Kinesin-1 is a dimeric motor that transports cargo along microtubules, taking 8.2-nm steps in a hand-over-hand fashion. The ATP hydrolysis cycles of its two heads are maintained out of phase by a series of gating mechanisms, which lead to processive runs averaging ∼1 μm. A key structural element for inter-head coordination is the neck linker (NL), which connects the heads to the stalk. To examine the role of the NL in regulating stepping, we investigated NL mutants of various lengths using single-molecule optical trapping and bulk fluorescence approaches in the context of a general framework for gating. Our results show that, although inter-head tension enhances motor velocity, it is crucial neither for inter-head coordination nor for rapid rear-head release. Furthermore, cysteine-light mutants do not produce wild-type motility under load. We conclude that kinesin-1 is primarily front-head gated, and that NL length is tuned to enhance unidirectional processivity and velocity.DOI: http://dx.doi.org/10.7554/eLife.07403.001

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or "gate," the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P i ) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P i state (by adding free P i ) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.single molecule | mechanochemistry | optical tweezers | molecular motors S ince its discovery nearly 30 years ago (1), kinesin-1-the founding member of the kinesin protein superfamily-has emerged as an important model system for studying biological motors (2, 3). During "hand-over-hand" stepping, kinesin dimers alternate between a two-heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one-head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the . A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2,3,(10)(11)(12)(13)(14)(15)(16)(17). This transition, called "NL docking," is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2,3,(10)(11)(12)(13)(14)(15)(16)(17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2,3,(10)(11)(12)(13)(14)(15)(16)(17). Prevailing models of the kinesin mechanochemical cycle (2,3,10,14,15,17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding....

Homodimeric KIF17 and heterotrimeric KIF3AB are processive, kinesin-2 family motors that act jointly to carry out anterograde intraflagellar transport (IFT), ferrying cargo along microtubules (MTs) toward the tips of cilia. How IFT trains attain speeds that exceed the unloaded rate of the slower, KIF3AB motor remains unknown. By characterizing the motility properties of kinesin-2 motors as a function of load we find that the increase in KIF3AB velocity, elicited by forward loads from KIF17 motors, cannot alone account for the speed of IFT trains in vivo. Instead, higher IFT velocities arise from an increased likelihood that KIF3AB motors dissociate from the MT, resulting in transport by KIF17 motors alone, unencumbered by opposition from KIF3AB. The rate of transport is therefore set by an equilibrium between a faster state, where only KIF17 motors move the train, and a slower state, where at least one KIF3AB motor on the train remains active in transport. The more frequently the faster state is accessed, the higher the overall velocity of the IFT train. We conclude that IFT velocity is governed by () the absolute numbers of each motor type on a given train, () how prone KIF3AB is to dissociation from MTs relative to KIF17, and () how prone both motors are to dissociation relative to binding MTs.

Eg5, a mitotic kinesin, has been a target for anticancer drug development. Clinical trials of small-molecule inhibitors of Eg5 have been stymied by the development of resistance, attributable to mitotic rescue by a different endogenous kinesin, KIF15. Compared with Eg5, relatively little is known about the properties of the KIF15 motor. Here, we employed single-molecule optical-trapping techniques to define the KIF15 mechanochemical cycle. We also studied the inhibitory effects of KIF15-IN-1, an uncharacterized, commercially available, small-molecule inhibitor, on KIF15 motility. To explore the complementary behaviors of KIF15 and Eg5, we also scored the effects of small-molecule inhibitors on admixtures of both motors, using both a microtubule (MT)-gliding assay and an assay for cancer cell viability. We found that () KIF15 motility differs significantly from Eg5; () KIF15-IN-1 is a potent inhibitor of KIF15 motility; () MT gliding powered by KIF15 and Eg5 only ceases when both motors are inhibited; and () pairing KIF15-IN-1 with Eg5 inhibitors synergistically reduces cancer cell growth. Taken together, our results lend support to the notion that a combination drug therapy employing both inhibitors may be a viable strategy for overcoming chemotherapeutic resistance.

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