Disulfide-crosslinking to constraint the neck linker in either the forward or backward pointing configuration.

(A) Three-dimensional structure of kinesin dimer on the microtubule in the two-head-bound intermediate state (modeled based on PDB# 4HNA and 4LNU). The motor domain, neck linker, and neck coiled-coil are shown in cyan, red, and green. Orange spheres represent the positions of cysteine residues as described in panel C. (B) Michaelis-Menten kinetics scheme for ATP-promoted dissociation of kinesin head from the microtubule, which includes ATP-binding (k+1), ATP-dissociation (k-1), and ATP-induced microtubule-dissociation (k2) steps. (C) Positions of the cysteine residues for disulfide-crosslinking of monomeric kinesin. Cys222 and Cys334 were introduced to constrain the neck linker in the forward, docked conformation (left), while Cys47 and Cys328 were introduced to constrain the neck linker in the backward-pointing configuration (right). (D) Disulfide crosslinking after oxidative treatment was analyzed by nonreducing SDS-PAGE. WT (without Cys introduction), C222/C334, and C47/C328 constructs were treated with a reducing reagent (DTT), none, or oxidizing reagents (Cu2+). Arrows and arrowheads indicate un-crosslinked and intramolecular crosslinked kinesins, respectively. The whole image of the gels is shown in Figure 1−figure supplement 1. (E) Microtubule-activated ATPase under reduced and oxidized conditions. kcat and Km(MT) were determined from the fit to a Michaelis-Menten equation of ATP turnover rates at various microtubule concentrations (Figure 1−figure supplement 2). Mean ± s.e.m. values were obtained from three independent assays.

Figure supplement 1. Estimation of disulfide-crosslinking efficiency.

Figure supplement 2. Steady-state microtubule-activated ATPase rates of monomeric kinesins before and after oxidative crosslinking.

Kinetic rate constants for disulfide-crosslinking monomers. Steady-state ATP-turnover rate (kcat and Km(MT)), pre-steady-state mant-ATP binding (k+1 and k-1) and microtubule dissociation rates (k2 and k2_SMF, measured by light scattering and single-molecule fluorescence) measured under reduced and oxidized conditions.

ATP-binding/dissociation kinetics of disulfide-crosslinked monomers.

(A) The rate of initial enhancement of the fluorescence after rapid mixing of kinesin-microtubule complex with mant-ATP (kobs; Figure 2−figure supplement 1) was plotted as a function of mant-ATP concentration. Solid lines represent a linear fit; the slope provides the on-rate of mant-ATP (k+1) and the off-set provides the off-rate of mant-ATP (k-1). (B) ATP binding rate k+1 and ATP dissociation rate k-1 of monomers before and after crosslinking obtained from the fit shown in pane A. Error bar shows the fit error. (C) Mant-dATP binding rate k+1 to wild-type (WT) and E236A monomers (kobs plots are shown in Figure 2−figure supplement 2). (D) Two typical traces of smFRET between the donor-labeled wild-type monomer and Alexa-ATP (500 nM). Fluorescence intensities of donor (blue) and acceptor (red), and the calculated FRET efficiency (green) recorded at 100 fps are shown. Left trace: the FRET efficiency transiently increases just before both dyes disappear (indicated by the red arrow), suggesting that the nucleotide-bound head detached from the microtubule (Figure 2−figure supplement 4A). Right trace: the FRET efficiency transiently increases, followed by the recovery of donor fluorescent (shown by the blue arrow), indicating the reversible dissociation of ATP. The black arrow indicates the photobleaching of the donor dye. The numbers of molecules observed for each type is shown at the top of the traces. (E) Typical trace of smFRET between the E236A monomer and Alexa-ATP (200 nM) recorded at 5 fps, demonstrating repetitive ATP binding and dissociation. (F) The dissociation rate k-1 of Alexa-ATP from wild-type and E236A monomers (histograms are shown in Figure 2−figure supplement 4B). The value for the wild-type may be underestimated due to the limited temporal resolution (10 ms).

Figure supplement 1. Pre-steady-state kinetics of ATP binding to monomeric kinesin-microtubule complex before and after crosslinking.

Figure supplement 2. ATP binding kinetics for E236A mutant monomer on microtubule. Figure supplement 3. Processive motility of wild-type kinesin dimer driven by Alexa 647 ATP.

Figure supplement 4. Dwell time of Alexa-ATP-bound state for wild-type and E236A monomers determined using smFRET.

Microtubule-detachment kinetics of disulfide-crosslinked monomers.

