Neck region-microtubule interactions direct counterclockwise stepping of kinesin-1.

The "neck" region of kinesin is a structurally conserved element critical for force generation, stepping directionality, and cargo transport along microtubules, yet its atomic-scale structure in a functional context remains unresolved. Here, we employ all-atom replica exchange molecular dynamics simulations to resolve a high-confidence neck-region conformation of the dimeric human kinesin-1 bound to a structurally realistic microtubule lattice and subsequently use this structure to simulate kinesin's initial stepping motion. Our simulations reveal that the neck coiled coil is oriented perpendicular to the microtubule's long axis and positioned in close proximity to the microtubule surface-a conformation consistent with earlier experimental proposals but previously lacking high-resolution validation. Importantly, simulations of kinesin's initial stepping indicate that interactions between the neck region and the microtubule surface decisively bias the stepping trajectory, directing the rear kinesin head to overtake the front head from the right side (counterclockwise stepping). These findings establish a direct structural mechanism by which neck region-microtubule interactions govern the directional bias of kinesin's initial stepping, providing fundamental new insight into the molecular basis of its motility.