A Model for the Microtubule-Ncd Motor Protein Complex Obtained by Cryo-Electron Microscopy and Image Analysis

Abstract

Kinesin motors convert chemical energy from ATP hydrolysis into unidirectional movement. To understand how kinesin motors bind to and move along microtubules, we fit the atomic structure of the motor domain of Ncd (a kinesin motor involved in meiosis and mitosis) into three-dimensional density maps of Ncd-microtubule complexes calculated by cryo-electron microscopy and image analysis. The model reveals that Ncd shares an extensive interaction surface with the microtubule, and that a portion of the binding site involves loops that contain conserved residues. In the Ncd dimer, the microtubule-bound motor domain makes intimate contact with its partner head, which is dissociated from the microtubule. This head-head interaction may be important in positioning the dissociated head to take a step to the next binding site on the microtubule protofilament.

Congruent Docking of Dimeric Kinesin and ncd into Three-dimensional Electron Cryomicroscopy Maps of Microtubule–Motor ADP Complexes

“…Similar predictions were reported by Sosa et al (1997), in orienting the monomeric ncd⅐ADP crystal structure into a map of ncd complexed to microtubules in the presence of AMP⅐PNP, and by Hoenger et al (1998), in docking the kinesin⅐ADP crystal structure into a map showing kinesin in the same state. The orientation of the directly bound heads appeared to be in agreement with most of the data obtained by biochemical and mutagenesis studies (Yang et al, 1989;Woehlke et al, 1997;Alonso et al, 1998).…”

“…The structures of microtubules fully decorated with monomeric kinesin or ncd constructs, as studied by different groups (Hirose et al, 1995(Hirose et al, , 1996Hoenger et al, 1995;Hoenger and Milligan, 1997;Kikkawa et al, 1995;Sosa et al, 1997), are mostly in good agreement and show a stoichiometry of one kinesin head per tubulin dimer. At the resolution of these studies (2-3.5 nm), there is no large difference in the structures of the kinesin-tubulin and ncd-tubulin complexes.…”

“…The position of the tethered heads of kinesin and ncd showed a clear difference; the unattached head of kinesin was closer to the plus end, whereas the unattached head of ncd was pointing toward the minus end of the microtubule. Although subsequent studies gave equivalent results for dimeric ncd (Arnal et al, 1996;Sosa et al, 1997), there has been disagreement over dimeric kinesin. The results of Arnal et al (1996) were similar to ours, but the structures obtained by Hoenger et al (1998) showed no additional density compared with the monomeric structure.…”

“…When one of the heads ("head B" in the crystal structure) of dimeric kinesin is docked in the orientation chosen by Sosa et al (1997) and Hoenger et al (1998), the coiled coil that dimerizes the two heads points into the microtubule surface. A similar docking with "head A" as the bound head was not possible, because the second head would then penetrate into the microtubule density.…”

“…Now, however, we show that 3-D images of both dimeric kinesin (newly presented here) and dimeric ncd (Hirose et al, 1998), complexed with ADP and attached to microtubules by one of their two heads, can agree with all the available crystal structures. The best fitting for the directly bound heads in these maps is rotated by ϳ60°, compared with the orientation with which Sosa et al (1997) and Hoenger et al (1998) fitted the heads into maps of unsuitable nucleotide states, and by ϳ90°in the other direction, compared with the orientation selected by Kozielski et al (1998) to fit into their map showing dimeric kinesin with ADP. Our orientation puts the predicted tubulin-binding loops L8, L11, and L12 toward the microtubule surface and also avoids a clash between the microtubule and the coiled coil of dimeric kinesin.…”