Kinetic regulation of kinesin’s two motor domains coordinates its stepping along microtubules

The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and the motor can take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for the head-head coordination; however, which kinetic step(s) in the chemomechanical cycle is “gated” by the neck linker remains unresolved. Here, we employed pre-steady state kinetics and single molecule assays to investigate how the neck linker conformation affects kinesin’s motility cycle. We show that the neck linker conformation in the front kinesin head confers higher affinity for microtubules, but does not change ATP affinity. In contrast, the neck linker conformation in the rear kinesin head increases ATP affinity by several thousand-fold compared to the front head but has little effect on microtubule affinity. These conformation-specific effects in combination favor ATP hydrolysis and dissociation of rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.