Figure 5. Unexpected antagonism between TOG: αβ-tubulin engagement and basic:lattice interactions.

(A) The structure of Stu2:TOG1 (slate) bound to yeast αβ-tubulin (α-tubulin in pink and β-tubulin in lime; PDB code 4FFB) is shown in cartoon representation. Red spheres indicate the approximate position of interface blocking mutations on the plus- (β:T175R,V179R) or minus-end (α:T350E) and of perturbing mutations on lateral interaction surfaces (α: H284A, β: F281A). All interface mutations are distant from the TOG-interacting surface. Orange spheres indicate the positions of the structured C-terminii of α- and β-tubulin that precede the charged ‘tails’. (B) Tubulin elements required to antagonize lattice binding by Stu2. Stu2:microtubule binding assays were monitored by TIRF and performed using stabilized microtubules as the substrate, 100 nM Stu2-eGFP, and 1 μM of tubulin mutants. Stu2 coats the stabilized microtubules when no unpolymerized tubulin in present (‘No tubulin’). Lattice-binding is substantially eliminated when wild-type tubulin is included as a competing binding partner (‘WT tubulin’). This tubulin-induced antagonism of lattice binding does not require the tubulin tails (‘∆tails tubulin’) or longitudinal contacts (‘end blocked tubulin’). Tubulin perturbed on the lateral interface does not effectively antagonize lattice binding by Stu2 (‘side blocked tubulin’), indicating that lateral contacts between TOG-bound tubulins are important. See also Figure 5—figure supplement 2. (C) Cartoon illustrating that lateral tubulin interactions (possibly transient) between TOG-bound tubulins antagonize interactions between the basic domain and the MT lattice. Tubulins bound to either the TOG1 or TOG2 domains are illustrated; other combinations of two tubulin-binding TOGs tested yield a similar result. (D) Control of lattice binding by unpolymerized tubulin is not an idiosyncratic property of Stu2. Zyg-9, a monomeric Stu2 family polymerase from C. elegans, also shows this tubulin-induced attenuation of microtubule lattice binding. 50 nM Zyg-9-mCherry was used with unlabeled GTPγS-stabilized yeast microtubules; 5 μM bovine tubulin was the competing binding partner.

Figure 5—figure supplement 1. Assays characterizing the assembly and TOG-binding properties of tubulin with a mutated lateral interface.

(A) ‘Spin-down’ assays for microtubule assembly reveal by SDS-PAGE that mutations on the lateral interface of β-tubulin (left, F281A) or α-tubulin (right, H284A) reduce the extent of microtubule assembly compared to wild-type. S = supernatant, p=pellet. Assays were performed using 1 μM tubulin. (B) A gel filtration binding assay demonstrate that a lateral tubulin mutant, Tub1-H284A, binds a TOG domain comparably to wild-type tubulin. Left: Overlaid gel filtration traces of WT tubulin alone (blue), TOG1 alone (red), and the tubulin + TOG mix (black). The shift to earlier elution volumes indicates formation of a larger complex. Right: Overlaid gel filtration traces of α-H284A tubulin alone (blue), TOG1 alone (red, repeated from left panel), and the tubulin + TOG mix (black). A similar shift in elution indicating the presence of a TOG:tubulin complex was observed. All samples contain 1 μM tubulin variant and 1 µM TOG1. (C) Analysis of TOG1:αβ-tubulin interactions by sedimentation velocity. The plot shows c(s) distributions (color coded by sample) for each control sample: TOG1 alone (2 µM, purple), WT tubulin (0.3 µM, blue), ‘LR1’ plus-end blocked tubulin (β-tubulin: T175R, V179R) (0.3 µM, red) and ‘F281A’ (β-tubulin: F281A) (0.3 µM, green). (D) Isotherms for Stu2 TOG1 domain binding to WT tubulin (black), plus-end blocked tubulin ‘LR1’ (β-tubulin:T175R,V179R) (blue) and side-block tubulin ‘F281A’ (β-tubulin:F281A) (red). TOG1 binds with comparable affinity to all three tubulin variants including the two tubulin mutants (plus-end and side-block) and WT tubulin. Fitted binding affinities and 1 σ confidence intervals for fitted affinities (in parentheses) are shown in the table on the right.

Figure 5—figure supplement 2. Additional data from assays using mutated tubulins.

More experiments testing whether tubulin mutated on the lateral or longitudinal tubulin interfaces can antagonize interactions between the basic region and the microtubule. Results are consistent with Figure 5 in showing that lateral interactions between TOG-bound tubulins are important: lattice binding by Stu2 is attenuated in the presence of plus-end blocked tubulin (β-tubulin:T175R,V179R, left); lattice binding by dimeric or monomeric forms of Stu2 is not attenuated in the presence of a mutation on the lateral interface (α-tubulin:H284A, middle and right). Samples contain 100 nM Stu2-eGFP variant and 1 μM tubulin mutant; unlabeled, GTPγS-stabilized yeast microtubules were used as the MT binding substrate.