Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation
- University of Chicago, United States
- University of Illinois at Urbana-Champaign, United States
- The Scripps Research Institute, United States
- Memorial Sloan Kettering Cancer Center, United States
- University of Illinois Urbana-Champaign, United States
- Texas A&M University, United States
Figures
Figure 1 with 1 supplement
Binding of the SRC3 coactivator to WT, Y537S, or D538G ERα LBD in the absence or presence of E2 or TOT. LBD, ligand-binding domain.
https://doi.org/10.7554/eLife.12792.004
Figure 1—figure supplement 1
Binding of the SRC3 coactivator to WT, Y537S, or D538G mutant ERα LBD with increasing concentrations of E2 or TOT.
https://doi.org/10.7554/eLife.12792.005
Figure 2
Determination of Kd values of estradiol (E2) binding to wild type, Y537S, and D538G LBDs, by a direct binding assay.
All slopes had an r2 of 0.95 or better; shown is a representative experiment. For details, see Methods. LBD, ligand-binding domain.
Figure 3
Relative binding affinity assay of wild type, Y537S, and D538G ligand-binding domains (LBDs), showing the TOT competition curves.
With all proteins, the E2 curve is set to 100% and is shown only once. For details, see Methods.
Figure 4 with 12 supplements
Conformational stability of WT and mutant ERα LBD H11-12 loop and H12.
(A) Proteolytic susceptibility of the WT, Y537S, and D538G ERα LBD mutants in the apo, E2-bound, and TOT-bound states. (B–C) Deuterium uptake plot for the C-terminus of H11 along with the H11-12 …
Figure 4—figure supplement 1
Complete differential amide HDX MS map of WT ERα LBD binding to E2.
https://doi.org/10.7554/eLife.12792.010
Figure 4—figure supplement 2
Complete differential amide HDX MS map of Y537S ERα LBD mutant binding to E2.
https://doi.org/10.7554/eLife.12792.011
Figure 4—figure supplement 3
Complete differential amide HDX MS map of D538G ERα LBD mutant binding to E2.
https://doi.org/10.7554/eLife.12792.012
Figure 4—figure supplement 4
Complete differential HDX perturbation maps comparing the apo WT versus apo Y537S ERα LBD.
https://doi.org/10.7554/eLife.12792.013
Figure 4—figure supplement 5
Complete differential HDX perturbation maps comparing the apo WT versus apo D538G ERα LBD.
https://doi.org/10.7554/eLife.12792.014
Figure 4—figure supplement 6
Complete differential HDX perturbation map of WT ERα LBD with SRC3-NRD.
https://doi.org/10.7554/eLife.12792.015
Figure 4—figure supplement 7
Complete differential HDX perturbation map of Y537S ERα LBD with SRC3-NRD.
https://doi.org/10.7554/eLife.12792.016
Figure 4—figure supplement 8
Complete differential HDX perturbation map of D538G ERα LBD with SRC3-NRD.
https://doi.org/10.7554/eLife.12792.017
Figure 4—figure supplement 9
Complete differential HDX perturbation map of WT ERα LBD with E2 and SRC3-NRD.
https://doi.org/10.7554/eLife.12792.018
Figure 4—figure supplement 10
Complete differential HDX perturbation map of Y537S ERα LBD with E2 and SRC3-NRD.
https://doi.org/10.7554/eLife.12792.019
Figure 4—figure supplement 11
Complete differential HDX perturbation map of D538G ERα LBD with E2 and SRC3-NRD.
https://doi.org/10.7554/eLife.12792.020
Figure 4—figure supplement 12
apo Y537S x-ray crystal structure (Yellow) (PDB: 2B23) superimposed with WT-E2 complex structure (White) (PDB: 1GWR).
https://doi.org/10.7554/eLife.12792.021
Figure 5 with 3 supplements
Stabilized D538G agonist state.
Superposition stereo-view image of the residues comprising the H11-12 loop (531–537) of monomer A of the D538G-E2 (cyan) overlaid with monomer A of the WT-E2 structure (PDB: 1GWR). E2 is represented …
Figure 5—figure supplement 1
Simulated annealing composite omit maps for the E2 (A) and TOT (B)-bound D538G ERα LBD crystal structures contoured to 1.5σ.
E2 and TOT are shown as sticks, helix 12 is highlighted in red, and electron density is shown as a blue cage. LBD, ligand-binding domain.
Figure 5—figure supplement 2
Y537 orientations for D538G and WT LBD.
(A) Y537 of the D538G-E2 structure rotates toward solvent and is replaced by a well ordered water molecule (sphere), location of the ligand-binding site is shown with estradiol as green sticks, …
Figure 5—figure supplement 3
Density of an unidentified small molecule in the ligand-binding site of the apo D538G x-ray crystal structure.
https://doi.org/10.7554/eLife.12792.026
Figure 6
Visualization of H11-12 loop dynamics.
(A) H11-12 loop of WT ERα LBD-E2 complex. (B) Superimposing the position of the phenolic oxygen of Y537 at 0.1-ns intervals for apo WT (red), WT-E2 (blue), and apo D538G mutant (green). (C) Mapping …
Figure 7 with 6 supplements
Alterations to the D538G and Y537S antagonist conformational states.
