Kinesin processivity is gated by phosphate release

Abstract

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....

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

“…S1), strongly suggesting that under physiological conditions, hydrolysis occurs before tethered head attachment. Milic and coworkers recently showed that processivity under assisting load is not increased in ATPγS but is increased by adding phosphate to the media, in support of ATP hydrolysis occurring before tethered head attachment (19,41). We extend that result by showing that in a zero-load assay the run length in ATPγS is not higher than the run length in ATP (Fig.…”

“…If hydrolysis can occur in either a 1HB or 2HB state, then run length should increase in ATPγS because the tethered head would have a much greater chance to bind before the bound head hydrolyzes its nucleotide and enters a vulnerable 1HB posthydrolysis state (19). The opposite was observed-in single-molecule Qdot-tracking experiments, run lengths were slightly shorter in saturating ATPγS than in saturating ATP (Fig.…”

“…After defining that the transition from the 2HB to the 1HB state principally occurs after ATP binding, we set out to determine whether completion of the 16.4-nm step, which is marked by the return to the 2HB state, occurs before or after ATP hydrolysis. Single-molecule tracking assays were repeated at 1,000 frames per second using saturating concentrations of ATPγS, a slowly hydrolyzable ATP analog (19,31,33). Motor velocity at 1 mM ATPγS was 13.6 ± 1.0 nm s −1 (SEM; n = 33 runs), roughly equal to the velocity for 1 μM ATP (13.3 ± 0.8 nm s ; n = 36 runs).…”

“…Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head-bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads-bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4,11,(14)(15)(16)(17)(18)(19)(20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks.…”

“…This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7,19,21,22) and single-molecule tracking studies (4,(8)(9)(10)(11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11,12,14,15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.…”