Image Reconstructions of Microtubules Decorated with Monomeric and Dimeric Kinesins: Comparison with X-Ray Structure and Implications for Motility

Abstract

We have decorated microtubules with monomeric and dimeric kinesin constructs, studied their structure by cryoelectron microscopy and three-dimensional image reconstruction, and compared the results with the x-ray crystal structure of monomeric and dimeric kinesin. A monomeric kinesin construct (rK354, containing only a short neck helix insufficient for coiled-coil formation) decorates microtubules with a stoichiometry of one kinesin head per tubulin subunit (α–β-heterodimer). The orientation of the kinesin head (an anterograde motor) on the microtubule surface is similar to that of ncd (a retrograde motor). A longer kinesin construct (rK379) forms a dimer because of the longer neck helix forming a coiled-coil. Unexpectedly, this construct also decorates the microtubule with a stoichiometry of one head per tubulin subunit, and the orientation is similar to that of the monomeric construct. This means that the interaction with microtubules causes the two heads of a kinesin dimer to separate sufficiently so that they can bind to two different tubulin subunits. This result is in contrast to recent models and can be explained by assuming that the tubulin–kinesin interaction is antagonistic to the coiled-coil interaction within a kinesin dimer.

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

“…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. The latter group have suggested this was because each head of a dimer was bound to separate sites on tubulin.…”

“…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).…”

“…On the other hand, attempts to relate the crystal structures of dimeric kinesin (Kozielski et al, 1997;Hoenger et al, 1998) to complexes seen by electron microscopy have been unsatisfactory. 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.…”

“…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.…”

“…A similar docking with "head A" as the bound head was not possible, because the second head would then penetrate into the microtubule density. With head B placed in the density of the bound head, the second head should extend from the top right of the bound head, as viewed from the outside of a microtubule with its plus end oriented upward (which we refer to as the "standard" view); however, the three-dimensional (3-D) map of Hoenger et al (1998) showed very little density here. In contrast, a map of dimeric kinesin complexed with ADP produced by Arnal and Wade (1998) did include extra density.…”