Fig. 6: Proposed scheme for kinesin stepping under load ATP binding populates the Await-Isomerisation (AI) state, by shifting the trailing, MT-attached head from an OPEN into a pre-CLOSED conformation.

a This shift triggers the tethered head to begin searching for its next site. In ATP, the AI state is transient at low load, but in ATPγS, the AI state is enriched, because ATPγS retards the isomerisation from pre-CLOSED to CLOSED. The AI state is incompetent to dock the NL and so tends to generate missteps. Under load, these are mostly backsteps (pink pathway). Isomerisation of the AI state allows entry to the Try-Forward era of the cycle (blue pathway). Full y docking the NL steers the tethered head to its next on-axis forwards binding site, maximally stabilising the CLOSED state and maximally potentiating hydrolysis. Hydrolysis can also proceed via partial NL docking, as shown (callouts), at a correspondingly slower rate. Competition between this slow background rate of hydrolysis and NL-accelerated hydrolysis creates a load-dependent time window for forward stepping. At high loads, NL docking increasingly fails to occur within this time window, so that slow flux into the No-steps (yellow) and Backslips (orange) pathways comes to dominate. Untethered backslips (orange) can evolve into full detachments, but more commonly are rescued via reattachment, reverting the motor to its ATP-waiting state and allowing forward stepping to be retried, at the expense of futile cycling. b–d illustrate specific scenarios, viewed en face. In b, ATPγS (S) binding triggers a single 8 nm backstep with reversion to the ATP-waiting state. This behaviour depends on the rear head remaining in its apo state whilst the lead head is recovered and so will dominate at 1 µM ATPγS. In c, ATPγS binds earlier, causing a second 8 nm backstep. This behaviour will dominate at 1 mM ATPγS. d is an 8 nm backslip, promoted by ADP rebinding to the front head. e is an 8 nm backstep, promoted by ADP rebinding to the front head at low ATPγS concentrations.