Backstepping Mechanism of Kinesin-1

Abstract

Kinesin-1 is an ATP-driven molecular motor that transports cellular cargo along microtubules. At low loads, kinesin-1 almost always steps forward, toward microtubule plus ends, but at higher loads, it can also step backward. Backsteps are usually 8 nm but can be larger. These larger backward 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 only these larger backsteps, but all kinesin-1 backsteps, are slips. We show first that kinesin waits before forward steps for less time than before backsteps and detachments; second, we show that kinesin waits for the same amount of time before backsteps and detachments; and third, we show 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, whereas 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. MATERIALS AND METHODS Kinesin beadsUnmodified 560-nm-diameter polystyrene beads (Polysciences, Warrington, PA) were mixed with purified recombinant full-length Drosophila

Force and Stepwise Movements of Gliding Motility in Human Pathogenic Bacterium Mycoplasma pneumoniae

“…In the infectious process, M. pneumoniae cells glide to the deep positions of respiratory systems and experience a large load at these positions (Prince et al, 2014). The load-dependent stepping behavior would be useful to glide against large loads because a load-independent stepping motor, kinesin, shows frequent back steps under large loads (Carter and Cross, 2006;Toleikis et al, 2020). Different step sizes under different loads have also been observed in M. mobile gliding (Kinosita et al, 2014;Mizutani et al, 2018).…”

Force and stepwise movements of gliding motility in human pathogenic bacterium Mycoplasma pneumoniae

“…In the infectious process, M. pneumoniae cells glide to the deep positions of respiratory systems and experience a large load at these positions (12). The load-dependent stepping manner would be useful to glide against large loads because a load-independent stepping motor, kinesin, shows frequent back steps under large loads (48, 49). Different step sizes under different loads have also been observed in M. mobile gliding (29, 31).…”

Force generation of KIF1C is impaired by pathogenic mutations

“…We proposed recently that backslips arise when phosphate is released before a forward step is completed, which converts the motor into a weakly bound state that is pulled backwards under the load of the trap. Backwards slipping is stopped when either of the two motor domains in ADP state re-engages at a binding site and releases nucleotide to start another cycle of trying to make a forward step (Toleikis et al, 2020). Kinesin-3s are about tenfold more processive and twice as fast as kinesin-1 (Soppina et al, 2014, Scarabelli et al, 2015.…”

“…To determine the force output of KIF1C, we used a classic single bead optical tweezer assay (Block et al, 1990, Carter andCross, 2005) to probe the force response of full-length recombinant human KIF1C (Siddiqui et al, 2019) and for comparison full-length Drosophila melanogaster kinesin heavy chain (KHC) (Coy et al, 1999, Toleikis et al, 2020. In this assay, a single motor protein pulls the bead against the force of the trap until it reaches stall force or detaches.…”

“…For KIF1CR169W-GFP data pooled from 3 recordings of 2 motile beads from 1 experiment. KHC data are a subset of a previously published dataset (Toleikis et al, 2020) with the exception that step analysis only included data acquired at a comparable trap stiffness to KIF1C data and data from superstall conditions were only included for the stall force analysis. Data analysis was carried out using custom code written in R. From the traces generated, a moving window t-test algorithm (Carter and Cross, 2005) was used to identify steps with the following parameters -t-test score threshold=30, minimum step size=5 nm, minimum force=1 pN, moving average, n=20 (1 ms).…”