SUMMARY Kinesin-1 is a two-headed motor that takes processive 8-nm hand-over-hand steps and transports intracellular cargos towards the plus end of microtubules. Processive motility requires a gating mechanism to coordinate the mechanochemical cycles of the two heads. Kinesin gating involves the neck linker (NL), a short peptide that interconnects the heads, but it remains unclear whether gating is facilitated by the NL orientation or tension. Using optical trapping, we measured the force-dependent microtubule release rate of kinesin monomers under different nucleotide conditions and pulling geometries. We find that pulling NL in the backward direction inhibits nucleotide binding and subsequent release from the microtubule. This inhibition is independent from the magnitude of tension (2–8 pN) exerted on NL. Our results provide evidence that the front head of a kinesin dimer is gated by the backward orientation of its NL until the rear head releases from the microtubule.

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steps. Structural dynamics of dynein's AAA þ ring domain during processive stepping are not well understood. We use a combination of polarized TIRF and sub-pixel particle tracking to measure the position and orientation of fluorescent nanorods rigidly attached to AAA5 and AAA6 of the individual dynein ring domains via biotin-NeutrAvidin linkage. We observe rotational changes of the ring and how they correlate with translocation steps. The dynein ring undergoes frequent, small rotations, typically less than 20 degrees, about twice as often as steps. Stepping and tilting both depend on ATP, although some ATP-independent rotations are observed. Rotations which accompany translocation steps have larger magnitudes than ones that are not correlated with steps. Not all steps are correlated with angle changes, either. A heterodimeric mutant construct, with one biotinylated ring carrying a quantum rod and one nonbiotinylated dead-head ring walks slowly, indicating that the rod does not destroy motility. Our results are inconsistent with a purely powerstroke stepping mechanism, analogous to that of myosin which would predict larger angle changes tightly coupled to stepping, but instead they support a winch-like mechanism that involves bending of the coiled-coil stalk under intermolecular torque between the heads in the double-headed bound state. Supported by NIH Grant P015GM087253.