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. 2011 Nov;9(11):e1001207.
doi: 10.1371/journal.pbio.1001207. Epub 2011 Nov 29.

Electrostatically Biased Binding of Kinesin to Microtubules

Free PMC article

Electrostatically Biased Binding of Kinesin to Microtubules

Barry J Grant et al. PLoS Biol. .
Free PMC article


The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Biased binding and unbiased binding frameworks for the kinesin minimal motor domain mechanism.
(Left) In biased binding models, the motor domain diffuses on a tether and diffusion-to-capture is directionally biased. (Right) In models with unbiased binding, diffusion-to-capture occurs with equal probability in both directions and progress is due to a subsequent conformational change. Conformational changes that follow binding in the progress direction contribute useful force, conformational changes that follow binding in the antiprogress direction do not.
Figure 2
Figure 2. Electrostatic analysis.
(A) Surface mapped electrostatic potentials for kinesin family representatives (see Movie S1 for additional mappings). Values are expressed as a color spectrum ranging from +5 kT/e (blue) to −5 kT/e (red). Note, despite the overall diversity in charge distribution, the consistent positive patch (blue) on the rear face of the motor domain (see also Movie S1). (B) Consensus electrostatic potential map of the kinesin family illustrating regions where 80% of structures have a potential of the same sign (see Movie S2 for additional consensus levels). (C) Electrostatic clustering of available kinesin structures. Structures are labeled with their PDB code and colored by sub-family.
Figure 3
Figure 3. Kinesin-tubulin BD simulations.
(A) Subfamily association rates from BD simulations. Two structures from each sub-family were simulated (PDB codes: 1bg2, 1goj, 1i6i, 1vfz, 1ii6, 2gm1, 1v8j, and 1v8k). Error bars represent 95% confidence intervals for the rate determination calculation. Note basal rates (dark bar) were determined in the absence of electrostatic forces for one subfamily representative only. (B) Occupancy maps highlight preferred association sites during BD simulations. Color coded sampling density (occupancy maps) of kinesin-3 about a tubulin heterodimer. Note the single preferred binding site and an apparent preferred path of approach to the bound configuration. (C) Kinesin-tubulin association center-of-mass distance versus relative torsion angle between kinesin and tubulin during successful approach trajectories. The insert plots the standard deviation of the relative torsion angle between kinesin and tubulin at a given separation distance during 200,000 trajectories.
Figure 4
Figure 4. Kinesin-microtubule BD simulations.
(A) Simulations utilized a microtubule model consisting of 7 protofilaments each with 5 tubulin heterodimer subunits. For kinesin dimer simulations, a flexible tether was placed between a freely diffusing head and a second immobile microtubule bound head (see methods). (B) Kinesin-1 monomer binding events. Each of the 35 potential binding sites is labeled and colored by the proportion of binding events at a given site. (C) Kinesin-1 un-tethered dimer binding events. Each simulation is commenced with the freely diffusing kinesin head within the tether distance of its immobile partner head. However, no spring constraint is applied. (D) Binding events for tethered kinesin-1 dimers. (E) Binding events for tethered kinesin-14 dimers and (F) uncharged kinesin-14 dimers.
Figure 5
Figure 5. Effects of charge neutralizing alanine mutations mapped to the kinesin-1 structure.
(A) Positions whose mutation to alanine decrease (negative: yellow and orange) and increase (positive: light blue and dark blue) calculated ΔΔGelec values. (B) The results of experimental mutagenesis on KmMT for microtubule-activated ATPase activity (sites in yellow increase, whilst those in blue decrease KmMT); see Woehlke et al. for details.
Figure 6
Figure 6. Experimental mutagenesis results.
(A) Motility assay. Sliding velocity for R326A is not significantly different from wild type. By contrast, mutant R326K shows ∼5-fold increase in microtubule sliding velocity over wild type. (B) ATPase activation curves for tubulin and for microtubules of two key mutants AAK (R326A) and KAK (R326K) in Nkin343 monomeric kinesin-1. Mutant R326A shows a ∼2.5-fold increase in Vmax for the microtubule-activated ATPase, with a ∼4-fold higher Km. Mutant R326K shows a modest decrease in Vmax for microtubule-activation, with a 3-fold higher Km.
Figure 7
Figure 7. Kinetic scheme.
In this 3-state scheme , mutagenesis that increases ΔΔGelec will over-populate the weakly bound state (state 2) by enhancing recruitment from the free motor population (increasing k+1 and decreasing k−1) and from the strongly bound state (state 3) (by increasing k−2 and decreasing k+2). Increasing the population of state 2 relative to state 3 will decrease internal drag in the motility assay, thereby increasing microtubule sliding velocity.

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