Microscopic evidence for a minus-end-directed power stroke in the kinesin motor ncd

Abstract

We used cryo-electron microscopy and image reconstruction to investigate the structure and microtubule-binding configurations of dimeric non-claret disjunctional (ncd) motor domains under various nucleotide conditions, and applied molecular docking using ncd's dimeric X-ray structure to generate a mechanistic model for force transduction. To visualize the alpha-helical coiled-coil neck better, we engineered an SH3 domain to the N-terminal end of our ncd construct (296-700). Ncd exhibits strikingly different nucleotide-dependent three-dimensional conformations and microtubule-binding patterns from those of conventional kinesin. In the absence of nucleotide, the neck adapts a configuration close to that found in the X-ray structure with stable interactions between the neck and motor core domain. Minus-end-directed movement is based mainly on two key events: (i) the stable neck-core interactions in ncd generate a binding geometry between motor and microtubule which places the motor ahead of its cargo in the minus-end direction; and (ii) after the uptake of ATP, the two heads rearrange their position relative to each other in a way that promotes a swing of the neck in the minus-end direction.

Fig. 1. A cryo-EM image of MTs decorated with dimeric ncd at subsaturating ratios reveals a cooperative binding mechanism in the axial direction, along individual protofilaments. While one of the MTs is mostly undecorated (I), partial decoration (III) and complete decoration (II) co-exist in the same area. A Fourier-filtered image of the MT and the motors in a side projection allows determination of the polarity of the MT according to the shape of the dimeric motor construct.

Fig. 2. Helical three-dimensional reconstructions of MTs decorated with dimeric ncd head domains under various nucleotide conditions. Left columns show the projected three-dimensional reconstructions of each condition, with the corresponding diffraction patterns in the second column from the left. Surface-rendered three-dimensional constructions are shown in the two columns on the right. (A) No significant decoration is achieved in the presence of excess ADP. (B) Dimeric ncd in the presence of AMP-PNP binds to tubulin with one of its heads (head 1) while the second head (head 2) locates on top of the bound one. (C) Absence of nucleotides creates a state similar to that in (B), but with head 2 rotated by ∼90°. (D) Only in the absence of nucleotide was it possible to detect a significant density coming from an SH3 domain (green volumes), which we engineered at the N-terminus of our construct to increase its visibility. This density was absent in reconstructions from tubes decorated with ncd in the presence of AMP-PNP, indicating an increased flexibility in the neck region.

Fig. 3. Molecular docking and modeling, fitting the atomic resolution X-ray structure from Sablin et al. (1998) into our EM-derived three-dimensional maps of MT–ncd complexes. The panels on the left show side views of a protofilament with the plus end to the left. The right panels show end-on views of protofilaments viewed from the plus end. (A) The conformation of dimeric ncd in the absence of nucleotides strongly resembles the ADP state found in the crystal structure. The neck was visualized by two SH3 domains engineered to the N-terminal ends of the neck helices. (B) The neck of the untagged construct was not visible in the presence of AMP-PNP due to an increased flexibility in that region. Nevertheless, our docking experiments revealed a large conformational change, rearranging heads 1 and 2 dramatically. Head 2 rotates around a radial axis located at the C-terminal end of the neck (the transition into the core). Thereby, the two heads approach each other and close the gap towards the plus end, thus moving the neck into the minus-end direction.

Fig. 4. The X-ray structure of dimeric ncd motor domains revealed a number of interactions between the motor cores (α1, loops 6, 10 and 13) and their corresponding neck helices (α0). (A) In our model, based on the docking shown in Figure 3, these interactions remain conserved for both heads in ncd dimers bound to MTs in the absence of nucleotide. (B) However, in the presence of AMP-PNP, these contacts may be only conserved for head 2, but not for head 1. Potential contacts now appear between loops 6 and 10 in head 1, and loop 2, β1a/b and the beginning of α1 in head 2. This indicates that ATP uptake triggers a conformational change within the motor core domain, which directly influences the neck–core interaction, and which may therefore constitute the underlying mechanism for ncd function.

Fig. 5. Working model for the generation of a minus-end-directed power stroke by ncd based on our cryo-EM data combined with the X-ray structure of Sablin et al. (1998). (A) In solution, the ncd dimer has ADP bound and displays low affinity for tubulin. The neck–core interactions (see Figure 4) are intact and form a stable complex. To bind successfully to MTs, the head domain has to be directed into a position ahead of the cargo in the minus-end direction. To achieve a binding position, the yellow arrow (tubulin surface) and red arrow (motor domain) need to assume a parallel position. (B) Once in contact with the MT surface, head 1 (yellow) releases ADP, thereby increasing its binding affinity. Head 2 (red) maintains an ADP-bound state and does not make contact with the MT surface. (C) Head 1 binds ATP, which triggers a 90° rotation of head 2 including the entire coiled-coil neck around a radial axis going through the C-terminal end of the neck. Thereby, the neck–core interactions between head 1 and its helix α0 are released. (D) Once ATP is hydrolyzed, the ncd dimer could be released from the MT surface, and the dimer reforms the stable complex between neck and both cores.