(A) Superposition of monomer A for the 538G-TOT structure with the WT (3ERT). TOT and residues 530–550 of the WT (blue) (PDB: 3ERT), TOT of D538G (green), residues 531–550 (red). (B) Predicted …
Figure 7—figure supplement 1
Complete differential amide HDX-MS map of WT ERα LBD binding to TOT.
https://doi.org/10.7554/eLife.12792.029
Figure 7—figure supplement 2
Complete differential amide HDX-MS map of Y537S ERα LBD mutant binding to TOT.
https://doi.org/10.7554/eLife.12792.030
Figure 7—figure supplement 3
Complete differential amide HDX-MS map of D538G ERα LBD mutant binding to TOT.
https://doi.org/10.7554/eLife.12792.031
Figure 7—figure supplement 4
Experiment comparison view comparing the differential HDX behavior of apo WT ERα LBD in the presence of various ligands or coactivator.
https://doi.org/10.7554/eLife.12792.032
Figure 7—figure supplement 5
Experiment comparison view comparing the differential HDX behavior of apo Y537S ERα LBD in the presence of various ligands or coactivator.
https://doi.org/10.7554/eLife.12792.033
Figure 7—figure supplement 6
Experiment comparison view comparing the differential HDX behavior of apo D538G ERα LBD in the presence of various ligands or coactivator.
https://doi.org/10.7554/eLife.12792.034
Figure 8
Model of Aberrent ERα Mutant Activity.
Upon hormone binding (E2), WT ERα sheds heat-shock/chaperone proteins (HSP), forms head-to-head homodimers, and recruits coactivator (CoA) to become active. By contrast, Y537S or D538G ERα mutants …
Tables
Table 1
SRC3 NRD and ligand recruitment affinities for the WT and mutant ERα LBDs. LBD, ligand-binding domain.
https://doi.org/10.7554/eLife.12792.003| SRC-3 NRD Kd (nM) | |
| WT apo | No Recruitment |
| Y537S apo | 13.6 ± 2.0 |
| D538G apo | 151 ± 20 |
| WT-E2 | 2.67 ± 0.5 |
| Y537S-E2 | 0.59 ± 0.1 |
| D538G-E2 | 3.65 ± 0.40 |
| E2 EC50 (nM) | |
| WT | 13.8 ± 0.9 |
| Y537S | 1.6 ± 1.2 |
| D538G | 2.2 ± 0.1 |
| TOT Ki (nM) | |
| WT | 1.82 ± 0.30 |
| Y537S | 6.7 ± 0.40 |
| D538G | 0.79 ± 0.04 |
Table 2
Ligand-binding affinities.
https://doi.org/10.7554/eLife.12792.007| Kd (nM) | |
| WT-E2 | 0.26 ± 0.13 |
| Y537S-E2 | 1.43 ± 0.55 |
| D537G-E2 | 1.30 ± 0.63 |
| Ki (nM) | |
| WT-TOT | 0.337 ± 0.018 |
| Y537S-TOT | 2.61 ± 0.60 |
| D538G-TOT | 3.42 ± 0.50 |
Table 3
Crystallographic data collection and refinement statistics.
https://doi.org/10.7554/eLife.12792.022| ERα LBD D538G Apo | ERα LBD D538G-E2 | ERα LBD D538G-4OHT | |
|---|---|---|---|
| Data collection | |||
| Space group | P21 | P21 | P212121 |
| a, b, c (Å) | 56.14, 82.66, 59.11 | 56.08, 84.18, 58.37 | 104.65, 104.65, 191.38 |
| α, β, γ (°) | 90.00, 111.05, 90.00 | 90.00, 108.83, 90.00 | 90.00, 90.00, 90.00 |
| Resolution range | 55.17-2.24 Å | 55.25-1.90 | 50.00-3.07 |
| Number of reflections | |||
| (all/unique) | 91,607/24,107 | 169,519/40,361 | 60,232/9,874 |
| I/σ (highest resolution) | 2.37 | 2.36 | 1.70 |
| Rmerge | 11.4 | 7.3 | 11.4 |
| Completeness (%) | 98.9 | 99.3 | 96.7 |
| Redundancy | 3.8 | 4.2 | 6.1 |
| Refinement | |||
| Rwork/Rfree | 19.8/24.9 | 17.9/21.4 | 21.6/28.3 |
| No. Residues/chain | |||
| ERα LBD D538G | 241 | 242 | 216 |
| GRIP peptide | 6 | 6 | 0 |
| Water | 16 | 44 | 2 |
| Ligand | 0 | 1 | 1 |
| RMSD | |||
| Bond lengths (Å) | 0.015 | 0.0170 | 0.0128 |
| Bond angles (°) | 1.76 | 1.5441 | 1.5356 |
| Chiral volume | 0.1117 | 0.1267 | 0.1036 |
| Ramachandran plot statistics | |||
| Preferred number (%) | 428 (96.40%) | 443 (98.88%) | 1,563 (95.42%) |
| Additional allowed (%) | 3.60 (3.6%) | 5 (1.12% ) | 75 (4.58%) |
| Outliers (%) | 0 | 0 | 0 |
Table 4
Protonation states of histidines for the structure used in MD simulations.
https://doi.org/10.7554/eLife.12792.036| HIS residue number | Monomer A | Monomer B |
|---|---|---|
| 356 | HSE | HSD |
| 373 | HSD | HSE |
| 377 | HSE | HSD |
| 398 | HSP | HSP |
| 474 | HSE | HSE |
| 476 | HSE | HSE |
| 488 | HSE | HSE |
| 501 | HSD | HSE |
| 513 | HSD | HSD |
| 516 | HSE | HSE |
| 524 | HSE | HSE |
| 547 | HSE | HSE |
