Mechanical control of the directional stepping dynamics of the kinesin motor

Among the multiple steps constituting the kinesin’s mechanochemical cycle, one of the most interesting events is observed when kinesins move an 8-nm step from one microtubule (MT)-binding site to another. The stepping motion that occurs within a relatively short time scale (100 microsec) is, however, beyond the resolution of current experiments, therefore a basic understanding to the real-time dynamics within the 8-nm step is still lacking. For instance, the rate of power stroke (or conformational change), that leads to the undocked-to-docked transition of neck-linker, is not known, and the existence of a substep during the 8-nm step still remains a controversial issue in the kinesin community. By using explicit structures of the kinesin dimer and the MT consisting of 13 protofilaments (PFs), we study the stepping dynamics with varying rates of power stroke (kp). We estimate that 1/kp < 20 microsec to avoid a substep in an averaged time trace. For a slow power stroke with 1/kp>20 microsec, the averaged time trace shows a substep that implies the existence of a transient intermediate, which is reminiscent of a recent single molecule experiment at high resolution. We identify the intermediate as a conformation in which the tethered head is trapped in the sideway binding site of the neighboring PF. We also find a partial unfolding (cracking) of the binding motifs occurring at the transition state ensemble along the pathways prior to binding between the kinesin and MT.

💡 Research Summary

The paper addresses a long‑standing gap in our understanding of kinesin’s 8‑nm stepping motion, which occurs on a sub‑100 µs timescale and is therefore invisible to most experimental techniques. The authors combine explicit atomic models of a kinesin dimer and a 13‑protofilament microtubule (MT) with a novel computational framework that treats the stepping process as a diffusion of the tethered head on a time‑dependent potential of mean force (PMF). Two extreme PMFs are generated: one for a fully disordered neck‑linker (λ = 0) and one for a fully docked neck‑linker (λ = 1). The λ = 1 landscape contains a deep basin at the forward binding site (e) and a broad, entropically driven basin (S) separated by modest barriers (1‑2 kBT in the x‑z and x‑y projections, 4‑5 kBT in y‑z). In contrast, the λ = 0 landscape favors a side‑binding site (c) on an adjacent protofilament, which offers only about one‑third of the native contacts compared with the correct site e, making it a metastable, poorly bound intermediate.

The stepping dynamics are modeled as a “reflected diffusion” where the neck‑linker docking (the power stroke) acts as a moving reflecting boundary that gradually restricts the accessible region for the tethered head. The transition from λ = 0 to λ = 1 is parameterized by a rate kp = τp⁻¹, and the combined PMF is interpolated linearly in time (Eq. 1). Brownian dynamics simulations of a quasi‑particle representing the head centroid are performed with an effective diffusion coefficient D_eff = 2 µm² s⁻¹, reproducing the experimental temporal resolution (~20 µs). By varying τp, the authors quantify how many trajectories become trapped in the side site c before reaching the forward site e.

Key findings: when τp ≤ 20 µs (kp ≫ kE, where kE is the diffusion‑exploration rate), >90 % of trajectories reach e directly, and the ensemble‑averaged position trace shows a single exponential rise—no substep appears. When τp > 20 µs (kp ≲ kE), a significant fraction of trajectories pause at c, producing a two‑phase rise in the averaged trace that matches the substep observed in recent high‑resolution single‑molecule experiments (≈4 nm displacement). The intermediate at c is destabilized by ~5 kBT during the docking transition, explaining why it disappears when the power stroke is fast.

In addition to the kinetic analysis, the study reveals a structural “cracking” phenomenon: during the transition state ensemble the MT‑binding motifs of the kinesin head partially unfold, reducing the entropic barrier for binding. This is quantified by the fractions of native contacts within the motifs (Q_p) and at the kinesin‑MT interface (Q_int). The transition state shows Q_p ≈ 0.65 versus Q_p ≈ 0.82 in the fully bound complex, and the free‑energy surface F(Q_p, Q_int) indicates a ~6 kBT barrier associated with this partial unfolding. Such cracking is reminiscent of mechanisms observed in other protein–protein association processes and suggests that structural flexibility facilitates rapid binding.

The discussion integrates these results with experimental observations. The authors argue that left‑diagonal stepping is prohibited by geometric constraints, and that a helical path arising from alternating side‑step and parallel‑step motions would contradict known kinesin trajectories, thereby ruling out the use of two parallel protofilaments for forward motion. They also emphasize that the existence of the side‑site intermediate depends critically on the relative speeds of power‑stroke docking and diffusive search; a fast power stroke eliminates the intermediate, while a slower stroke allows its detection as a substep.

Overall, the paper provides a coherent, semi‑quantitative framework that links the molecular architecture of kinesin and the microtubule lattice to the observable stepping dynamics. By identifying the critical timescale (~20 µs) that separates substep‑free from substep‑containing motion, and by uncovering the role of partial unfolding in facilitating binding, the work advances our mechanistic understanding of how chemical energy is converted into directed mechanical motion in this essential molecular motor.