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

Kinesin is a prototypical molecular motor that converts the chemical energy of ATP hydrolysis into directed mechanical motion along microtubules (MTs), advancing in discrete 8‑nm steps. While ensemble experiments have established that each step occurs within roughly 100 µs, this timescale is beyond the resolution of most single‑molecule techniques, leaving the real‑time structural dynamics of the step largely unknown. Two central questions have persisted: (1) how fast is the power‑stroke (the neck‑linker undocking‑to‑docking transition) that drives the step, and (2) does a sub‑step or transient intermediate exist during the 8‑nm transition?

In this work the authors address both questions by constructing an explicit atomistic model of a kinesin dimer interacting with a 13‑protofilament (PF) microtubule lattice. Using a hybrid approach that couples coarse‑grained molecular dynamics with a kinetic Monte‑Carlo description of the ATP‑driven chemical cycle, they introduce a tunable parameter kp that represents the rate of the power‑stroke. By varying kp they generate ensembles of stepping trajectories and compute averaged time traces that mimic experimental read‑outs.

The simulations reveal a clear bifurcation in stepping behavior governed by the power‑stroke time constant τ = 1/kp. When τ ≤ 20 µs (fast power‑stroke), the tethered (forward) head docks directly onto the next forward binding site on the same PF. The averaged trace shows a single, smooth 8‑nm displacement with no detectable sub‑step, reproducing the classic “single‑step” picture obtained from low‑resolution measurements.

Conversely, when τ > 20 µs (slow power‑stroke), the forward head does not immediately find the canonical binding site. Instead, it slides laterally along the MT surface and becomes transiently trapped in a side‑binding pocket on an adjacent PF. This intermediate generates a pronounced sub‑step in the averaged trace; the magnitude and duration of the sub‑step increase with τ. The authors identify this side‑binding configuration as the structural basis for the “intermediate” observed in recent high‑resolution single‑molecule experiments, thereby reconciling theory with the latest data.

A detailed analysis of the transition‑state ensemble (TSE) along the binding pathway uncovers partial unfolding—or “cracking”— of key binding motifs (β‑sheets and loop regions) at the moment of MT engagement. This localized destabilization lowers the free‑energy barrier for docking and is especially prominent when the power‑stroke is slow, suggesting that the motor exploits controlled structural flexibility to facilitate binding under sub‑optimal kinetic conditions.

The study also highlights the functional relevance of the 13‑PF lattice. The presence of neighboring PFs creates alternative, lower‑affinity side‑binding sites that are inaccessible in simplified one‑PF models. The ability of the motor to explore these sites when the power‑stroke is delayed provides a mechanistic explanation for the observed sub‑step and underscores the importance of the full MT geometry in kinesin’s stepping dynamics.

From a broader perspective, the work demonstrates that the power‑stroke rate is a mechanical “knob” that directly tunes the continuity and precision of kinesin stepping. Cellular factors that modulate ATP concentration, load force, or regulatory protein interactions could thereby adjust kp, shifting the motor between a fast, processive regime (no sub‑step) and a slower, more exploratory regime (with a side‑binding intermediate). This mechanistic insight has implications for the design of kinesin‑targeted drugs, synthetic molecular machines, and for interpreting how intracellular conditions influence cargo transport efficiency.

In summary, by integrating explicit structural models of the kinesin dimer and a realistic 13‑PF microtubule lattice, the authors provide the first computational demonstration that the rate of the neck‑linker power‑stroke determines whether a sub‑step appears during the 8‑nm transition. They identify the side‑binding intermediate and the partial unfolding of binding motifs as key structural features of the transition state, linking these microscopic events to experimentally observed stepping behavior. This work bridges the gap between kinetic models and structural dynamics, offering a comprehensive framework for understanding the mechanical control of kinesin’s directional stepping.