Mechanism of Microtubule Stabilization by Kinesin-5

Abstract

In addition to their capacity to slide apart antiparallel microtubules during spindle formation, the mitotic kinesin-5 motor Eg5 has been shown to pause at microtubule plus-ends and enhance microtubule polymerization (Chen and Hancock, Nature Comm. 2015:8160). The goal of the present work is to understand the Eg5 microtubule polymerase mechanism by studying how the motor alters the lateral and longitudinal tubulin-tubulin interactions that stabilize the microtubule lattice. Transient kinetics and single-molecule tracking experiments demonstrate that dimeric Eg5 motors reside predominantly in a two-head-bound strong-binding state while stepping along the microtubule (Chen et al., JBC 2016:291(39), 20283-94). This suggests that when Eg5 pauses at a growing microtubule plus-end, the motor acts as a two-head-bound “staple” to stabilize the longitudinal bonds of incoming tubulin dimers. The on-rate for Eg5 binding to free tubulin is slow, suggesting that end-bound Eg5 motors do not bind free tubulin in solution; rather they stabilize incoming tubulin dimers that have bound to the plus-end. Because tubulin in the microtubule lattice resides in a “straight” conformation, while free tubulin resides in a “kinked” conformation, a second (non-mutually exclusive) model is that Eg5 stabilizes the straight conformation of tubulin. Consistent with this, monomeric Eg5 motors, which bind to the microtubule lattice without crosslinking tubulin dimers, also stabilize microtubules against depolymerization. Furthermore, the affinity of Eg5 for free tubulin is reduced in the presence of “wedge inhibitor” drugs that stabilize the kinked conformation of tubulin; conversely Eg5 binding to tubulin diminishes drug binding. Thus, we propose a microtubule polymerase mechanism in which binding by one Eg5 motor domain at the microtubule plus-end straightens tubulin dimers and stabilizes lateral tubulin-tubulin bonds, while the second Eg5 head binds incoming tubulin and acts as a staple to stabilize longitudinal tubulin-tubulin bonds and enhance microtubule growth.