An Allosteric Gear Shift Mechanism in Eg5 Enhances Mechanochemical Coupling and Shifts the Force-Velocity Landscape

Abstract

Microtubule motors control a diverse collection of physiologies, including cell division, organelle traffic, and microtubule dynamics. Fourteen kinesin family members are involved in mitosis. The best studied of these is Eg5, which is a poorly processive motor that can occupy a two head-strongly-bound state on the microtubule. It works primarily in ensembles of tetramers, cross-linking parallel and anti-parallel microtubules in the maturing spindle. Chemical ablation of Eg5 arrests mitosis. Proteomic screens have identified post-translational modifications as potential modifiers of Eg5 activity. We investigated a novel modification, acetylation of lysine 146 by mutating this residue in Eg5's α2 helix to a glutamine. Molecular dynamics simulations demonstrate that modifications of this lysine disrupts critical ionic interactions between the α2 and α1 helices, increasing the allosteric coupling between the nucleotide binding site and Eg5's mechanical element, the neck-linker. We tested these predictions using structural kinetics measurements made by transient time-resolved FRET detection of neck-linker docking and switch-1 closure during ATP binding to rigor Eg5 attached to microtubules. We also investigated the mechanical consequences of this charge mutant by investigating processive motility of fluorescently labeled Eg5 dimers in the presence and absence of applied load. These tests show that the K146Q acetylation-mimic mutation increases the coupling between the neck-linker and nucleotide binding site, accelerates ATP driven neck-linker docking, tightens the force dependence of stepping, and increases processive run-length and stall force in the presence of load, all indicating that the modification enhances mechanochemical coupling in Eg5. After modification of this residue, Eg5 behaves much more like kinesin-1, a processive motor with head-head gating. We are investigating the physiologic implications for these changes.