ATPγS substantially defeats the biasing mechanism for kinesin steps

TL;DR

This study shows how ATPγS affects kinesin motor proteins, causing them to take backward steps and revealing a new state in their movement mechanism.

Contribution

The paper identifies a new ATP-bound state in kinesin that influences its directional bias and stepping behavior under load.

Findings

ATPγS at 1 mM causes kinesin to take slow backsteps by overpopulating the AI state.

Load-dependent neck-linker docking potentiates exit from the AI state and promotes forward stepping.

The AI state is pivotal in the biasing mechanism that maximizes forward movement under load.

Abstract

Kinesin-1 microtubule motors are ATP-fuelled, twin-headed cargo transporters that step processively along microtubules, with a load-dependent directional bias. Here we show using single molecule optical trapping that 1 mM ATPγS, a slowly-hydrolysed analogue, substantially defeats the biasing mechanism, whereas 1 µM ATPγS supports it. Our data argue that nucleotide binding puts kinesin into a previously unrecognised Await-Isomerisation (AI) state that is overpopulated by ATPγS and generates slow backsteps. In the working model we propose, exit from this AI state establishes hydrolytic competence and is potentiated by load-dependent neck-linker docking, which steers the tethered head towards its next on-axis binding site. By overpopulating the AI state, ATPγS reveals its pivotal role in the biasing mechanism, whose control logic maximises forward stepping under load in ATP by coupling steered diffusion-to-capture of the leading kinesin head to load-dependent neck linker docking and nucleotide hydrolysis on the trailing head.

ATPγS, a slowly-hydrolysed ATP analogue, causes kinesin to take extra backsteps under load. Here, the authors examine single-molecule mechanics and propose a state in which forward steps and coupled ATP hydrolysis are turned off, but unsteered steps are allowed.

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