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The role of tubulin–tubulin lattice contacts in the mechanism of microtubule dynamic instability

Nature Structural & Molecular Biology volume 25, pages 607–615 (2018)Cite this article

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Abstract

Microtubules form from longitudinally and laterally assembling tubulin α–β dimers. The assembly induces strain in tubulin, resulting in cycles of microtubule catastrophe and regrowth. This ‘dynamic instability’ is governed by GTP hydrolysis that renders the microtubule lattice unstable, but it is unclear how. We used a human microtubule nucleating and stabilizing neuronal protein, doublecortin, and high-resolution cryo-EM to capture tubulin’s elusive hydrolysis intermediate GDP•Pi state, alongside the prehydrolysis analog GMPCPP state and the posthydrolysis GDP state with and without an anticancer drug, Taxol. GTP hydrolysis to GDP•Pi followed by Pi release constitutes two distinct structural transitions, causing unevenly distributed compressions of tubulin dimers, thereby tightening longitudinal and loosening lateral interdimer contacts. We conclude that microtubule catastrophe is triggered because the lateral contacts can no longer counteract the strain energy stored in the lattice, while reinforcement of the longitudinal contacts may support generation of force.

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Fig. 1: Tubulin GTPase cycle and MT structures stabilized by DCX.
Fig. 2: GTPase-dependent structural transitions strengthen the longitudinal MT lattice contacts.
Fig. 3: GTPase-dependent structural transitions weaken the lateral MT lattice contacts.
Fig. 4: Effect of Taxol binding to GDP-DCX-MT.
Fig. 5: Mechanisms and implications of MT catastrophe and the tubulin GTPase cycle.

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Acknowledgements

We thank A. Roberts and M. Steinmetz for invaluable discussions about this work. S.W.M. and C.A.M. are supported by the Medical Research Council, UK (MR/J000973/1 and MR/R000352/1).

Author information

Authors and Affiliations

  1. Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK

    Szymon W. Manka & Carolyn A. Moores

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  1. Szymon W. Manka
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Contributions

S.W.M. conceived experimental strategies, designed and carried out experiments and computations, processed data, interpreted results, and wrote the manuscript; C.A.M. proposed and supervised research, interpreted results, and wrote the manuscript.

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Correspondence to Szymon W. Manka or Carolyn A. Moores.

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Integrated supplementary information

Supplementary Figure 1 Average and local resolution of DCX-MT reconstructions and the relative DCX decoration of different MT lattices.

a, Average resolution estimation using 0.143 cut-off criterion in the plot of Fourier Shell Correlation (FSC) between two independent half-reconstructions. b, Relative extent of DCX decoration on different MT lattices, measured as described in Methods. The error bars indicate standard deviation between 3 non-overlapping groups of data. c, Local resolution estimation in Å using ResMap (Kucukelbir, A. et al., Nat. Methods 11, 63–65, 2013). DCX density in the GMPCPP lattice is shown with lower threshold (dotted ovals) due to lower decoration of the extended GMPCPP lattice with DCX.

Supplementary Figure 2 Difference mapping between ligand densities in different nucleotide states.

Pairwise comparison of nucleotide binding sites in different reconstructions provide additional support for conclusions about the nucleotide states captured in this study, including the loss of the metal ion between GMPCPP and GDP•Pi. The GDP•Pi vs GTPγS comparison shows the similarity of the density corresponding to these nucleotides despite their different effects on MT conformation (Supplementary Fig. 3).

t

Supplementary Figure 3 Tubulin conformation in GTPγS-DCX-MT resembles that in GDP-DCX-MT.

a, Backbone front view comparisons of tubulin conformation in the GTPγS lattice against those in the GDP•Pi or GDP lattices, based on pairwise superposition on the β1 subunit (underlined); coloured by the degree of displacement or as follows: GTPγS, orange; GDP•Pi, orange, GDP, red. b, β1 E-site comparisons in the cryo-EM map of the GTPγS lattice: tubulin density, sky blue; nucleotide density, orange. Tubulin model chains are shown as ribbons: GTPγS state, skye blue; GDP•Pi state, slate blue; GDP state, grey, and their associated nucleotides (GTPγS, orange/heteroatom; GDP•Pi and GDP, light grey) and selected side chains (coloured by heteroatom) are shown as sticks.

Supplementary Figure 4 Details of the longitudinal interdimer interface in different DCX-MT lattices.

View from MT+ end at: a, α-tubulin residues having at least one atom within 4 Å distance from any atom in the interfacing β-tubulin, shown as sticks coloured by heteroatom in their cryo-EM density. The density for the catalytic E254 residue is captured only in the GMPCPP state (arrows); b, superposition of both α and β faces of the interface shown in different representations with two anchor points (A1 and A2) indicated, to aid relating positions of the interfacing residues; α-face residues (shown in a with IDs) are connected by white backbone, to visualize secondary structural context (labelled); the atoms within 4 Å distance from the interfacing β-tubulin are shown as spheres; E254 residues are pointed with arrows; β-face atoms within 4 Å distance from the interfacing α-tubulin are shown as model-derived surfaces coloured by element (C, grey; N, blue; O, red); c, β-tubulin residues having at least one atom within 4 Å distance from any atom of the bound α-tubulin; the residues are shown as sticks coloured by element (see d for ID labels), and the atoms within 4 Å distance from the interfacing α-tubulin are shown as spheres together with model-derived surface coloured by element; secondary structures to which particular residues belong and two anchor points (A1 and A2) are indicated; d, cryo-EM density and ID labels for the residues shown in c.

