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 conformation of the neck region in dimeric human kinesin-1 bound to a realistic microtubule lattice, and 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 axis and positioned near its surface—a conformation consistent with earlier proposals but lacking high-resolution validation. Importantly, simulations indicate that neck–microtubule interactions bias the stepping trajectory, directing the rear kinesin head to overtake the front head from the right (counterclockwise stepping). These findings establish a mechanism by which neck–microtubule interactions govern directional bias in kinesin’s initial step, offering new insight into the molecular basis of its motility.