Skip to main content
NCBI home page
Acta Crystallographica Section F: Structural Biology Communications logoLink to Acta Crystallographica Section F: Structural Biology Communications
. 2023 Mar 30;79(Pt 4):95–104. doi: 10.1107/S2053230X23002224

A partially open conformation of an androgen receptor ligand-binding domain with drug-resistance mutations

Selom K Doamekpor a, Panfeng Peng b, Ruo Xu b, Liandong Ma b, Youzhi Tong b, Liang Tong a,*
Editor: S Sheriffc
PMCID: PMC10071832  PMID: 36995121

A partially open conformation of the human androgen receptor ligand-binding domain containing four drug-resistance mutations, L702H/H875Y/F877L/T878A, was observed. Of these mutations, both L702H and F877L are important for this conformation, which may affect ligand binding as well as resistance to antagonists.

Keywords: androgen receptors, ligand-binding domain, DHT, drug resistance, prostate cancer, COVID-19, pruxelutamide, enzalutamide

Abstract

Mutations in the androgen receptor (AR) ligand-binding domain (LBD) can cause resistance to drugs used to treat prostate cancer. Commonly found mutations include L702H, W742C, H875Y, F877L and T878A, while the F877L mutation can convert second-generation antagonists such as enzalutamide and apalutamide into agonists. However, pruxelutamide, another second-generation AR antagonist, has no agonist activity with the F877L and F877L/T878A mutants and instead maintains its inhibitory activity against them. Here, it is shown that the quadruple mutation L702H/H875Y/F877L/T878A increases the soluble expression of AR LBD in complex with pruxelutamide in Escherichia coli. The crystal structure of the quadruple mutant in complex with the agonist dihydrotestosterone (DHT) reveals a partially open conformation of the AR LBD due to conformational changes in the loop connecting helices H11 and H12 (the H11–H12 loop) and Leu881. This partially open conformation creates a larger ligand-binding site for AR. Additional structural studies suggest that both the L702H and F877L mutations are important for conformational changes. This structural variability in the AR LBD could affect ligand binding as well as the resistance to antagonists.

1. Introduction

The androgen receptor (AR) is a nuclear receptor for the male hormone testosterone and its derivative dihydrotestosterone (DHT; Fig. 1 ▸ a). Recognition of these androgen agonists by the ligand-binding domain (LBD) of AR facilitates its nuclear translocation and dimerization and the activation of AR transcriptional targets. The LBD contains ∼250 residues and is located at the C-terminus of AR. It is connected to the DNA-binding domain (DBD; ∼100 residues) by a short hinge. The N-terminal transactivation domain of AR (∼550 residues) is mostly disordered.

Figure 1.

Figure 1

Open in a new tab

Drug-resistance mutations increase the soluble expression of AR LBD with antagonists. (a) Chemical structures of DHT, bicalutamide, enzalutamide, apalutamide and pruxelutamide. (b) The L702H/H875Y/F877L/T878A quadruple mutation increases the soluble expression of AR LBD in the presence of pruxelutamide (Prux) in bacteria. The W742L mutation increases the soluble expression in the presence of bicalutamide (Bic) but not pruxelutamide. The LBD is expressed as a SUMO fusion protein. Ins, insoluble fraction of cell lysate. Sol, soluble fraction after nickel affinity chromatography.

Among its many functions, AR is critical in the development of prostate cancer, and antagonists of AR have been developed over the years as successful prostate cancer therapies. The second-generation nonsteroidal antagonists enzalutamide (Tran et al., 2009 ▸) and apalutamide (Clegg et al., 2012 ▸) (Fig. 1 ▸ a) are approved treatments for prostate cancer, with improved efficacy compared with first-generation antagonists. Many other AR inhibitors, such as pruxelutamide (also known as proxalutamide and GT0918; Tong et al., 2014 ▸; Qu et al., 2020 ▸; Zhou et al., 2020 ▸; Fig. 1 ▸ a), are currently in various stages of clinical trials. However, resistance mutations have been identified for many of these antagonists, including L702H, W742C, H875Y, F877L and T878A (Watson et al., 2015 ▸). These mutations often give rise to AR activation in the presence of the antagonists, effectively turning them into full or partial agonists. Different combinations of these and other mutations have also been observed in cells (Ledet et al., 2020 ▸; Sumiyoshi et al., 2019 ▸). For example, the F877L/T878A double mutation causes even stronger agonist behavior of enzalut­amide than the single F877L mutation (Joseph et al., 2013 ▸; Korpal et al., 2013 ▸). On the other hand, pruxelutamide has a higher binding affinity for AR LBD than enzalutamide and is insensitive to the F877L and F877L/T878A mutations, maintaining its inhibitory activity against them (Zhou et al., 2020 ▸).

