A kinetic dissection of the fast and superprocessive kinesin-3 KIF1A reveals a predominant one-head-bound state during its chemomechanical cycle

Abstract

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to wild-type KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and-2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate limiting transition in the KIF1A stepping cycle. Thus, KIF1A’s activity can be explained by a fast rear head detachment rate, a rate-limiting step of tethered head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly-bound post-hydrolysis state.

Pathogenic mutations in the kinesin-3 motor KIF1A diminish force generation and movement through allosteric mechanisms

“…S3 I). Given the recent finding that ATP hydrolysis triggers forward stepping of KIF1A (Zaniewski et al, 2020), these analyses are consistent with the MD simulations that predict allosteric effects on reduced catalytic site closure and reduced ATP hydrolysis (Fig. 5).…”

“…While such a behavior has also been reported for kinesin-1 (Khataee and Howard, 2019;Pyrpassopoulos et al, 2020), KIF1A appears to be more sensitive to vertical forces than single kinesin-1 motors, which appear to resist detachment under load better (Brenner et al, 2020;Carter and Cross, 2005;Ramaiya et al, 2017;Svoboda and Block, 1994). A high loaddependent detachment rate may be due to the fact that KIF1A spends most of its mechanochemical cycle in a one-head-bound state, at least under unloaded conditions (Zaniewski et al, 2020), and provides a mechanism for why KIF1A gives up easily when forced to compete with kinesin-1 motors in driving cargo transport (Arpag et al, 2019;Arpag et al, 2014;Norris et al, 2014). Interestingly, kinesin-2 (KIF3A/KIF3B) and kinesin-5 (Eg5) motors also have a tendency to detach at moderate forces in optical trap assays (Andreasson et al, 2015;Korneev et al, 2007;Milic et al, 2017;Schroeder et al, 2012;Shimamoto et al, 2015;Valentine and Block, 2009;Valentine et al, 2006) and to give up easily when in competition with kinesin-1 (Arpag et al, 2014).…”

“…Recent studies have shown that several members of the kinesin-3 family have striking motility properties, as they are exceptionally fast and superprocessive and have high MT landing rates (ability to productively engage with MTs; Hammond et al, 2009;Huckaba et al, 2011;Lessard et al, 2019;Okada et al, 1995;Rogers et al, 2001;Scarabelli et al, 2015;Siddiqui et al, 2019;Tomishige et al, 2002;Zaniewski et al, 2020). However, little is known about the ability of kinesin-3 motors to generate and sustain force.…”

Cellular functions of actin- and microtubule-associated septins

“…SEPT9_i1 might provide additional attachment sites for KIF1A through its amino-terminal acidic domains and/or induce conformational changes at the microtubule-kinesin interface. The latter may enhance microtubule docking of the lagging unbound motor domain of the KIF1A dimer, a rate limiting-step in KIF1A motility 142 , and/or weaken the intramolecular interactions between the microtubule-bound motor domain and the neck linker, which would accelerate docking of the lagging motor domain 143 . Of note, the neck linker of KIF1A is uniquely shorter than other kinesin motors 143,144 .…”

KIF1A is kinetically tuned to be a super-engaging motor under hindering loads

“…A recent biochemical study exploring the mechanochemical adaptations of KIF1A suggested that rear-head detachment is an order of magnitude faster than found for kinesins-1 or -2, and that this feature helps to explain its rapid stepping rate. This kinetic feature also results in a predominant steady-state intermediate that is bound via a single "weakly-bound" post-hydrolysis motor domain through electrostatic interactions with the microtubule (14). This single-head microtubule interaction may result in a molecule that is vulnerable to detachment under mechanical load.…”