High-Speed Observations Of The Unbinding/Binding Motions Of The Leading Head Of Kinesin-1 From/To Microtubule Provide Kinetic Evidence For The Front-Head Gating Mechanism

Kinesin-1 is a motor protein that moves processively along microtubule by alternately moving two motor domains (“heads”). To move in the hand-over-hand manner, ATP hydrolysis cycles of the two heads should be coordinated such that the trailing head hydrolyses ATP and detaches from the microtubule before the leading head does. To explain such coordination, the front-head gating model has been proposed that ATP binding or hydrolysis in the leading head is prohibited due to the backward tension posed through the neck linker. However, it has been technically difficult to directly measure the detachment rate of the leading head. Here, we utilized a heterodimer composed of the wild-type head and a E236A mutant head that hydrolyses ATP very slowly. Single molecule FRET observations between dyes attached to two heads showed that the wild-type head spent majority of time in the leading position under saturating ATP condition. Then we attached a 40 nm-ϕ gold particle specifically to the wild-type head and observed its motion using total internal reflection-based dark-field microscopy at 50 μs temporal resolution. At this resolution, we could clearly distinguish microtubule-bound and -unbound states by the difference in the magnitude of fluctuation of the gold probe. The dwell time of the leading head in the microtubule-bound state was 160 ms on average at 1 mM ATP condition, which was significantly larger than the dwell time of a head of wild-type homodimer in the bound state at trailing position (10 ms; Isojima et al. Nat. Chem. Biol. 2016). These results provide kinetic evidence for the front-head gating model.