AR also promotes the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, and transmembrane protease, serine 2 (TMPRSS2), which is critical for the entry of SARS-CoV-2 into cells. Therefore, AR is an attractive target for COVID-19 treatment, and pruxelutamide has shown efficacy in clinical trials (Cadegiani et al., 2021 ▸; Hou et al., 2022 ▸).

A large number of crystal structures are currently available of wild-type and mutant AR LBD in complex with agonists or antagonists that exhibit agonist behavior with the mutants (Matias et al., 2000 ▸, 2002 ▸; Sack et al., 2001 ▸; Bohl, Gao et al., 2005 ▸; Pereira de Jésus-Tran et al., 2006 ▸; Wang et al., 2006 ▸; Bohl et al., 2007 ▸, 2008 ▸; Cantin et al., 2007 ▸; Duke et al., 2011 ▸; Saeed et al., 2016 ▸; Aikawa et al., 2017 ▸; Radaeva et al., 2022 ▸). The agonists are bound in a large cavity in the center of the LBD and are generally inaccessible to the solvent. The resistance mutations line the surface of this cavity or are located close to it. In comparison, no structural information on AR LBD in an inhibited conformation is currently available, and the molecular mechanism for AR inhibition is still not well understood.

In our attempts to produce soluble samples of AR LBD in complex with the antagonists pruxelutamide or enzalutamide by bacterial expression, we introduced four drug-resistance mutations into the LBD: L702H/H875Y/F877L/T878A. Unexpectedly, our crystal structure of this quadruple mutant in complex with the agonist DHT reveals a partially open conformation of the LBD. The loop connecting helices H11 and H12 (the H11–H12 loop) undergoes a large conformational change, which enlarges the ligand-binding cavity in the LBD and connects it to the bulk solvent. We examined various combinations of the four mutations and found that the L702H and F877L mutations are important for this partially open conformation.

2. Materials and methods

2.1. Protein expression and purification

Human AR LBD (residues 663–920) in pET-28a-SUMO was used to transform Escherichia coli BL21 (DE3) Rosetta cells (Table 1 ▸). AR LBD containing DHT was produced and purified as reported previously (Matias et al., 2000 ▸; Pereira de Jésus-Tran et al., 2006 ▸). His-SUMO-AR LBD production was induced with 0.05 mM isopropyl β-d-thio­galactopyranoside at 16°C for 18 h in the presence of 0.05 mM DHT. The cells were lysed in lysis buffer consisting of 20 mM Tris pH 7.5, 150 mM NaCl, 20 mM imidazole, 5 mM β-mercapto­ethanol (BME), 10%(v/v) glycerol, 0.5%(w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which was supplemented with 1 mM phenylmethanesulfonylfluoride (PMSF), 1 mg ml−1 chicken egg-white lysozyme, 2 U ml−1 DNase I, 10 mM MgCl2 and 0.05 mM DHT and treated with three freeze–thaw cycles. The lysate was incubated with 1 ml Ni–NTA Superflow resin (Qiagen) with nutation for 1 h, AR LBD was eluted with 5 ml lysis buffer supplemented with 250 mM imidazole and the His-SUMO tag was removed by incubation with Ulp1 overnight at 4°C. AR LBD was further purified by cation-exchange chromatography (HP SP, GE Healthcare) with elution in buffer consisting of 20 mM Tris pH 7.5, 50–1000 mM NaCl, 1 mM DTT, 10%(v/v) glycerol, 0.1%(w/v) n-octyl-β-glucoside (BOG), 0.01 mM DHT after dilution to low salt. The buffer was exchanged to 50 mM HEPES pH 7.5, 150 mM Li2SO4, 10 mM DTT, 10%(v/v) glycerol, 0.1%(w/v) BOG, 0.02 mM DHT and AR LBD was concentrated to 4 mg ml−1 before being frozen in liquid nitrogen.

Table 1. Macromolecule-production information.

