Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5–Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance

Ottilie Von Loeffelholz,Alejandro Peña,Douglas Robert Drummond,Robert Cross,Carolyn Ann Moores

doi:10.1016/j.jmb.2019.01.011

Ottilie Von Loeffelholz, Alejandro Peña + Show 3 more

Open Access

https://doi.org/10.1016/j.jmb.2019.01.011

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Abstract

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor–microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.

Highlights

Members of the kinesin superfamily of microtubule (MT)-based, ATP-driven molecular motors play multiple essential roles in cell division, reflecting the dynamic complexity of the machinery required for accurate chromosome segregation
Purified native fission yeast tubulin contains a mixture of α-tubulin1/2 isoforms (87% sequence identity), a single β-tubulin isoform and no post-translational modifications [11]
We recently showed that the structural properties of fission yeast tubulin are distinct from mammalian brain tubulin (Mam_tub) with respect to the angle of protofilament skew within the MT lattice and the different structural response to tubulin GTPase activity [15]

Summary

Introduction

Members of the kinesin superfamily of microtubule (MT)-based, ATP-driven molecular motors play multiple essential roles in cell division, reflecting the dynamic complexity of the machinery required for accurate chromosome segregation. We used cryoelectron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding.

Results

Conclusion