(A) ATP-induced dissociation rate from microtubule k2 measured by turbidity change after the rapid mixing of kinesin-microtubule complex with 1 mM ATP. The turbidity time traces are shown in Figure 3−figure supplement 1. (B) Kymographs showing the binding and dissociation of GFP-fused kinesin under reduced and oxidized conditions on the microtubule. C47/C328 showed an extended dwell on the microtubule after crosslinking. (C) ATP-induced dissociation rate from the microtubule measured using single-molecule observations of GFP-fused kinesin. k2_SMF was determined as an inverse of the mean dwell time of the fluorescent kinesin on the microtubule in the presence of 1 mM ATP. The dwell time histograms are shown in Figure 3−figure supplement 2B. (D) Positions of Cys4 and Cys330 residues for disulfide-crosslinking of a monomer between the N-terminal cover strand (CS; green) and the neck linker (NL; red) (left). Positions of Cys47 and Cys335 residues for disulfide-crosslinking of a monomer to constrain the neck linker in the backward-pointing configuration allowing partial docking of the neck linker (right; Figure 3−figure supplement 3). The ATP-induced dissociation rates from the microtubule of C47/C335 monomer before and after crosslinking, measured by turbidity change (k2) and single-molecule fluorescence (k2_SMF), are shown in Figure 3A, C. Histograms are displayed in Figure 3−figure supplement 4E. (E) Mant-ATP binding rate k+1 and dissociation rate k-1 of C4/C330 monomer before and after crosslinking (data for C222/C334 are included from Figure 2B for comparison). The kobs plots are shown in Figure 3−figure supplement 4B.

Figure supplement 1. ATP-induced dissociation kinetics of crosslinked kinesin from microtubule measured using light scattering.

Figure supplement 2. ATP-dependent microtubule-binding and -dissociation of crosslinked kinesins observed by single-molecule fluorescence microscopy.

Figure supplement 3. Locations of the C47/C328 and C47/C335 residues when the labeled head is in the front or rear of dimeric kinesin.

Figure supplement 4. Kinetics measurements of ATP-binding and microtubule-dissociation of C4/C330 and C47/C335 crosslinked monomeric kinesins.

ATP-binding/dissociation kinetics of the E236A-WT heterodimer.

(A) Diagram showing the positions of H100 residue (highlighted in blue) labeled with Alexa 488 dye. This label quenches the mant-ATP signal of either the rear W236A head (upper; used to measure ATP-binding to the front head) or the front wild-type head (lower; used to measure ATP-binding to the rear head). (B) kobs plots for mant-ATP binding to the front wild-type and rear E236A heads of the E236A-WT heterodimer (typical fluorescent transients are shown in Figure 4−figure supplement 2). Solid lines indicate a linear fit. The fit parameters are k+1 = 5.3 ± 0.5 μM-1s-1 and k-1 = 92 ± 16 s-1 for WT front head and k+1 = 4.4 ± 0.2 μM-1s-1 and k-1 = −3.3 ± 5.1 s-1 for E236A rear head. (C) ATP-binding rate (k+1) for front and rear heads of the E236A-WT heterodimer, as determined by fitting in panel B. Diagram showing the position of Cys43 residue of E236A chain for labeling with donor (Cy3; blue) fluorophore, and the Alexa 647 ATP (red) bound to the rear E236A head (upper) or the front wild-type head (lower) of the E236A-WT heterodimer. High FRET efficiency is expected when the Alexa-ATP binds specifically to the rear E236A head. (E) A representative trace of fluorescence intensities of donor (Cy3; blue) on the E236A rear head and acceptor (Alexa 647; red) fluorophores, with calculated FRET efficiency (green), for the E236A-WT heterodimer at 200 nM Alexa-ATP recorded at 5 fps. The black arrow indicates the dissociation of Alexa-ATP, accompanied by the recovery of donor fluorescent. ATP remained bound to the rear head significantly longer compared to the E236A monomer (Figure 2E). (F) ATP dissociation rate (k-1) for front and rear heads of the E236A-WT heterodimer. Note that these rates were measured using different methods (stopped flow for the WT font head (Figure 4B) and smFRET for the E236A rear head (Figure 4E)) and thus cannot be directly compared.

Figure supplement 1. Single-molecule FRET between two heads of E236A-WT heterodimer. Figure supplement 2. ATP binding kinetics for front and rear heads of E236A-WT heterodimer.

Figure supplement 3. Single-molecule FRET observation between donor dye on one of the head of E236A-WT heterodimer and acceptor-labeled ATP.

Microtubule-detachment kinetics of the front WT head of E236A-WT heterodimer.

(A) A diagram illustrating the kinetic transitions for the detachment of the gold-labeled WT head of the E236A-WT heterodimer. Upon binding to the microtubule, the front head releases ADP, becoming a nucleotide-free state (indicated as ϕ; pre-ATP binding). The detachment kinetics from this state can be described using the same scheme as the monomer (Figure 1B). (B) Typical trace for the centroid positions of the gold probe attached to the WT head of the E236A-WT heterodimer in the presence of 1 mM ATP (light red lines), toward the microtubule long axis (on axis) and perpendicular to the microtubule axis (off axis). Red and blue lines depict the median-filtered trace (with a window size of 51 frames) for the bound and unbound states, respectively. Lower panels display the standard deviation (s.d.) of on- and off-axis positions for each time frame t (calculated as [t – 20, t + 20]). (C) Histogram of the dwell time in the bound state. The solid line shows the fit with an exponential function. The number represents the average dwell time (± s.e.m.) determined from the fit. (D) Typical trace for the centroid positions of the gold probe attached to the WT head of E236A-WT heterodimer in the presence of 10 µM ATP. (E) Mean dwell times in the bound state under various ATP concentrations as determined from the fit of the histograms of the dwell times (Figure 5−figure supplement 3).