Structure L702H/H875Y/F877L/T878A L702H/H875Y/F877L H875Y/F877L/T878A F877L/T878A L702H/H875Y
Source organism Homo sapiens
DNA source cDNA
Forward cloning primer GGCCTGGTGCCGCGCGGCAGCTCACACATTGAAGGCTATGAA
Forward mutagenesis primer Primer 1, CTATTGCGAGAGAGCTGTATCAGCTGGCGTTTGACCTGCTAATCAAG Primer 1, CCTATTGCGAGAGAGCTGTATCAGCTGACTTTTGACCTGCTAATC CTATTGCGAGAGAGCTGTATCAGCTGGCGTTTGACCTGCTAATCAAG TGCGAGAGAGCTGCATCAGCTGGCGTTTGACCTGCTAATCAAGTC Primer 1, CTATTGCGAGAGAGCTGTATCAGTTCACTTTTGAC
Primer 2, CTCCTTTGCAGCCTTGCATTCTAGCCTCAATGAAC Primer 2, CTCCTTTGCAGCCTTGCATTCTAGCCTCAATGAAC     Primer 2, CTCCTTTGCAGCCTTGCATTCTAGCCTCAATGAAC
Reverse cloning primer GTGGTGGTGGTGGTGCTCGAGTCAGGTGTGGAAATAGATGGGCTT
Reverse mutagenesis primer Primer 1, CTTGATTAGCAGGTCAAACGCCAGCTGATACAGCTCTCTCGCAATAG Primer 1, GATTAGCAGGTCAAAAGTCAGCTGATACAGCTCTCTCGCAATAGG CTTGATTAGCAGGTCAAACGCCAGCTGATACAGCTCTCTCGCAATAG GACTTGATTAGCAGGTCAAACGCCAGCTGATGCAGCTCTCTCGCA Primer 1, GTCAAAAGTGAACTGATACAGCTCTCTCGCAATAG
Primer 2, GTTCATTGAGGCTAGAATGCAAGGCTGCAAAGGAG Primer 2, GTTCATTGAGGCTAGAATGCAAGGCTGCAAAGGAG     Primer 2, GTTCATTGAGGCTAGAATGCAAGGCTGCAAAGGAG
Cloning vector pDNR-Lib
Expression vector pET-28a-SUMO
Expression host E. coli BL21 (DE3) Rosetta
Complete amino-acid sequence of the construct produced SHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALHSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELYQLAFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ SHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALHSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELYQLTFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ SHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELYQLAFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ SHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALLSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELHQLAFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ SHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALHSSLNELGERQLVHVVKWAKALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRMYSQCVRMRHLSQEFGWLQITPQEFLCMKALLLFSIIPVDGLKNQKFFDELRMNYIKELDRIIACKRKNPTSCSRRFYQLTKLLDSVQPIARELYQFTFDLLIKSHMVSVDFPEMMAEIISVQVPKILSGKVKPIYFHTQ
Open in a new tab

2.2. Protein crystallization

AR LBD crystals were obtained by the sitting-drop vapor-diffusion method (Bohl, Miller et al., 2005 ▸; Table 2 ▸). AR LBD crystals were flash-cooled in liquid nitrogen for diffraction analysis and data collection at 100 K.

Table 2. Crystallization.

Method† Vapor diffusion
Plate type Sitting drop
Temperature (K) 293
Protein concentration (mg ml−1) 4
Buffer composition of protein solution 50 mM HEPES pH 7.5, 150 mM Li2SO4, 10 mM DTT, 10%(v/v) glycerol, 0.1%(w/v) BOG, 0.02 mM DHT
Composition of reservoir solution 0.1 M HEPES pH 7.5, 500–800 mM sodium citrate, 20%(v/v) ethylene glycol
Volume and ratio of drop 0.75 µl protein solution:0.75 µl reservoir solution
Volume of reservoir (µl) 100
Open in a new tab
†

The crystallization method was the same for all five structures reported here.

2.3. Data collection and structure determination

X-ray diffraction data were collected and the diffraction images were processed and scaled using XDS (Kabsch, 2010 ▸; Table 3 ▸). The crystals, with one molecule in the asymmetric unit, were nearly isomorphous to those reported previously (Matias et al., 2000 ▸; Pereira de Jésus-Tran et al., 2006 ▸), with the c axis roughly 2 Å longer (71 versus 73 Å). The structures were determined by molecular replacement using an AR LBD structure (PDB entry 5vo4; Unwalla et al., 2017 ▸) as the search model, and structure refinement was performed using Phenix (Liebschner et al., 2019 ▸; Table 4 ▸). Manual rebuilding of the atomic model was carried out with Coot (Emsley et al., 2010 ▸). The Ramachandran plot statistics are from MolProbity (Chen et al., 2010 ▸).

Table 3. Data collection and processing.

