High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis

Abstract

Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces.

Figure 1. High-resolution cryo-EM structures of dynamic and stabilized microtubules

A) Cartoon of the αβ–tubulin dimer, which spontaneously exchanges bound GDP for GTP in solution. B) Cartoon illustrating structural intermediates of microtubule polymerization and depolymerization. C) Cryo-EM maps of GMPCPP (left panel, 4.7 Å resolution), GDP (middle panel, 4.9 Å resolution) and taxol-stabilized (right panel, 5.6 Å resolution) microtubules, viewed from inside the microtubule lumen. α-tubulin, green, β-tubulin, blue, GMPCPP/GTP, orange, GDP, pink, taxol, yellow. Maps are contoured at 1.1 σ. (See also Table S1a, Figures S1 and S2 and Movies S1, S2 and S3)

Figure 2. Rosetta Modeling of the GMPCPP Microtubule

A) The low energy 1% GMPCPP ensemble is shown in cartoon representation, colored as in Fig. 1. Bound nucleotides are shown in stick representation and colored by heteroatom, as are magnesium ions (green) and coordinating water molecules. The map is displayed as a transparent grey isosurface. Regions of high-variability in the Rosetta ensemble (above an RMSF threshold of 0.89) are colored in shades of purple. B) β-tubulin C-terminal helices from the energy minimized consensus, all-atom Rosetta model, colored as in Fig. 1. Map is displayed as in A. C) Individual beta strands in the α-tubulin intermediate domain. (See also Tables S1b and S1c and Figures S3 and S4.)

Figure 3. Hydrolysis results in a compression of the E-site at the interdimer interface

A) Cα traces of two adjacent tubulin dimers from the GMPCPP (gold) and GDP (light purple) consensus models, superimposed at the underlined β tubulin. View is tangential to the microtubule lumen. Nucleotides from the consensus models are shown in orange (GTP) and pink (GDP). B) Displacement vectors between Cα coordinates from the consensus models of the GMPCPP state to the GDP state, superimposed as in A, are displayed as narrow cylinders. The chain-trace displayed corresponds to the GMPCPP consensus model: α-tubulin is light grey and β-tubulin dark grey. For clarity, vectors are only displayed for every other Cα pair. Vectors are colored by subdomain unless otherwise noted: N-terminal domain, light blue, intermediate domain, purple, C-terminal domain, red; vector length has been scaled 1.5 fold to aid visualization throughout. Selected structural elements along with associated vectors are highlighted: β-tubulin nucleotide binding loops, dark blue, α-tubulin T7-H8, green, α-tubulin intermediate domain beta sheet, purple, α-tubulin H7, yellow. Nucleotides are displayed as in A. C) View of the E-site structural unit, colored as in B. The RGB values of vector colors correspond to angular displacements relative to a Cartesian coordinate system, i.e. vectors of similar color point in a similar direction. D) View of the α-tubulin intermediate domain and H7 in the GMPCPP model (gold), GDP model (light purple), and 1SA0 (dark red), superimposed on the beta sheet of the α-tubulin N-terminal domain. (See also Figure S5 and Movies S1, S3, S4 and S5)

Figure 4. Rearrangements upon hydrolysis alter MAP binding sites on the microtubule surface

Rearrangements on the microtubule surface. Vectors are colored by subdomain as in Fig. 3. Binding sites for key MAPs are indicated. (See also Movie S6)

Figure 5. Taxol binding restores the GDP lattice to a GMPCPP-like extended state

A) Analogous to Fig. 2A, but comparing the GMPCPP (gold) to the GDP–taxol (light blue) state. B) View from the microtubule lumen of the superimposed GDP (light purple) and GDP–taxol (light blue) models. Note the swelling of the taxol binding site (1), opening of the E-site interface (2), and reversal of the longitudinal displacement of the α-tubulin intermediate domain (3). Nucleotides from the GDP-taxol consensus models are displayed and colored as in Fig. 3. Taxol is yellow. (See also Figure S6 and Movies S2 and S7)

Figure 6. Lateral contacts are highly similar between stable and unstable states

View from the microtubule lumen of homotypic inter-protofilament lateral contacts. The GDP–taxol map is displayed as a transparent grey isosurface. A) α-tubulin is colored light grey, β-tubulin, dark grey. Consensus models are displayed with lateral contacts colored (GMPCPP, gold, GDP, light purple, and GDP–taxol, light blue). Key residues mediating lateral contacts are displayed in stick representation, as is taxol. Taxol and its associated density are colored yellow. B) Superpositions of β-tubulin subunits from the electron crystallographic structure of taxol-bound tubulin (PDB 1JFF, dark blue)(Lowe et al., 2001) the structure of unassembled, inhibited zampanolide-bound tubulin (PDB 4I4T, dark red) (Prota et al., 2013), and the GDP-taxol model from this study (light blue, taxol is yellow). Note that Y283 is only in position to mediate the lateral contact in the GDP-taxol model, and that a clash is present with the laterally adjacent subunit in the zampanolide model (asterisk).

Figure 7. Proposed model of destabilizing and stabilizing structural transitions in the microtubule lattice

Cartoon of conformational transitions colored as in Fig. 1, except the α-tubulin intermediate domain is purple. Left, nucleotide hydrolysis and phosphate release leads to compaction of the E-site and rearrangement of the α-tubulin intermediate domain (middle), generating destabilizing strain while tubulin remains within the constraints of the microtubule lattice. Taxol binding (right, top), allosterically leads to a reversal of E-site compaction and the α-tubulin rearrangement; unraveling the detailed mechanism of this transition will require structural analysis at near-atomic resolution. Subtle structural changes could be propagated across the E-site interdimer interface (up arrow), within the dimer (down arrow), or both. In the absence of binding by a stabilizing agent, strain would be dissipated by tubulin bending during catastrophe (right, bottom), when the α-tubulin intermediate domain (and β-tubulin intermediate domain, dark blue) are capable of undergoing rotation due to the relief of steric constraints imposed by lateral contacts.