Kinesin's front head is gated by the backward orientation of its neck linker

Abstract

Kinesin-1 is a two-headed motor that takes processive 8-nm hand-over-hand steps and transports intracellular cargos toward the plus-end of microtubules. Processive motility requires a gating mechanism to coordinate the mechanochemical cycles of the two heads. Kinesin gating involves 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 of 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.

Figure 1. Force-dependent release of kinesin from MTs

(A) (Top) Schematic of a kinesin dimer in a 2HB state. The NL (yellow) of the front head is oriented backwards and that of the rear head is oriented forward. (Bottom) Orientation of the NLs or tension between them (black arrows) may prevent ATP binding to the front head, or accelerate the nucleotide hydrolysis and subsequent MT release of the rear head to facilitate coordinated movement. (B) NL orientation of the front and rear heads can be mimicked by pulling a kinesin monomer from its NL via a short DNA tether using an optical trap (not to scale). (C) A trapped bead is oscillated between two positions 250 nm apart along the MT long axis. (1) When a monomer binds to the MT, (2) the movement of the bead to the next trap position is restricted. In this state, the trap exerts a constant force as a function of bead-trap separation (Δx) on the motor until it releases from the MT (Δt). (3) When the monomer releases from the MT (red arrowhead), the bead resumes following the trap. (D) Cumulative probability distributions (solid circles) represent the dwell time data for kinesin monomers pulled from the head towards the plus-end in the absence of nucleotide at different force ranges. N = 200 for each histogram. The release rates (k₁ and k₂) at a given force range were calculated by a two-exponential-decay fit (solid curves). (E) Model of kinesin-MT interaction shows two distinct binding modes in the apo state. k₁ and k₂ represent force-induced release rates from the weak and strong states, respectively. See also Figures S1 and S2.

Figure 2. MT release rates of head- and NL-pulled kinesins under different nucleotide conditions

(A) Without nucleotide, monomers release in response to external force (left). k₁ (middle) and k₂ (right) of kinesin monomers pulled from the head and NL increase with force in both forward (positive forces) and backward directions. (B) With 1 mM ATP in solution, kinesin can release by force or hydrolysis of the bound nucleotide (left). MT release rates of the headand NL-pulled kinesins increase compared to the apo condition. (C) At 1 mM ADP, kinesin can release from MTs by force or ADP binding (left). NL-pulled kinesins show slower release under backward forces. Error bars represent 95% confidence intervals. See also Figures S3 and S4.

Figure 3. Nucleotide binding to a kinesin head is inhibited when the NL is oriented backward

(A) k₁ values of the apo condition were subtracted from that of 1 mM ADP to calculate the nucleotide-binding induced MT release rate from the weakly-bound state. k_1-ADP − k_1-apo of NL-pulled kinesins was 35 s⁻¹ at −1.5 pN, and decreased to ~0 s⁻¹ at higher negative forces. (B) k₂ values of the apo condition were subtracted from that of 1 mM ADP to calculate the nucleotide-binding induced MT release rate from the strongly-bound state. k_2-ADP − k_2-apo of NL-pulled kinesins remained nearly constant at 3.0 s⁻¹ under negative forces, whereas k_2-ADP − k_2-apo of head-pulled kinesins increased from 3.4 s⁻¹ at −1.8 pN to 14.2 s⁻¹ at −6.7 pN.

Figure 4. Front-head gating model for kinesin

(1) In the ATP waiting state, the rear head is ADP-bound and weakly interacting with MT. (2) ATP binding to the front head triggers NL docking that pulls the rear head forward. (3) The unbound head releases ADP and rebinds MT ahead of its partner head. (3–4) In 2HB state, the NL of the front head (red) is oriented backward and ATP binding to this head is inhibited until the rear head hydrolyzes ATP and releases from MT.