In the almost two decades since the bipolar, homotetrameric structure was proposed for the mitotic motor, kinesin-5 (refs 9
), progress has been made in the structural analysis of its motor domain31
but very little has been learned about how it adopts its unique architecture. Thus, the most significant advance of the current study is in providing an advance in that direction, by suggesting how the BASS domain of kinesin-5 can specify how its four subunits assemble into this bipolar organization, which is thought to underlie the sliding filament mechanism by which kinesin-5 functions during mitosis.
The idea that the BASS domain forms a four-strand bundle consisting of two antiparallel CC, superficially resembling a vimentin tetramer, is supported by sequence analysis, EM, mass spectrometry, hydrodynamics, native PAGE and site-directed spin label EPR spectroscopy. The results are all incorporated into the summary showing the homology model () superimposed on STEM images of single molecules of FL-kinesin-5 (left) and the BASS domain (right), decorated on their N-termini with 2
nm Nanogold (). The BASS domain forms a structure that fits very well into the four-strand antiparallel α-helical CC rod of the FL-kinesin-5 homotetramer, but in the EM, the α-helical C-terminal overhangs are apparently not resolved (discussed below). A rigorous test of this model versus alternative 4-strand helical bundle motifs would benefit from an atomic structure. For this it is conceivable that the tetrameric BASS-core, identified herein via predictions based on the BASS domain model, might be a useful target for crystallization. However, the BASS-core displays evidence of instability and under conditions used for EM it tended to form aggregates. Thus, we feel more work must be done to better stabilize this protein in its tetrameric state, before undertaking such detailed structural studies.
Previously, we reported that native kinesin-5 purified from Drosphila
embryos and examined by rotary shadow EM displayed overall length=96
nm (versus 79
nm herein) with a 61
nm rod (versus 57
nm herein). Moreover, in that study the motor and tail domains appeared collapsed into a single 22
nm globular domain and bipolarity was inferred based on 46 of 70 molecules being decorated on both ends by motor domain-specific IgG10
. In the current study, the higher yield of purified baculovirus-expressed motor combined with the use of superior UF-negative staining allowed >10-fold more decorated and undecorated molecules, manipulated in various ways, to be examined (for example, for FL-kinesin-5; 530 of 663 molecules were decorated on both ends) and the motor domains could be clearly resolved in favourable images (; Supplementary Fig. S1
). The revised dimensions and constructs reported here may prove useful for advancing our understanding of kinesin-5 structure and function in the spindle by EM. For example, experiments are in progress to test the prediction that functionally replacing the native KLP61F
gene with the gene expressing the Bonsai construct will decrease the length of putative kinesin-5 crossbridges between MTs in embryo mitotic spindles from 60 to 30
nm (ref. 33
One unresolved question is why the C-terminal overhangs are not visible in EM images of the BASS domain, where one might expect them to project as single CC rods from the tetrameric BASS-core, and of the Bonsai construct, where they might extend beyond the motor domains on the N-terminal end of the antiparallel neighbouring CCs (). The EM techniques we use should resolve single CCs, so we suspect that, following BASS domain purification, in the absence of their antiparallel neighbour, these overhangs become unstable, disordered and splay apart, making them difficult to visualize by TEM. This speculation is supported by analysis of the purified BASS domain by CD spectroscopy, which yields only 50% α-helix and by SDSL-EPR spectroscopy which fails to detect the predicted CC segments in the overhangs. It is possible that the CD spectrum could underestimate the true α-helical content of the purified BASS domain, much like the kinesin-1 rod, which is estimated to be 65–70% α-helical by CD spectroscopy at physiological temperatures, but is predicted to be 90% α-helix based on sequence analysis23
. However, this would not be inconsistent with our proposal that the rod of full-length kinesin-5 is largely an alpha helical, antiparallel CC rod, whose CC conformation is preserved in the overlap zone of the BASS domain following its purification (), but not in the overhangs, which become disordered, accounting for the CD, EPR and EM data. In the case of Bonsai, the overhangs also contain the C-terminal tail domain, which we suspect interact with the antiparallel CC’s N-terminal heads, further obscuring their visualization.
Indeed, interactions between the C-terminal tails on one CC with the N-terminal heads on the neighbouring CC at each end of the FL-kinesin-5 could explain the curious observation that the tail domains of kinesin-5 are required for MT–MT-cross-linking and sliding by kinesin-5 (ref. 34
). As the tail domains appear to have MT-binding activity35
, one explanation for this result is that the kinesin-5 subunits are arranged in parallel and crosslink and slide MTs using a similar geometry to kinesin-1 and kinesin-14 (refs 36
), but a head-tail interaction within the kinesin-5 homotetramer suggests that the tails could mediate an allosteric activation of MT binding by the heads instead34
. As a precedent, head-tail interactions are well established in the case of the better characterized kinesin-1 (ref. 38
). By EM, the ML-kinesin-5 tetramer is much longer than the rod of FL-kinesin-5, again consistent with the idea that the tail may fold up as a result of its interactions with the motor domain, and the idea that this may have a stabilizing effect is supported by our inability to purify and characterize the ‘tailless’ kinesin-5 complex (this report)13
Although this study provides strong support for the bipolar, homotetrameric architecture of kinesin-5 by providing a structural explanation for the organization of the BASS domain, it is important to notice that we also detected other oligomeric states of kinesin-5 and its subfragments in some experiments. For example, the BASS-Δ subfragment only formed monomers, whereas monomer, dimer and tetramer forms of the BASS domain were detected by MS, and in routine hydrodynamic analyses we found some BASS domain dimers co-existing with tetramers. By EM a significant fraction (20% or less) of mono- or un-decorated molecules are seen. Also, in native PAGE and AUC, 2–3% higher order aggregates were detected. Although it is plausible that these are experimental artifacts, it is also possible that they represent functionally distinct oligomeric states that merit further investigation. For example, the formation of higher order filaments analogous to muscle thick filaments might be relevant to the proposal that kinesin-5 acts in concert with a ‘spindle matrix’6
and dimeric or monomeric kinesin-5 could mediate chromosome congression,40
or contribute to protein synthesis on the ribosome41
processes for which the functional advantage of a bipolar homotetrameric architecture is not obvious.
In summary, this paper reports a major advance in our understanding of how kinesin-5 acquires its unique bipolar architecture. Thus, it represents a significant advance towards the ultimate goal of an atomic resolution understanding of the kinesin-5-dependent sliding filament mechanism that is proposed to underlie key aspects of mitosis and chromosome segregation, processes that are crucial to the formation and maintenance of all known living organisms.