Figure supplement 1. Long-term trace of the gold-labeled E236A-WT heterodimer exhibiting very slow stepping motion.

Figure supplement 2. The unbound state of the gold-labeled WT head of the E236A-WT heterodimer.

Figure supplement 3. Distributions of the dwell time in the bound and unbound states of the leading WT head of E236A-WT heterodimer.

Effect of the tension applied to the neck linker on the gating examined using the neck-linker extended E236A-WT heterodimer.

(A) Diagram showing the position of the poly-Gly residues (blue) inserted between the neck linker (red) and the neck coiled-coil (green). The Cys55 residue for gold-labeling is represented by an orange sphere. (B) kcat for the microtubule-activated ATPase of E236A-WT heterodimers without (termed as G0) and with neck-linker extensions (G7 and G12) (Figure 6−figure supplement 1). (C) Typical trace for the centroid positions of the gold probe attached to the WT head of the E236A-WT heterodimer with a 7 poly-Gly insertion in the presence of 1 mM ATP. (D) The inverse of the mean dwell time in the bound state was plotted as a function of ATP concentration, as determined from the fit of the histograms of the dwell times (Figure 6−figure supplement 2, 3). Error bars represent s.e.m. The solid lines show the fit with the Michaelis-Menten equation. The fit parameters are kcat (or k2) = 6.3 ± 0.2 s-1 and Km (ATP) = 43 ± 6 µM for G0, kcat = 7.0 ± 0.5 s-1 and Km (ATP) = 12 ± 3 µM for G7, and kcat = 9.3 ± 0.7 s-1 and Km (ATP) = 6.1 ± 2.1 µM for G12. The Km (ATP) decreases as the insertion length increases.

(D) The kcat value, which represents the ATP-induced detachment rate of the front head k2, obtained from the fit shown in panel d. The error bars represent s.e.m. (F) The ATP dissociation rates k-1 for the E236A rear head of the wild-type (G0), G7 and G12 E236A-WT heterodimer were determined using smFRET. The k-1 was calculated as the inverse of the mean dwell time for the high FRET state (see the typical trace and dwell time histograms for the G7 and G12 heterodimer in Figure 6−figure supplement 5). For comparison, data for the E236A monomer head (referred to as MH) is included from Figure 2F. Figure supplement 1. Microtubule-activated ATPase rates of the E236A-WT heterodimer with and without neck-linker extension.

Figure supplement 2. Distributions of the dwell time in the bound and unbound states of the leading WT head of E236A-WT heterodimer with 7 poly-Gly insertion (G7).

Figure supplement 3. Distributions of the dwell time in the bound and unbound states of the leading WT head of E236A-WT heterodimer with 12 poly-Gly insertion (G12).

Figure supplement 4. Binding to the rear-tubulin binding site observed for the G12 E236-WT heterodimer.

Figure supplement 5. Single molecule FRET between donor-labeled E236A head of E236A-WT heterodimer with extended neck linker and acceptor-conjugated ATP.

Model to explain how the tension exerted by the stretched neck linker in dimeric kinesin coordinates the microtubule detachment of the two heads.

This model is based on the proposal that the transition between ATP-bound open and closed conformations of the head is regulated by the neck linker strain (Figure 7−figure supplement 2). The open and closed conformational states of the head are indicated in blue and red, respectively, with the α6 helix, which connects to the neck linker, highlighted as a rod. The microtubule-detached ADP-bound state is shown in orange. The neck linker is depicted in green, while the α4 helix, which directly interacts with the microtubule, and the neck coiled-coil are shown in yellow. In the two-head-bound state, the front head remains in the open state because the backward strain prevents it from transitioning to the closed state. In this state, ATP can weakly bind to the nucleotide-pocket but often dissociates. Conversely, the rear head is stabilized in the closed state because the transition to the open state is suppressed by the forward strain. ATP is tightly bound to the closed nucleotide-pocket, causing the rear head to hydrolyze ATP and detach from the microtubule before the front head does. In the one-head-bound state, the microtubule-bound head can transition between the open and closed states. However, once the tethered head binds preferentially to the forward tubulin-binding site, the strain built up between the two microtubule-bound heads stabilizes the rear head in the closed conformation and the front head in the open conformation.

Figure supplement 1. Structural difference between the open and closed conformational states of the kinesin head.

Figure supplement 2. Schematic model showing that the open-closed conformational transition (isomerization) is gated by the neck linker strain.