Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron Microscopy

Alejandro Peña,Aaron Sweeney,Alexander D Cook,Julia Locke,Maya Topf,Carolyn A Moores

doi:10.1016/j.str.2020.01.013

Alejandro Peña, Aaron Sweeney + Show 4 more

Open Access

https://doi.org/10.1016/j.str.2020.01.013

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Abstract

SummaryKinesin-5 motors are vital mitotic spindle components, and disruption of their function perturbs cell division. We investigated the molecular mechanism of the human kinesin-5 inhibitor GSK-1, which allosterically promotes tight microtubule binding. GSK-1 inhibits monomeric human kinesin-5 ATPase and microtubule gliding activities, and promotes the motor's microtubule stabilization activity. Using cryoelectron microscopy, we determined the 3D structure of the microtubule-bound motor-GSK-1 at 3.8 Å overall resolution. The structure reveals that GSK-1 stabilizes the microtubule binding surface of the motor in an ATP-like conformation, while destabilizing regions of the motor around the empty nucleotide binding pocket. Density corresponding to GSK-1 is located between helix-α4 and helix-α6 in the motor domain at its interface with the microtubule. Using a combination of difference mapping and protein-ligand docking, we characterized the kinesin-5-GSK-1 interaction and further validated this binding site using mutagenesis. This work opens up new avenues of investigation of kinesin inhibition and spindle perturbation.

Highlights

Kinesins are ATP-dependent motors that move along microtubules (MTs), organize them, and modify their dynamics
Our HsK5 construct was inhibited by GSK-1 with a half maximal inhibitory concentration (IC50) of 0.8 nM (Figure 1B), consistent with previous reports (Luo et al, 2007)
In a multi-motor MT gliding assay, where HsK5 exhibited an average uninhibited gliding velocity of 26 nm/s, HsK5 activity was inhibited by GSK-1 with an IC50 of 1.8 nM (Figure 1C)

Summary

Introduction

Kinesins are ATP-dependent motors that move along microtubules (MTs), organize them, and modify their dynamics. Kinesin-5s are important for the assembly and maintenance of spindle bipolarity. They are dumbbellshaped tetramers with pairs of motor domains at either end (Goulet and Moores, 2013). This molecular layout enables them to crosslink and slide MTs by moving toward their plus ends. The molecular mechanisms by which kinesin-5s couple their ATPase activity to nucleotidedependent conformational changes that drive motility and MT sliding are increasingly well understood (Goulet and Moores, 2013; Mann and Wadsworth, 2019)

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