced by shortening the neck linker domain from 17 to 14 residues. In single-molecule assays in ADP, the motor dissociates at 2.1 s-1, 20-fold slower than the stepping rate, demonstrating the presence of rear-head gating. In microtubule pelleting assays the KDMt is similar in ADP and ATP. The data and accompanying simulations suggest that, rather than KIF3A processivity resulting from strain-dependent regulation of nucleotide binding (front-head gating), the motor spends a significant fraction of its hydrolysis cycle in a low affinity state, but dissociates only slowly from this state. This work provides a mechanism to explain differences in the load-dependent properties of kinesin-1 and kinesin-2.