A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd

Abstract

Kinesins are microtubule-based motor proteins that power intracellular transport. Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that govern this directional preference have been described. By contrast, the nature and timing of the structural changes underlying the minus-end-directed motility of Kinesin-14 motors (such as Drosophila Ncd) are less well understood. Using cryo-electron microscopy, here we demonstrate that a coiled-coil mechanical element of microtubule-bound Ncd rotates approximately 70 degrees towards the minus end upon ATP binding. Extending or shortening this coiled coil increases or decreases velocity, respectively, without affecting ATPase activity. An unusual Ncd mutant that lacks directional preference shows unstable nucleotide-dependent conformations of its coiled coil, underscoring the role of this mechanical element in motility. These results show that the force-producing conformational change in Ncd occurs on ATP binding, as in other kinesins, but involves the swing of a lever-arm mechanical element similar to that described for myosins.

Figure 1. 3D maps of Ncd–microtubule complexes by cryo-EM

a– c,Wild-type Ncd in the absence of nucleotide (a) and in the presence of the nucleotide analogues Mg²⁺-AMPPNP (5 mM, b) and Mg²⁺-$\text{ADP}-{\text{AIF}}_4^{-}$ (5 mM, c). Putative density for the bound head, unbound head and neck region are marked as H_B, H_U and N, respectively. Arrows indicate the rotation of the unbound head and neck between the nucleotide-free and ATP-like states. d–f, Ncd crystal structures docked into wild-type maps. Shown is the Ncd-ADP structure docked into the nucleotide-free maps (d). The NcdN600K structure (e) and the NcdN600K structure with a 10° rotation of the unbound head and neck about Gly 347 (the residue marking the boundary between the neck and the catalytic core) (f), are shown docked to the Mg²⁺-$\text{ADP}-{\text{AIF}}_4^{-}$ maps. g, h, Cryo-EM maps for the bidirectional mutant NcdN340K, without nucleotide (g) and with 5mM Mg²⁺-$\text{ADP}-{\text{AIF}}_4^{-}$ (h). 5mM Mg²⁺-AMPPNP produces an equivalent map to 5mM Mg²⁺-$\text{ADP}-{\text{AIF}}_4^{-}$ (Supplementary Fig. 3). i, Density map generated by averaging the Ncd wild-type nucleotide-free and Mg²⁺-$\text{ADP}-{\text{AIF}}_4^{-}$ data. Detached density in NcdN340K nucleotide-free map (g) and in Ncd average map (i) circled in red. All figures are oriented so that the plus end of the microtubule axis is at the top of the page. Figs were generated with Pymol (Delano Scientific).

Figure 2. Ncd mutants with truncated or extended necks

a, Gliding assay velocities and ATPase k _cat (in ATP molecules per head per s). Velocity data are the mean ± s.d. (n > 150); ATPase k_cat data are the weighted average and errors obtained from the fits of two independent ATPase experiments from at least two protein preparations. Numbers in the construct name represent the starting residue for the native Ncd residues in the construct. N-terminal LZ extensions (29 residues) and the flexible Gly-Ser linker (12 residues) are labelled LZ and GS, respectively (see Methods). The domain structures of the constructs are shown on the left with motor cores in black, native neck residues in red, LZ extensions in dark blue, the flexible linker in light blue, and the biotin tag in orange. b, c, Velocities of gliding movement (mean ± s.d; b) and ATP k _cat (weighted average and errors; c) plotted against predicted neck length of each construct. Data points representing constructs containing only native neck residues are red, and data from constructs with LZ extensions are blue.

Figure 3. Model of the Ncd motility cycle

The model is based on surface representations of the docked Ncd structures (Fig. 1d, f). The microtubule is oriented so that the plus end is on the right. In this model, ATP binding causes a rotation of the neck (coloured red in the nucleotide-free and yellow in the ATP state) that leads to a minus-end displacement along the microtubule (indicated by the red arrow). After this lever-arm rotation, the motor releases from the microtubule after nucleotide hydrolysis but probably before phosphate release,. The released motor then returns to its pre-powerstroke position so that the cycle can repeat. Images rendered from atomic structures by Graham Johnson (fiVth media: 〈http://www.FiVth.com〉).