Structure L702H/H875Y/F877L/T878A L702H/H875Y/F877L H875Y/F877L/T878A F877L/T878A L702H/H875Y
PDB code 8fgy 8fgz 8fh0 8fh1 8fh2
Diffraction source 24-ID-C, APS 24-ID-E, APS 24-ID-C, APS 24-ID-C, APS 24-ID-C, APS
Wavelength (Å) 0.97895 0.97918 0.97918 0.97918 0.97918
Temperature (K) 100 100 100 100 100
Detector Dectris EIGER2 16M Dectris EIGER2 16M Dectris EIGER2 16M Dectris EIGER2 16M Dectris EIGER2 16M
Crystal-to-detector distance (mm) 230 180 200 230 200
Rotation range per image (°) 0.2 0.2 0.2 0.2 0.2
Total rotation range (°) 140 200 200 200 250
Exposure time per image (s) 0.2 0.2 0.2 0.2 0.2
Space group P212121 P212121 P212121 P212121 P212121
a, b, c (Å) 56.41, 66.10, 73.38 56.74, 66.08, 73.07 56.52, 65.98, 73.14 56.35, 66.06, 73.17 56.88, 65.86, 72.65
α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90
Mosaicity (°) 0.25 0.14 0.11 0.14 0.11
Resolution range (Å) 50–2.19 (2.33–2.19) 50–1.61 (1.70–1.61) 50–1.59 (1.69–1.59) 50–1.69 (1.80–1.69) 50–1.59 (1.69–1.59)
Total No. of reflections 75325 266841 271984 224108 338075
No. of unique reflections 14426 36469 37224 30925 37114
Completeness (%) 98.8 (99.2) 99.7 (98.7) 99.8 (99.2) 99.5 (98.1) 99.9 (99.5)
Multiplicity 5.2 (5.5) 7.3 (6.9) 7.3 (7.4) 7.2 (7.6) 9.1 (9.2)
〈I/σ(I)〉 8.8 (2.6) 25.4 (1.9)† 22.8 (2.6) 20.9 (2.0) 19.5 (2.6)
R meas (%) 19.8 (88.5) 3.5 (103) 4.0 (71.6) 4.4 (105) 6.2 (104)
CC1/2 0.992 (0.786) 1.00 (0.785) 0.999 (0.909) 0.999 (0.865) 0.999 (0.872)
Overall B factor from Wilson plot (Å2) 26 29 28 31 27
Open in a new tab
†

Note: I/σ(I) = 3.7 at 1.70 Å resolution.

Table 4. Structure solution and refinement.

Structure L702H/H875Y/F877L/T878A L702H/H875Y/F877L H875Y/F877L/T878A F877L/T878A L702H/H875Y
PDB code 8fgy 8fgz 8fh0 8fh1 8fh2
Resolution range (Å) 49.11–2.20 (2.28–2.20) 49.01–1.61 (1.65–1.61) 48.99–1.59 (1.63–1.59) 49.03–1.69 (1.74–1.69) 48.79–1.59 (1.63–1.59)
Completeness (%) 99.6 99.7 99.8 99.5 99.9
σ Cutoff F > 1.35σ(F) F > 1.35σ(F) F > 1.35σ(F) F > 1.35σ(F) F > 1.35σ(F)
No. of reflections, working set 14408 (1291) 36412 (2372) 37154 (2426) 30878 (1965) 37056 (2447)
No. of reflections, test set 1426 (142) 2000 (138) 2000 (138) 1998 (136) 1999 (139)
Final R cryst 0.195 (0.2175) 0.197 (0.3304) 0.196 (0.3078) 0.199 (0.3507) 0.194 (0.2599)
Final R free 0.246 (0.2955) 0.220 (0.3581) 0.208 (0.2931) 0.229 (0.3420) 0.221 (0.3088)
Coordinate error (Å) 0.23 0.19 0.18 0.20 0.18
No. of non-H atoms
 Total 2084 2137 2137 2088 2176
 Protein 1967 1999 1973 1971 2023
 Ligand 51 51 51 51 51
 Ion 0 10 5 5 5
 Water 66 77 108 61 97
R.m.s. deviations
 Bond lengths (Å) 0.008 0.007 0.006 0.009 0.007
 Angles (°) 0.9 0.9 0.9 1.1 1.1
Average B factors (Å2)
 Overall 26 36 34 41 33
 Protein 26 36 33 41 33
 Ligand 21 33 28 36 29
 Ion — 67 49 62 55
 Water 32 43 43 45 41
Ramachandran plot
 Favored (%) 97.9 97.5 98.3 98.3 97.9
 Allowed (%) 2.1 2.1 1.7 1.3 2.1
 Outliers (%) 0.0 0.0 0.0 0.4 0.0
Open in a new tab

3. Results and discussion

3.1. Resistance mutations increase the soluble expression of AR LBD in complex with pruxelutamide

AR resistance mutations can convert antagonists into agonists. Structural studies of AR with antagonists have been hampered by the insolubility of AR LBD with antagonists, which is presumably due to the conformational changes that occur upon antagonist binding. Soluble expression of AR with antagonists can be achieved in some cases by co-expressing an antagonist with mutation(s) in the LBD that convert it to an agonist, such as W742L with bicalutamide (a first-generation antagonist; Fig. 1 ▸ a; Bohl, Gao et al., 2005 ▸), which we have also observed (Fig. 1 ▸ b). Therefore, we introduced mutations into the LBD and found that the quadruple mutation L702H/H875Y/F877L/T878A was able to produce some soluble expression in the presence of pruxelutamide or enzalutamide (Fig. 1 ▸ b). In comparison, wild-type LBD and the W742L mutation were not able to produce soluble expression with pruxelutamide. We succeeded in purifying small amounts of the quadruple mutant in complex with pruxelutamide or enzalutamide, but we were unable to obtain any crystals.

