A TOG:αβ-tubulin complex structure reveals conformation-based mechanisms for a microtubule polymerase

Abstract

Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use αβ-tubulin-interacting tumor overexpressed gene (TOG) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin, but the mechanism by which they do so has remained unclear. Here, we report the structure of the TOG1 domain from Stu2p bound to yeast αβ-tubulin. TOG1 binds αβ-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a curved conformation of αβ-tubulin that cannot be incorporated into microtubules, contacting α- and β-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with αβ-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin and how they selectively recognize the growing end of the microtubule.

Figure 1. Structure of a TOG1:αβ-tubulin complex, revealing significant contacts with α- and β-tubulin

A Cartoon representation of the complex (pink: α-tubulin, lime: β-tubulin, slate: TOG1). Contacts probed by mutagenesis are represented as spheres (colored to match panel C and Fig. S3), as are GTPs. (inset) mF₀-DF_c omit electron density map contoured at 3.5 σ and computed from a model without nucleotides: β-tubulin is bound to GTP. B Size-exclusion chromatography assay for TOG1:αβ-tubulin interactions. C TOG1:αβ-tubulin binding assay using interface mutants on Loop 1 (see Fig. S3). D TOG1 (left) and TOG2 (right) (grey) each form a 1:1 complex (red) with αβ-tubulin (black) as detected by analytical ultracentrifugation (Table S4). Curves are shown from S=1 to eliminate a slowly sedimenting contaminant in the TOG2 run.

Figure 2. Disruptive point mutations on the tubulin-binding interfaces of TOG1 or TOG2 affect Stu2p function in vivo

A Yeast carrying plasmid-based rescue constructs of Stu2p were plated at serial dilutions on media containing DMSO (control) or 500 μM CuSO₄ (to deplete endogenous Stu2p) plus 20 μg/ml benomyl (to cause microtubule stress). TOG1 or TOG2 impaired for αβ-tubulin interactions only partially compensate for the depletion of endogenous Stu2p. B Fluorescence images one hour after release from hydroxyurea arrest (green: αβ-tubulin; red: DNA) of yeast depleted of endogenous Stu2p and rescued with wild-type (left) or R200A (right) Stu2p. Spindle elongation is compromised when TOG1:αβ-tubulin interactions are impaired (R200A).

Figure 3. αβ-tubulin-GTP adopts a curved conformation

A Superposition of yeast α (pink) and β-tubulin (lime) shows similar positioning of the H6-H7 segment (represented with darker colors). B Superposition of yeast α- and β-tubulin onto curved (α: orange, β: yellow) and straight (α: maroon, β: dark blue) structures shows the H6-H7 segments of yeast tubulins arranged as in prior curved structures. C Pairwise Cα r.m.s. coordinate deviations between yeast α- and β-tubulin and prior structures, computed for the subdomains indicated (Table S3). D The quaternary structure of yeast αβ-tubulin (pink and green) resembles that of the “curved” form (grey, left), and differs from the straight form (grey, right).

Figure 4. TOG1:αβ-tubulin interactions are conformation-selective

A The structure of the TOG1:αβ-tubulin complex (left) and a docked model with straight αβ-tubulin (right) illustrates how TOG1-contacting epitopes on α- and β-tubulin move relative to each other in the two conformations. B Microtubule assembly reactions (15 μM animal αβ-tubulin) containing 3 μM TOG1 (red) or TOG2 (blue) are inhibited relative to control reactions (black and grey) that received only buffer. C Microtubule co-sedimentation showing that TOG1 or TOG2 do not appreciably bind microtubules, even though the TOG-interacting epitopes are accessible on the outside of the microtubule (Fig. S7). S: supernatant, P: pellet. D Size distributions showing that substoichiometric concentrations of TOG1-TOG2 mixed with αβ-tubulin (red) behave as a complex that sediments faster than αβ-tubulin alone (black) and the TOG1-TOG2:(αβ)₁ complex that results when TOGs are in molar excess over αβ-tubulin (blue). E Minimal cartoon model illustrating how conformation-selective TOG:αβ-tubulin interactions contribute to function. ‘+++’ denotes a basic region that provides microtubule affinity. TOG1 can efficiently capture unpolymerized αβ-tubulin in its naturally curved state (left). The straight/straighter conformation of αβ-tubulin in the MT has lower affinity interactions with TOG1 (right) and may be recognized by TOG2.