ATPγS substantially defeats the biasing mechanism for kinesin steps.

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.