Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus-end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. Additionally, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein’s stepping motion at high interhead separations. We developed a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.

Cytoplasmic dynein is an AAA+ motor responsible for intracellular cargo transport and force generation along microtubules (MTs). Unlike kinesin and myosin, dynein contains multiple ATPase subunits, with AAA1 serving as the primary catalytic site. ATPase activity at AAA3 is also essential for robust motility, but its role in dynein’s mechanochemical cycle remains unclear. Here, we introduced transient pauses in Saccharomyces cerevisiae dynein motility by using a slowly hydrolyzing ATP analog. Analysis of pausing behavior revealed that AAA3 hydrolyzes nucleotide an order of magnitude slower than AAA1 and the two sites do not coordinate. ATPase mutations to AAA3 abolish the ability of dynein to modulate MT release. Nucleotide hydrolysis at AAA3 lifts this “MT gate” to fast motility. These results suggest that AAA3 acts as a switch that repurposes cytoplasmic dynein for fast cargo transport and MT anchoring tasks in cells.

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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