Structural insight into the stabilization of microtubules by taxanes
- Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Switzerland
- Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas, Spain
- State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, China
- Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Switzerland
- Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Japan
- Department of Biomedical Sciences, University of Alcalá, Spain
- University of Basel, Switzerland
Figures
Figure 1
Structures of tubulin and ligands employed in the work.
(A) Tubulin heterodimer (α-tubulin in gray and β-tubulin in white) in ribbon representation, where nucleotide binding sites have been highlighted in sphere representation (B) Structural features of …
Figure 2
T2R-TTL structures in complex with baccatin III, 2a, and 2b.
Overall view of the T2R-TTL-baccatin III (PDB ID 8BDE) (A), the T2R-TTL-2a (PDB ID 8BDF) (B), and the T2R-TTL-2b (PDB ID 8BDG) crystal structures. The α- and β-tubulin chains are colored in dark and …
Figure 3
Crystal structure of T2R-TTL-baccatin III (PDB ID 8BDE) and T2R-TTL-2a (PDB ID 8BDF) complexes.
(A) Close-up view of the interaction network observed between baccatin III (lemon) and β-tubulin (light gray). Interacting residues of tubulin are shown in stick representation and are labeled. …
Figure 4
Comparison of taxane binding to unassembled curved versus assembled straight tubulin.
(A) Close-up view of the superimposed baccatin III bound (ligand in lemon; protein in gray ribbon and sticks) to curved tubulin (PDB ID 8BDE) and paclitaxel bound to straight tubulin as found in a …
Figure 5
Conformational changes induced by taxane binding to unassembled, curved tubulin.
(A) Conformational changes on the backbone atoms (dark blue) of the β-tubulin chain induced by baccatin III upon binding to curved tubulin. The tubulin chains are in ribbon representation and are …
Figure 6
Schematic representation of subtle domain movements observed from apo to baccatin III- to 2a-bound curved tubulin.
The three structures were superimposed onto their central helices βH7 to highlight better the subtle domain movements relative to each other. The individual domains are colored according to their …
Figure 7
Fiber diffraction patterns of microtubules.
Microtubules assembled from guanosine triphosphate (GTP)-tubulin and paclitaxel (A), GTP-tubulin and docetaxel (B), GTP-tubulin and 2a (C), GTP-tubulin and 2b (D), and GTP-tubulin and baccatin III (E…
Figure 8
Flexibility of β subunit and βM loop during the αβ-tubulin dimer molecular dynamics (MD) simulation.
(A) Mass-weighted positional fluctuations (or root-mean-square fluctuations, Å) by residue for atoms in the β subunit of the αβ-tubulin dimer over the course of 0.6 µs of MD simulation, in the apo …
Figure 9
Molecular dynamics (MD) simulation of tubulin-taxane complexes.
(A,B) MD simulations of the free αβ-tubulin dimer. (A) Initial stage of the simulation, starting from a βM loop (residues β275-β286; yellow) organized as an α-helix akin to what is observed in a …
Figure 10
Solvent-corrected interaction energies between individual β2-tubulin residues and ligands throughout the molecular dynamics (MD) simulations of the minimalist representation of a microtubule.
(A) The interfacial site 1 between neighboring protofilamentes. (B) The solvent-exposed site 2. These per-residue energies, which together represent a ‘binding fingerprint’, were calculated by means …
Figure 11
Molecular dynamics (MD simulations of minimalist) representations of a microtubule ((α1-β1-α2)/(α1’-β1’-α2’)) in complex with the ligands employed.
Baccatin III (green, A), 2a (red, B), paclitaxel (blue, C). α- (dark gray) and β-tubulin (light gray) are displayed as ribbons, with the βM loop colored in yellow and the side chain of Tyr283 as …
Figure 12
Surface representations of liganded taxane sites in both the curved and straight tubulin conformational states.
(A) Curved tubulin; (B) straight tubulin. The structures of 2a (white) and paclitaxel (slate) bound to microtubules (PDB ID 6WVR) were superimposed onto their central helices βH7. The side chains of …
Scheme 1
Reagents and conditions.
(a) TESCl, triethylamine (TEA), 4-dimethylaminopyridine (DMAP), LiBr, THF, RT to 70°C, 84%; (b) LHMDS, THF, –45°C, 79%; (c) 10% Pd/C, H2, MeOH, 64%; (d) DCC, DMAP, acid, DCM, 0°C to RT; (e) HF, Py, …
Videos
Video 1
Conformational transition from apo to baccatin III-bound, unassembled tubulin state.
Top view on β-tubulin (onto the ‘plus end’ in the context of a microtubule).
Video 2
Conformational transition from apo to baccatin III-bound, unassembled tubulin state.
Luminal view on β-tubulin (view from the lumen in the context of a microtubule).
Video 3
Conformational transition from apo to 2a-bound, unassembled tubulin state.
Top view on β-tubulin (onto the ‘plus end’ in the context of a microtubule).
Video 4
Conformational transition from apo to 2a-bound, unassembled tubulin state.
Luminal view on β-tubulin (view from the lumen in the context of a microtubule).
