Kinesin moves by an asymmetric hand-over-hand mechanism

Abstract

Kinesin is a double-headed motor protein that moves along microtubules in 8-nanometer steps. Two broad classes of model have been invoked to explain kinesin movement: hand-over-hand and inchworm. In hand-over-hand models, the heads exchange leading and trailing roles with every step, whereas no such exchange is postulated for inchworm models, where one head always leads. By measuring the stepwise motion of individual enzymes, we find that some kinesin molecules exhibit a marked alternation in the dwell times between sequential steps, causing these motors to "limp" along the microtubule. Limping implies that kinesin molecules strictly alternate between two different conformations as they step, indicative of an asymmetric, hand-over-hand mechanism.

Fig. 1

Representative high-resolution stepping records of position against time, showing the single-molecule behavior of kinesin motors under constant 4 pN rearward loads. (A) Limping motion of the recombinant kinesin construct, DmK401. The dwell intervals between successive 8-nm steps alternate between slow and fast phases, causing steps to appear in pairs, as indicated by the ligatures. (B) Nonlimping motion of native squid kinesin, LpK. No alternation of steps is apparent; vertical lines mark the stepping transitions. Slow and fast phase assignments, as described in the text, are indicated in color on the uppermost trace of each panel (blue and red, respectively), and the corresponding dwell intervals are numbered. All traces were median filtered with a 2.5-ms window.

Fig. 2

Stepping statistics for DmK401, LpK, and a computer simulation. (A) Experimental dwell time distributions for the slow (blue) and fast (red) phases of DmK401 motion, with statistical errors as indicated. Solid lines show single-exponential fits to the data, with time constants τ_slow = 136 ± 6 ms and τ_fast = 24 ± 1 ms. The first bin and bins with < 7 counts were not included in fits (these points are displayed without error bars); 2,948 intervals from 278 individual records are represented. (B) Experimental distribution of the limp factor, L, for individual records of DmK401 movement (black). (Inset) Histograms of the mean durations of the slow (blue) and fast (red) phases for individual records. (C) Same as in (A), but for LpK. The time constants are τ_slow = 90 ± 4 ms and τ_fast = 54 ± 2 ms. 2,523 dwells from 208 records are represented. (D) Same as in (B), but for LpK. Outliers with L > 6 (arrows) are caused by rare, limping LpK molecules. (E) Same as in (A), but for a computer simulation of an ideal Poisson stepper with a single characteristic stepping time (τ = 71 ms) that produced the time constants τ_slow = 87 ± 3 ms and τ_fast = 56 ± 2 ms. 2,230 dwells from 220 records are represented. (F) Same as in (B), but for the simulation.

Fig. 3

The effect of construct length on kinesin limping. (A) Cartoons showing the named structural features of kinesin dimers used in this work, with stalk lengths displayed approximately to scale and numbers of amino acids shown. (B) The mean limp factors, $\overline{L}$, for DmKHC constructs (black circles), full-length native LpK (open circle), and for a nonlimping Poisson stepper under conditions similar to our experiments (dashed lines, showing 63% confidence intervals), as a function of the construct length (number of amino acids). Points show the means and estimated errors [evaluated by bootstrapping ()] for each distribution of not less than 141 limp factors. (C) The average durations of the slow (blue circles) and fast stepping phases (red triangles) as functions of the construct length for DmKHC derivatives (solid symbols) and native LpK (open symbols).

Fig. 4

Candidate models for limping in kinesin. (A) The misregistration model. Left (in register): The two-heads-bound state for native, non-limping kinesin, shown as it moves to the right along the microtubule lattice (tubulin α–β subunits, spaced pairwise by 8 nm, are indicated in gray and yellow). The coils of both heavy chains (blue and red ribbons) are in correct register. Middle and right (misregistered): A shift in registration of the coiled-coil by a single heptad changes the relative lengths of the head-neck linker regions by up to 1 nm. On alternate steps, this shift places one head (dark gray) farther from its binding site on the microtubule, reducing the stepping rate when this head attempts to take the lead (middle). When the partner head (light gray) steps, the shift is accommodated by slack in the longer tether (right). (B) The winding model. When one head (dark gray) steps forward, the neck coiled-coil (red and blue) is overwound relative to the relaxed state. When the partner head (light gray) leads, the coiled-coil is underwound. Asymmetry in torsional compliance slows the stepping associated with overwinding.