3.2. A partially open conformation of AR LBD with resistance mutations

To assess how the mutations affect the structure of AR LBD, we determined the crystal structure of the L702H/H875Y/F877L/T878A quadruple mutant in complex with the agonist DHT at 2.2 Å resolution (PDB entry 8fgy; Table 4 ▸). The crystals belonged to the same space group as previously reported for wild-type AR LBD in complex with DHT (PDB entries 1e3g and 2ama; Matias et al., 2000 ▸; Pereira de Jésus-Tran et al., 2006 ▸), with nearly the same unit-cell parameters and crystal packing as the previous crystals.

The overall structure of this quadruple mutant is similar to that of wild-type AR LBD, with 11 α-helices and two short two-stranded β-sheets (Figs. 2 ▸ a and 2 ▸ b). DHT is bound in a mostly hydrophobic cavity in the LBD that is inaccessible to the solvent in wild-type AR-LBD (Fig. 2 ▸ c). However, the root-mean-square (r.m.s.) distance between the structure of this mutant and wild-type AR LBD in complex with DHT (Pereira de Jésus-Tran et al., 2006 ▸; PDB entry 2ama) is 0.8 Å for 239 equivalent Cα atoms. This is due to the loop connecting helices H11 and H12 (the H11–H12 loop) assuming a different conformation in the mutant structure (Figs. 2 ▸ a and 2 ▸ b). This loop has clearly defined electron density in the mutant, indicating that it is well ordered. If the residues in this loop (884–892) are excluded from the overlay, the r.m.s. distance is 0.3 Å, indicating that the rest of the mutant structure, including the H11 and H12 helices themselves, is essentially the same as in wild-type AR LBD. Some difference is observed for residues 759–761 in the loop after H6, but these residues have weaker electron density.

Figure 2.

Figure 2

Open in a new tab

The human AR LBD L702H/H875Y/F877L/T878A quadruple mutant in complex with DHT is in a partially open conformation. (a) Overlay of the structure of the human AR LBD L702H/H875Y/F877L/T878A quadruple mutant (PDB entry 8fgy) in complex with DHT (cyan) with that of wild-type AR LBD in complex with DHT (gray; PDB entry 2ama; Pereira de Jésus-Tran et al., 2006 ▸). (b) Another view of the structural overlay, emphasizing the conformational changes of the H11–H12 loop. (c) Semi-transparent surface of the ligand-binding cavity in wild-type AR LBD. It is inaccessible to the solvent. (d) Surface of the ligand-binding cavity in the quadruple mutant. It is connected to the solvent (at the top in this view) due to the conformational changes. The H12 helix is behind the cavity in this view. The mutated residues are labeled in red. All structure figures were produced with PyMOL (http://www.pymol.org).

The quadruple mutant is in a partially open conformation, and the structural changes for residues 884–892 create a small channel from the solvent into the DHT binding site (Fig. 2 ▸ d), although the binding mode of DHT is not affected (Fig. 2 ▸ a). Residues 885–888 extend helix H11 by another turn at the C-terminus in the quadruple mutant structure, and the H11–H12 loop is shorter as a consequence (Fig. 2 ▸ b). In the wild-type AR LBD structure residues 884–888 form a one-turn helix at the end of H11, but the axis of this short helix is perpendicular to that of H11.

To understand how the mutations may have promoted the conformational changes, we examined each of the mutation sites in detail. To the best of our knowledge, the structure of the quadruple mutant represents the first experimentally determined structure of AR-LBD containing the F877L mutation. This mutation (in helix H11) reduces the size of the side chain, and the Leu881 side chain in the next turn of the helix assumes a different rotamer to maintain contact with the new F877L side chain in this structure (Fig. 3 ▸ a). The Leu881 side chain interacts with Val888 and Val890 in the H11–H12 loop in the wild-type AR LBD structure. These two side chains move by 5–6 Å as part of the conformational changes in the mutant and can no longer interact with Leu881. The Cδ2 atom of the F877L side chain points away from the Phe877 side chain in the wild type and induces a conformational change in the side chain of Met781 (Fig. 3 ▸ b), which is also near the DHT.

Figure 3.

Figure 3

Open in a new tab

Conformational changes in the human AR LBD L702H/H875Y/F877L/T878A quadruple mutant in complex with DHT. (a) Overlay of the structure of the human AR LBD L702H/H875Y/F877L/T878A quadruple mutant in complex with DHT (PDB entry 8fgy; cyan) with that of wild-type AR LBD in complex with DHT (gray; PDB entry 2ama; Pereira de Jésus-Tran et al., 2006 ▸) near the DHT binding site and the H11–H12 loop. (b) Another view of the overlay, showing the interactions at the H875Y mutation site in more detail. (c) Overlay of the structure of human wild-type AR LBD in complex with DHT (gray; PDB entry 2ama) with that of the T878A mutant in complex with cyproterone acetate (CPA; yellow; PDB entry 2oz7; Bohl et al., 2007 ▸). (d) Overlay of the structure of the human AR LBD L702H/H875Y/F877L/T878A quadruple mutant in complex with DHT (PDB entry 8fgy; cyan) with that of the T878A mutant in complex with cyproterone acetate (CPA; yellow).

