Backstepping mechanism of kinesin-1

Kinesin-1 is an ATP-driven molecular motor that transports cellular cargo along microtubules. At low loads, kinesin-1 almost always steps forwards, towards microtubule plus ends, but at higher loads, it can also step backwards. Backsteps are usually 8 nm, but can be larger. These larger backwards events of 16 nm, 24 nm or more are thought to be slips rather than steps, because they are too fast to consist of multiple, tightly-coupled 8 nm steps. Here we propose that not just these larger backsteps, but all kinesin-1 backsteps, are slips. We show firstly that kinesin waits before forward steps for less time than before backsteps and detachments; secondly that kinesin waits for the same amount of time before backsteps and detachments and thirdly that by varying the microtubule type we can change the ratio of backsteps to detachments, without affecting forward stepping. Our findings indicate that backsteps and detachments originate from the same state and that this state arises later in the mechanochemical cycle than the state that gives rise to forward steps. To explain our data, we propose that in each cycle of ATP turnover, forward kinesin steps can only occur before Pi release, whilst backslips and detachments can only occur after Pi release. In the scheme we propose, Pi release gates access to a weak binding K.ADP-K.ADP state that can slip back along the microtubule, re-engage, release ADP and try again to take an ATP-driven forward step. We predict that this rescued detachment pathway is key to maintaining kinesin processivity under load. Significance statementKinesin-1 molecular motors are ATP-driven walking machines that typically step forward, towards microtubule plus ends. But they can also step backwards, especially at high load. Backsteps are currently thought to occur by directional reversal of forwards walking. To the contrary, we propose here that kinesin backsteps are not steps, but slips. We show that backwards translocations originate from a different and later state in the kinesin mechanism than the state that generates forward steps. To explain this, we propose that following ATP binding, kinesin molecules that fail to step forward within a load-dependent time window convert to a state that can slip back, rebind to the microtubule, and try again to step forward.