Animation 1
Molecular dynamics (MD) movie of the apo tubulin dimer showing the occupancy of the taxane site by the βM loop.
1 µs simulation, 1 snapshot every 5 ns, βM loop in yellow.
Animation 2
Simulation of ligand exit and entry using targeted molecular dynamics (MD) for baccatin III unbinding from and binding to αβ-tubulin.
Animation 3
Simulation of ligand exit and entry using targeted molecular dynamics (MD) for paclitaxel unbinding from and binding to αβ-tubulin.
Tables
Table 1
X-ray data collection and refinement statistics.
| T2R-TTL-BacIII | T2R-TTL-2a | T2R-TTL-2b | |
|---|---|---|---|
| Data collection | |||
| Space group | P212121 | P212121 | P212121 |
| Cell dimensions | |||
| a, b, c (Å) | 104.1, 157.2, 179.2 | 104.8, 157.9, 179.1 | 105.3, 158.6, 179.2 |
| Resolution (Å) | 49.2–1.9 (1.95–1.90) | 49.3–1.95 (2.00–1.95) | 49.4–2.35 (2.41–2.35) |
| Rmerge(%) | 10.7 (491.9) | 13.3 (516.6) | 17.4 (403.5) |
| Rmeas (%) | 11.1 (513.1) | 13.6 (526.1) | 17.7 (410.8) |
| Rpim (%) | 3.3 (147.5) | 2.9 (102.9) | 2.6 (57.7) |
| I/σI | 16.5 (0.5) | 20.1 (0.7) | 20.1 (0.9) |
| CC half | 100 (17.8) | 100 (31.4) | 99.9 (46.6) |
| Completeness (%) | 100 (99.8) | 100 (100) | 100 (100) |
| Redundancy | 13.5 (12.4) | 27.3 (27.8) | 28.5 (28.3) |
| Refinement | |||
| Resolution (Å) | 49.2–1.9 | 49.3–1.95 | 49.4–2.35 |
| No. unique reflections | 229654 | 215774 | 125168 |
| Rwork/Rfree | 19.2/21.8 | 18.9/21.6 | 18.3/21.4 |
| No. atoms | |||
| Protein | 17555 | 17389 | 17227 |
| Ligand | 42 | 120 | |
| Water | 861 | 883 | 166 |
| Average B-factors (Å2) | |||
| Protein | 59.0 | 62.9 | 76.1 |
| Ligand (chain B/D) | n.a. / 109.2 | 111.4/102.8 | 146.6/144.9 |
| Water | 56.2 | 60.3 | 59.4 |
| Wilson B-factor | 41.7 | 43.1 | 56.9 |
| R.m.s. deviations | |||
| Bond lengths (Å) | 0.003 | 0.003 | 0.002 |
| Bond angles (°) | 0.642 | 0.655 | 0.550 |
| Ramachandran statistics | |||
| Favored regions (%) | 98.1 | 98.1 | 98.0 |
| Allowed regions (%) | 1.8 | 1.8 | 2.0 |
| Outliers (%) | 0.1 | 0.1 | 0 |
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For each structure, data were collected from a single crystal. Values in parentheses are for highest-resolution shell.
Table 2
Structural parameters of microtubules assembled in the presence of different nucleotides and drugs*.
| Paclitaxel-pre microtubules | Paclitaxel- post microtubules | Paclitaxel- GDP tubulin | GDP-microtubules | GMPCPP-microtubules | Docetaxel-microtubules | Baccatin III-microtubules | 2a-microtubules | 2b-microtubules | |
|---|---|---|---|---|---|---|---|---|---|
| Microtubule radius (nm) | 10.97±0.10 | 11.04±0.51 | 10.98±0.47 | 11.42±0.10 | 11.63±0.10 | 11.53±0.10 | 11.06±0.35 | 11.27±0.57 | 11.60±0.36 |
| Avg. PF number | 12.21±0.10 | 12.28±0.71 | 12.23±0.65 | 12.91±0.10 | 13.29±0.10 | 12.90±0.10 | 12.29±0.39 | 12.63±0.72 | 12.99±0.40 |
| Inter-PF distances (nm) | 5.58±0.01 | 5.59±0.33 | 5.57±0.29 | 5.50±0.03 | 5.45±0.03 | 5.57±0.01 | 5.61±0.18 | 5.55±0.31 | 5.56±0.17 |
| Avg. monomer length (nm) | 4.18±0.01 | 4.18±0.01 | 4.18±0.01 | 4.06±0.01 | 4.18±0.010 | 4.18±0.01 | 4.16±0.03 | 4.15±0.03 | 4.13±0.03 |
| 1 nm band peak position (nm–1) | 6.02±0.01 | 6.02±0.01 | 6.02±0.01 | 6.19±0.01 | 6.02±0.01 | 6.02±0.01 | 6.04±0.5 | 6.06±0.05 | 6.08±0.05 |
| 8 nm band | Yes | Yes | Yes | No | Yes | Yes | Yes | Yes | Yes |
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*
Errors are SE of three independent.
