A tethered delivery mechanism explains the catalytic action of a microtubule polymerase
- UT Southwestern Medical Center, United States
- Howard Hughes Medical Institute, UT Southwestern Medical Center, United States
Figures
Figure 1
TOG2 binds to curved αβ-tubulin analogously to TOG1.
(A) Structure of the TOG2:αβ-tubulin complex (TOG2: slate, α-tubulin: pink, β-tubulin: green), with the important binding residues W341 and R519 represented as spheres. The semi-transparent gray …
Figure 2
TOG1 and TOG2 bind αβ-tubulin with comparable affinity.
(A) Sedimentation velocity analytical ultracentrifugation of polymerization competent yeast αβ-tubulin does not show signs of self-association between 80 nM and 1 μM concentration. The main plot …
Figure 3
In TOG1-TOG2, the two TOG domains bind two αβ-tubulins without positive cooperativity.
(A) Cartoons illustrating three different possible arrangements of a TOG1-TOG2:(αβ)2 complex: independent (left) denotes that an αβ-tubulin:αβ-tubulin interface does not provide additional stability …
Figure 4
Two TOG domains are required for Stu2 function, but they do not have to be different.
(A) Yeast carrying plasmid-based rescue constructs coding for dimerization-competent variants of Stu2p were plated at serial dilutions on media that was unmodified (control) or that contained 500 μM …
Figure 5
Stu2p function tolerates variation in the primary sequence and in the length of the TOG1-TOG2 linker.
Rescue assays were performed as in Figure 4, using dimerization-impaired rescue constructs. (A) Stu2p variants with ‘shuffled’ (randomized) linker sequences rescue the depletion of endogenous Stu2p …
Figure 6
A tethering model for the polymerase function that incorporates our structural and biochemical observations.
The model posits that MT plus-end recognition occurs through TOG-mediated recognition of curved (black outline, ‘kinked’ αβ-tubulin cartoon), not straight (gray outlines), αβ-tubulin on the MT end, …
Figure 7 with 1 supplement
An implicit model can recapitulate the catalytic nature of polymerase activity.
We developed a kinetic model for microtubule elongation and altered it to explore models for the polymerase. (A) Cartoon of the cylindrical microtubule (left; pink and green spheres represent α- and …
Figure 7—figure supplement 1
An implicit model as in Figure 7 but trained against different measured growth rates.
(A) A grid search identified parameters capable of recapitulating the concentration dependence of microtubule elongation rates measured by Walker et al. (1988). The black line summarizes the trend …
Figure 8
Schematic cartoons illustrating the origin of catalytic action.
The microtubule end has multiple sites where αβ-tubulin can associate, but elongation is largely dominated by additions into the few, high-affinity ‘corner’ sites (left panel) because pure …
Tables
Table 1
Data collection and refinement statistics
| Data collection | |
| Space group | C2 |
| Cell dimensions | |
| a, b, c (Å) | 111.91, 89.57, 135.51 |
| β (°) | 112.31 |
| Resolution (Å) | 50.0–2.81 (2.92–2.81)* |
| Rsym | 0.143 (0.924) |
| <I>/<σI> | 9.6 (1.1) |
| Wilson B-value (Å) | 48.9 |
| Anisotropy (Å) relative to best direction (001) | |
| ΔB in (100) direction, ΔB in (010) direction | +29.95, +8.38 |
| CC1/2 in high resolution shell | 0.542 |
| Completeness (%) | 98.2 (91.3) |
| Redundancy | 4.1 (3.2) |
| Refinement | |
| Resolution (Å) | 2.81 |
| No. reflections | 26,235 |
| Completeness(%) | 86.5† (35.3) |
| Rwork/ Rfree (%) | 21.8/25.9 (33.0/41.3) |
| Maximum likelihood estimated coordinate error (Å) | 0.42 |
| No. atoms | 8524 |
| Protein (non-hydrogen) | 8437 |
| Ligand/ion | 66 |
| Water | 21 |
| B-factors | |
| Protein | 44.7 |
| Ligand/ion | 52.0 |
| Water | 27.3 |
| Rms deviations | |
| Bond lengths (Å) | 0.003 |
| Bond angles (°) | 0.66 |
| Ramanchandran plot | |
| Favored (%) | 95.0 |
| Allowed (%) | 4.25 |
| Disallowed (%) | 0.75 |
| Rotamer outliers (%) | 3.2 |
| Molprobity clash score | 1.5 |
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*
Highest resolution shell is shown in parenthesis.
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†
The data were corrected for anisotropy in HKL2000. This treatment eliminated weak reflections and reduced the completeness of the data used for refinement compared to the completeness reported for data collection.
