Processivity of the kinesin-2 KIF3A results from rear head gating and not front head gating.

The kinesin-2 family motor KIF3A/B works together with dynein to bidirectionally transport intraflagellar particles, melanosomes, and neuronal vesicles. Compared with 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 multimotor assays to characterize the entire kinetic cycle of a KIF3A homodimer that exhibits motility similar to that of full-length KIF3A/B. Upon first encounter with a microtubule, the motor rapidly exchanges both mADP and mATP. When adenosine 5'-[(ß,γ)-imido]triphosphate 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 KD(Mt) 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.

Kinesin-5 Promotes Microtubule Nucleation and Assembly by Stabilizing a Lattice-Competent Conformation of Tubulin.

Besides sliding apart antiparallel microtubules during spindle elongation, the mitotic kinesin-5, Eg5, promotes microtubule polymerization, emphasizing its importance in mitotic spindle length control. Here, we characterize the Eg5 microtubule polymerase mechanism by assessing motor-induced changes in the longitudinal and lateral tubulin-tubulin bonds that form the microtubule lattice. Isolated Eg5 motor domains promote microtubule nucleation, growth, and stability; thus, crosslinking tubulin by pairs of motor heads is not necessary for polymerase activity. Eg5 binds preferentially to microtubules over free tubulin, which contrasts with microtubule-depolymerizing kinesins that preferentially bind free tubulin over microtubules. Colchicine-like inhibitors that stabilize the bent conformation of tubulin allosterically inhibit Eg5 binding, consistent with a model in which Eg5 induces a curved-to-straight transition in tubulin. Domain swap experiments establish that the family-specific loop11-helix 4 junction, which resides near the nucleotide-sensing switch-II domain, is necessary and sufficient for the polymerase activity of Eg5. Thus, we propose a microtubule polymerase mechanism in which Eg5 at the plus-end promotes a curved-to-straight transition in tubulin that enhances lateral bond formation and thereby promotes microtubule growth and stability. One implication is that regulation of Eg5 motile properties by regulatory proteins or small molecule inhibitors could also have effects on intracellular microtubule dynamics.