Biology subjects

Tomishige, M.

Publications and source records attributed to Tomishige, M..

3 recordsLinked to original sources

bioRxiv2025

Kinetic asymmetry drives kinesin-1's unidirectional and processive movement

Kinesin-1 is a dimeric motor protein that uses ATP hydrolysis energy to move along microtubules in a hand-over-hand manner1. The unidirectional movement of kinesin-1 has traditionally been explained by an ATP-dependent power stroke action of the neck linker2-4, connecting its two catalytic domains (heads), that biases the diffusional motion forward (biased-diffusion). However, recent studies on synthetic molecular motors have supported a Brownian ratchet mechanism based on kinetic asymmetry between two locations (biased- binding)5-9, and which of these mechanisms applies to biological motors remains debated10-12. Here, we engineered a two-headed kinesin that alternately uses these mechanisms to step forward, allowing us to investigate how they contribute to unidirectional movement. The tethered head that uses biased-diffusion frequently rebound to the rear-binding site but eventually stepped forward, as the front head remained securely bound to the microtubule. The biased-binding mechanism proved more efficient by preventing rebinding of the detached head and was independent of ATP binding. Instead, ATP hydrolysis energy is primarily consumed to ensure preferential detachment of the rear head. These findings demonstrate that kinesin-1 functions as an information ratchet based on kinetic asymmetry in microtubule-binding and detachment of the heads, while power strokes serve to enhance movements under load.

biophysics↗

bioRxiv2024

Tension-induced suppression of allosteric conformational changes explains coordinated stepping of kinesin-1

The dimeric motor protein kinesin-1 walks along microtubules by alternating ATP hydrolysis and movement of its two motor domains ("head"). The detached head preferentially binds to the forward tubulin-binding site after ATP binds to the microtubule-bound head, but the mechanism preventing premature binding to the microtubule while the partner head awaits ATP remains unknown. Here, we examined the role of the neck linker, the segment connecting the two heads, in this mechanism. High-resolution structural analyses of the nucleotide-free head revealed a bulge just ahead of the neck linkers base that creates an asymmetric constraint on its mobility. While the neck linker can stretch freely backward, it must navigate around this bulge to extend forward. Based on this finding, we hypothesized that premature binding of the tethered head is suppressed by an intolerable increase in neck linker tension. Molecular dynamic simulations and single-molecule fluorescent assays supported this model. These findings demonstrate a tension-based regulation mechanism where off-pathway conformational transitions are thermodynamically suppressed through entropy loss associated with neck linker stretching, suggesting that neck linker tension influences the allosteric conformational transition rather than directly affecting the nucleotide state.

biophysics↗

bioRxiv2024

Kinetic regulation of kinesin's two motor domains coordinates its stepping along microtubules

The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for the head-head coordination; however, which kinetic step(s) in the chemomechanical cycle is "gated" by the neck linker remains unresolved. Here, we employed pre-steady state kinetics and single molecule assays to investigate how the neck linker conformation affects kinesins motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubules, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.

biophysics↗