The L702H mutation (in helix H3) forms a hydrogen bond to the main-chain carbonyl of Ser779, and the side chain of L702H also makes van der Waals contacts with that of Leu881 in its new position (Fig. 3 ▸ a). In addition, the L702H side chain introduces a polar moiety into the mostly hydrophobic ligand-binding pocket. The Leu702 side chain in wild-type AR LBD assumes a rotamer that is rarely observed, suggesting that its conformation is somewhat strained by the local environment. In comparison, the L702H side chain assumes a common rotamer.

The bulkier Tyr side chain of the H875Y mutation (in helix H11) fills a cavity in the structure of AR LBD, and the hydroxyl group replaces a water molecule in the structure of wild-type AR LBD and is hydrogen-bonded to the main-chain carbonyl of Tyr740 (Askew et al., 2007 ▸), which is one of two exposed main-chain carbonyls in the kink between helices H5 and H6 (Fig. 3 ▸ b). This mutation is therefore likely to provide extra stabilization of AR LBD. The T878A mutation (in helix H11) loses a hydrogen bond to the hydroxyl group on C17 of DHT (Fig. 3 ▸ a), but does not cause any structural changes in this region (Fig. 3 ▸ b). The smaller T878A mutant side chain increases the size of the ligand-binding pocket.

Conformational changes for the H11–H12 loop region were observed in the crystal structure of the T878A mutant in complex with the antagonist cyproterone acetate (CPA; PDB entry 2oz7; Bohl et al., 2007 ▸). This compound has bulkier substitutions near the C17 position of DHT (Fig. 3 ▸ c). In particular, the acetate group clashes with the position of Leu702 in the wild-type AR-LBD structure, which undergoes a conformational change that in turn clashes with Val890 in the H11–H12 loop. Therefore, the conformational changes of this loop are necessary for the binding of this bulkier antagonist. The conformation of the H11–H12 loop in the CPA complex has some resemblance to that in the quadruple mutant (Fig. 3 ▸ d), although the cavity remains inaccessible to the solvent and the loop has weak electron density for residues 887–892 and is mostly disordered. Nonetheless, these observations provide evidence that conformational variability of the H11–H12 loop contributes to ligand binding to AR LBD.

Conformational changes of the H11–H12 loop region have also been observed in molecular-dynamics simulations with second-generation antagonists (Balbas et al., 2013 ▸; Gim et al., 2021 ▸; Kocak & Yildiz, 2022 ▸). In the docked poses, part of enzalutamide can be positioned near the Phe877 side chain and the H11–H12 loop region, suggesting that the conformation of this region could affect ligand binding. On the other hand, the H11–H12 loop region is distant from the dimerization interface of AR LBD (Nadal et al., 2017 ▸; Wasmuth et al., 2022 ▸), the binding site of coactivator motifs (activation function 2, AF2 site; He et al., 2004 ▸; Hur et al., 2004 ▸; Askew et al., 2007 ▸; Estébanez-Perpiñá et al., 2005 ▸; Zhou et al., 2010 ▸; Axerio-Cilies et al., 2011 ▸; Nique et al., 2012 ▸; Hsu et al., 2014 ▸) and the allosteric regulatory site (binding function 3, BF3 site) for binding of this coactivator (Estébanez-Perpiñá et al., 2007 ▸; Lack et al., 2011 ▸).

3.3. The L702H and F877L mutations are important for conformational changes

To assess which residues are important for the conformational changes observed in the quadruple mutant, we determined the structures of various combinations of these mutations in complex with DHT (Tables 4 ▸ and 5 ▸). For the triple mutants, we found that the L702H/H875Y/F877L mutant (PDB entry 8fgz) is also in a partially open conformation (r.m.s. distance of 0.2 Å to the quadruple mutant), while the H875Y/F877L/T878A mutant (PDB entry 8fh0) is in a closed conformation (r.m.s. distance of 0.3 Å to wild-type AR LBD) (Fig. 4 ▸ a). These data suggest that the T878A mutation is not required, while the L702H mutation plays a critical role in the partially open conformation.

Table 5. Summary of AR LBD mutant structures with DHT.