Supplementary Figure 5 Select views at particular changes in the lateral contacts of an MT lattice during the GTPase transitions.

Related to and extending Fig. 3 to provide additional depiction of selected stable and vulnerable connections, according to measurements performed on our models of MT nucleotide states. Top panels focus on one inter-α-subunit connection (solid line) likely to be affected (dotted line) from the first transition. Bottom panels focus on one inter-β-subunit connection likely to be preserved (solid lines) and two connections likely to be affected (dotted lines) during the second transition. Atomic models are shown with experimental densities and coloured accordingly or by heteroatom. The interacting atoms are named according to the PDB convention.

Supplementary Figure 6 Changes in PF skew accompanying nucleotide state transitions in the DCX-MT lattice.

a, Cartoon illustration of PF skew in selected DCX-MT lattices and the plot showing mean value (~) of the skew angle measured as described in Methods. Error bars represent standard deviations. b, Statistical analysis of the skew angles of all DCX-MT lattices by ordinary one-way ANOVA using Prism 6 software (see Methods). Top, box and whiskers plot combined with scatter dot plot showing all n data points collected for each lattice (n = sample size). The whiskers are defined by the minimum and the maximum value, the box shows the interquartile range (between the 25th and 75th percentile) and the central line indicates the median value (50th percentile). Significance has been verified through multiple comparisons (see below): **** - significant, p < 0.0001; ns - not significant. Bottom, Multiple comparisons: comparing every mean with every other mean by Tukey-Kramer method, which is suitable for unequal sample sizes between compared groups. Plotted are the differences between the mean skew angle values of the compared lattices (colour-coded circles) together with 95 % confidence intervals (CIs) for the difference between the two means. Multiplicity adjusted P values (accounting for multiple comparisons) are also reported.

Supplementary Figure 7 Comparisons of bent (solution) and straight (MT lattice-associated) tubulin conformations and their mechanistic consequences.

a, Backbone side view of β1-subunit (underlined) superposition of different bent tubulin X-ray structures with our straight tubulin structures. Left, comparison of tubulin conformation in the extended GTP-like MT lattice (GMPCPP) with that of a less bent tubulin-RB3 stathmin-like domain complex, 60% occupied by GTP (PDB code: 3RYF), and a more bent, GDP-bound tubulin-eribulin complex (5JH7). Right, comparison of tubulin conformation in the compacted GDP-MT lattice with bent, GDP-bound conformations in: tubulin-pironetin complex (5LA6), tubulin-stathmin-TTL-vinblastin complex (5BMV), and 5JH7. b, Close-up lumenal view of the superposition shown on the right in panel a. During the bending of GDP-tubulin after catastrophe, the M-loops - main contributors to MT lateral contacts - become drastically displaced and incompatible with forming MT lattice-like contacts. Taxol® binding (at the indicated site) prevents M-loop displacement in the context of the lattice and therefore stabilises it (see Fig. 4 for details). Moreover, the conformation of helices βH6, βH7 and their connecting loop in the bent tubulin would clash with αS8-H10 loop in the straight tubulin (asterisk). Thus, bending of tubulin dimers weakens not only lateral, but also longitudinal inter-dimer interactions (small arrows). c, Inter-dimer interface area changes during tubulin GTPase cycle computed by PDBePISA (www.ebi.ac.uk/pdbe/pisa/). The areas from 4I4T≈1026 Å2 (crystal structure of tubulin-RB3-TTL-Zampanolide complex), 5BMV≈898 Å2, 5LA6≈877 Å2 and 5JH7≈832 Å2 were averaged (908 ± 83 Å2) as GDP-bound (Esite:GDP) bent tubulin structures, and the area of 3RYF≈1188 Å2 provided a single value for the only available 60% GTP-bound (Esite:GTP) tubulin structure. Results for “MT lattice” are as shown in Fig. 2d. Error bars represent standard deviation of the mean where applicable. d, Superposition of tubulin in the extended GTP-like MT lattice state (GMPCPP) with 60% GTP-bound bent tubulin (3RYF) on helix αH8, to visualise the increased distance between the catalytic E254 residue and the γ-P of GTP in the bent tubulin conformation, compared to the straight conformation. The acidic side chain of E254 polarises water molecule making it ready for nucleophilic attack on the positively charged γ-P. Tubulin straightening in the MT lattice seems to facilitate (catalyse) GTP hydrolysis, by shortening the distance between the polarised water and its target; conversely, tubulin bending seems to result in a conformation suboptimal for GTP hydrolysis, potentially preventing premature hydrolysis in the associating bent PFs (MT precursors).

