Komori, Y. | bioRxiv Science

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↗

bioRxiv2019

Structural basis for two-way communication between dynein and microtubules

The movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain of dynein via an a-helical coiled-coil stalk, but the structural basis of this communication remains elusive. Here, we regulated MTBD either in high-affinity or low-affinity states by introducing a disulfide bond between the coiled-coils and analyzed the resulting structures by NMRand cryo-EM. In the MT-unbound state, the affinity changes of MTBD were achieved by sliding of the N-terminal -helix by one half-turn, which suggests that structural changes propagate from the ATPase-domain to MTBD. In addition, cryo-EM analysis showed that MT binding induced further sliding of the N-terminal -helix even without the disulfide bond, which suggests the MT-induced conformational changes propagate toward the ATPase domain. Based on differences in the MT-binding surface between the high- and low-affinity states, we propose a potential mechanism for the directional bias of dynein movement on MT tracks.

biophysics↗