Mutation PDB code Solubility Structure H11–H12 loop Leu881
Wild type   Yes Yes Closed Rotamer 2
L702H/H875Y/F877L/T878A 8fgy Yes Yes Open Rotamer 1
L702H/H875Y/F877L 8fgz Yes Yes Open Rotamer 1
H875Y/F877L/T878A 8fh0 Yes Yes Closed Rotamer 2
L702H/H875Y 8fh2 Yes Yes Closed Rotamer 2
L702H/F877L   Low No — —
F877L/T878A 8fh1 Yes Yes Closed Rotamer 2
L702H   Low No — —
Open in a new tab

Figure 4.

Figure 4

Open in a new tab

Closed conformation of the H875Y/F877L/T878A and F877L/T878A mutants. (a) Overlay of the structures of the human AR LBD H875Y/F877L/T878A triple mutant in complex with DHT (PDB entry 8fh0; orange) and the AR LBD F877L/T878A double mutant in complex with DHT (PDB entry 8fh1; pink) with that of wild-type AR LBD in complex with DHT (gray; PDB entry 2ama; Pereira de Jésus-Tran et al., 2006 ▸). (b) Semi-transparent surface of the ligand-binding cavity in the F877L/T878A double mutant.

For the double mutants, we found that the L702H/H875Y mutant (PDB entry 8fh2) is in the closed conformation (r.m.s. distance of 0.3 Å to wild-type AR LBD). A previous structure of the L702H/T878A mutant (PDB entry 1gs4; Matias et al., 2002 ▸) was also in the closed conformation (r.m.s. distance of 0.3 Å to wild-type AR LBD). These data suggest that the L702H mutation itself is not sufficient to induce the partially open conformation.

We found that the F877L/T878A double mutant (PDB entry 8fh1) is also in the closed conformation (r.m.s. distance of 0.3 Å to wild-type AR LBD; Fig. 4 ▸ a), suggesting that the F877L mutation alone is not sufficient to induce the partially open conformation. Despite the smaller size of the side chain, Leu881 does not change its conformation to maintain contact with F877L, but instead maintains contacts with Val888 and Val890. This creates a small cavity between the F877L and Leu881 side chains, expanding the size of the ligand-binding cavity in this double mutant (Fig. 4 ▸ b) as well as in the H875Y/F877L/T878A triple mutant. The F877L mutation is sufficient to convert second-generation antagonists such as enzalut­amide and apalutamide into agonists (Joseph et al., 2013 ▸; Korpal et al., 2013 ▸). Consistent with our observations on Leu881, an L881Q mutation has been observed in prostate cancer (Lallous et al., 2021 ▸).

The observations with the triple and double mutants suggest that both L702H and F877L are important for the partially open conformation of AR LBD. We attempted to determine the crystal structure of this double mutant, but found that the protein was expressed with very low solubility (Table 5 ▸) even in the presence of DHT in the medium. We also tried to determine the structure of the L702H single mutant and also found low solubility for this mutant. This is consistent with the lower level of DHT binding observed previously for this mutant (Matias et al., 2002 ▸). However, combining L702H with H875Y rescues the expression of the mutant, similar to the combination of L702H with T878A (Matias et al., 2002 ▸).

4. Conclusions

Overall, our structures demonstrate how different combinations of drug-resistance mutations can affect the conformation of AR LBD. The F877L mutation, possibly combined with a T878A and/or an H875Y mutation, shows that extra space is created in the ligand-binding site. The F877L mutation combined with an L702H mutation generates a conformational change of the H11–H12 loop, together with a change in the side chain of Leu881. These conformational changes create a solvent-accessible channel to the ligand-binding site. While they are observed in the structures of AR LBD bound to the agonist DHT, they could also have implications for the binding of antagonists to AR LBD.

Supplementary Material

PDB reference: human androgen receptor ligand-binding domain, L702H/H875Y/F877L/T878A mutations, 8fgy

PDB reference: L702H/H875Y/F877L mutations, 8fgz

PDB reference: H875Y/F877L/T878A mutations, 8fh0

PDB reference: F877L/T878A mutations, 8fh1

PDB reference: L702H/H875Y mutations, 8fh2

Acknowledgments

We thank K. Perry, S. Banerjee, D. Neau, I. Kourinov, N. Sukumar and C. Salbego for access to the NE-CAT 24-ID-C and 24-ID-E beamlines at the Advanced Photon Source (APS). LT is an independent non-executive director at Kintor. This work used Northeastern Collaborative Access Team beamlines (GM103403) and a PILATUS detector (RR029205) at the APS (DE-AC02-06CH11357).

Funding Statement

This work was supported by a grant from Kintor Pharmaceuticals (to LT).