Supplementary information

Supplementary Figures 1–7

Reporting Summary

Supplementary Table 1

Supplementary Video 1

GTPase-driven MT lattice transitions. Related to Fig. 2A. Backbone representation of two tubulin dimers (minimal PF) viewed from the front (as indicated in Fig. 2A) morphs through 3 distinct tubulin conformations, based on β1-subunit superposition (framed label), pausing for a few seconds between them: starting with the GTP-DCX-MT state (α-tubulin, light pink; β-tubulin, dark pink), through the intermediate GDP•Pi-DCX-MT state (α-tubulin, light slate blue; β-tubulin, dark slate blue), finishing with the GDP-DCX-MT state (α- tubulin, light grey; β-tubulin, dark grey). Nucleotides and Mg2+ ion are displayed during the short pauses between the morphs (GTP, yellow sticks; GDP•Pi, orange sticks; GDP, red sticks; Mg2+, green sphere). Anchor points (A1 and A2) are indicated in helices αH8 and αH10. Next the sequence of transitions is repeated for the lumenal view of the minimal PF.

Supplementary Video 2

Conformational changes at the catalytic E-site during GTP hydrolysis. Related to Fig. 2B. Starts with ribbon representation of two tubulin dimers (minimal PF) in the GTP-DCX-MT state (α- tubulin, light pink; β-tubulin, dark pink; GTP, yellow sticks; Mg2+, green sphere), viewed from the front. The view zooms into the Esite, occupied by GTP (colored yellow and by heteroatom) and an Mg2+ ion. Selected residues contacting GTP (solid lines) fade in as sticks colored by heteroatom. Next, the structure morphs through GDP•Pi- (slate blue) to GDP-DCX-MT (grey) state, based on β1- subunit superposition (framed label at the start of the movie), with a longer pause during the first (GTP to GDP•Pi) transition, presenting marked displacement of the resultant GDP (orange/heteroatom colored sticks) together with αS9 strand and the βT5 loop. New pattern of connections is revealed. Cryo-EM density for βD177 and βT178 side chains is unclear in the GDP•Pi and GDP states (Fig. 2B), so information about their exact position is missing, but these residues are less likely to interact with the nucleotide after hydrolysis. The second transition (Pi release) shows subtle conformational change. Approximate location of anchor points (A1 and A2) is indicated.

Supplementary Video 3

GTPase-driven reinforcement of MT longitudinal interdimer contact. Related to Fig. 2C. Starts with front backbone view of two tubulin dimers (minimal PF) in the GTP-DCX-MT state (α-tubulin, light pink; β-tubulin, dark pink; GTP, yellow sticks; Mg2+, green sphere). After the β1-subunit representation changes to surface, the viewing position moves to +end and the view focuses on the interdimer interface showing superposition of the α2-subunit on the β1- subunit. The regions of the α2-subunit (labeled) that are near the β1-subunit are shown as backbone and colored according to nucleotide state (GTP, light pink; GDP•Pi, light blue; GDP, light grey); this includes the catalytic E254 residue side chain, the position of which is shown as sticks colored by heteroatom at GTP hydrolysis step. β1-surface atoms that are within 4Å distance from any atoms of the α2-subunit are colored with the same color scheme. The contact area between the subunits increases at structural transitions 1 and 2, which follow nucleotide changes (GTP, yellow/heteroatom; GDP•Pi, orange/heteroatom; GDP, red/heteroatom). Morphing between tubulin conformations is sequential, with β1-surface following α2-backbone.

Supplementary Video 4

GTPase-driven MT lattice transitions. Related to Fig. 3. Starts with front backbone view of two PFs in the GTP-DCX-MT state (α- tubulin, light pink; β-tubulin, dark pink; GTP, yellow sticks; Mg2+, green sphere). The structure rotates and the view switches to lumenal, focusing on two homotypic lateral contacts: one α-α and one β-β. The α-α lateral contact is zoomed into and selected resides are shown as sticks colored by heteroatom and with connections (putative H-bonds) depicted between them with solid lines. The view is tilted and rocks for clearer inspection before the structure morphs into the GDP•Pi-DCX-MT state (α-tubulin, light slate blue; β-tubulin, dark slate blue), based on β1-subunit superposition (framed label at the start of the movie). This first transition reveals likely loss of 2 connections. For the subsequent (GDP•Pi to GDP) transition, the view focuses on the β - β lateral contact and the likely loss of additional 2 connections during this step is visualised analogously. Next, the PFs - now in the GDP-DCX-MT state (α - tubulin, light grey; β-tubulin, dark grey; GDP, orange sticks) - return to the initial view, and catastrophe is simulated by tubulin morphing into the bent conformation of the GDP-bound tubulin–eribulin complex (PDB code: 5JH7), based on α1-subunit superposition (framed label).

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Manka, S.W., Moores, C.A. The role of tubulin–tubulin lattice contacts in the mechanism of microtubule dynamic instability. Nat Struct Mol Biol 25, 607–615 (2018). https://doi.org/10.1038/s41594-018-0087-8

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