References

  1. Aikawa, K., Asano, M., Ono, K., Habuka, N., Yano, J., Wilson, K., Fujita, H., Kandori, H., Hara, T., Morimoto, M., Santou, T., Yamaoka, M., Nakayama, M. & Hasuoka, A. (2017). Bioorg. Med. Chem. 25, 3330–3349. [DOI] [PubMed]
  2. Askew, E. B., Gampe, R. T. Jr, Stanley, T. B., Faggart, J. L. & Wilson, E. M. (2007). J. Biol. Chem. 282, 25801–25816. [DOI] [PMC free article] [PubMed]
  3. Axerio-Cilies, P., Lack, N. A., Nayana, M. R., Chan, K. H., Yeung, A., Leblanc, E., Guns, E. S., Rennie, P. S. & Cherkasov, A. (2011). J. Med. Chem. 54, 6197–6205. [DOI] [PubMed]
  4. Balbas, M. D., Evans, M. J., Hosfield, D. J., Wongvipat, J., Arora, V. K., Watson, P. A., Chen, Y., Greene, G. L., Shen, Y. & Sawyers, C. L. (2013). eLife, 2, e00499. [DOI] [PMC free article] [PubMed]
  5. Bohl, C. E., Gao, W., Miller, D. D., Bell, C. E. & Dalton, J. T. (2005). Proc. Natl Acad. Sci. USA, 102, 6201–6206. [DOI] [PMC free article] [PubMed]
  6. Bohl, C. E., Miller, D. D., Chen, J., Bell, C. E. & Dalton, J. T. (2005). J. Biol. Chem. 280, 37747–37754. [DOI] [PMC free article] [PubMed]
  7. Bohl, C. E., Wu, Z., Chen, J., Mohler, M. L., Yang, J., Hwang, D. J., Mustafa, S., Miller, D. D., Bell, C. E. & Dalton, J. T. (2008). Bioorg. Med. Chem. Lett. 18, 5567–5570. [DOI] [PMC free article] [PubMed]
  8. Bohl, C. E., Wu, Z., Miller, D. D., Bell, C. E. & Dalton, J. T. (2007). J. Biol. Chem. 282, 13648–13655. [DOI] [PMC free article] [PubMed]
  9. Cadegiani, F. A., Zimerman, R. A., Fonseca, D. N., Correia, M. N., Muller, M. P., Bet, D. L., Slaviero, M. R., Zardo, I., Benites, P. R., Barros, R. N., Paulain, R. W., Onety, D. C., Israel, K. C. P., Gustavo Wambier, C. & Goren, A. (2021). Cureus, 13, e20691. [DOI] [PMC free article] [PubMed]
  10. Cantin, L., Faucher, F., Couture, J.-F., Pereira de Jésus-Tran, K., Legrand, P., Ciobanu, L. C., Fréchette, Y., Labrecque, R., Singh, S. M., Labrie, F. & Breton, R. (2007). J. Biol. Chem. 282, 30910–30919. [DOI] [PubMed]
  11. Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12–21. [DOI] [PMC free article] [PubMed]
  12. Clegg, N. J., Wongvipat, J., Joseph, J. D., Tran, C., Ouk, S., Dilhas, A., Chen, Y., Grillot, K., Bischoff, E. D., Cai, L., Aparicio, A., Dorow, S., Arora, V., Shao, G., Qian, J., Zhao, H., Yang, G., Cao, C., Sensintaffar, J., Wasielewska, T., Herbert, M. R., Bonnefous, C., Darimont, B., Scher, H. I., Smith-Jones, P., Klang, M., Smith, N. D., De Stanchina, E., Wu, N., Ouerfelli, O., Rix, P. J., Heyman, R. A., Jung, M. E., Sawyers, C. L. & Hager, J. H. (2012). Cancer Res. 72, 1494–1503. [DOI] [PMC free article] [PubMed]
  13. Duke, C. B., Jones, A., Bohl, C. E., Dalton, J. T. & Miller, D. D. (2011). J. Med. Chem. 54, 3973–3976. [DOI] [PubMed]
  14. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501. [DOI] [PMC free article] [PubMed]
  15. Estébanez-Perpiñá, E., Arnold, L. A., Nguyen, P., Rodrigues, E. D., Mar, E., Bateman, R., Pallai, P., Shokat, K. M., Baxter, J. D., Guy, R. K., Webb, P. & Fletterick, R. J. (2007). Proc. Natl Acad. Sci. USA, 104, 16074–16079. [DOI] [PMC free article] [PubMed]
  16. Estébanez-Perpiñá, E., Moore, J. M., Mar, E., Delgado-Rodrigues, E., Nguyen, P., Baxter, J. D., Buehrer, B. M., Webb, P., Fletterick, R. J. & Guy, R. K. (2005). J. Biol. Chem. 280, 8060–8068. [DOI] [PubMed]
  17. Gim, H. J., Park, J., Jung, M. E. & Houk, K. N. (2021). Sci. Rep. 11, 15887. [DOI] [PMC free article] [PubMed]
  18. He, B., Gampe, R. T. Jr, Kole, A. J., Hnat, A. T., Stanley, T. B., An, G., Stewart, E. L., Kalman, R. I., Minges, J. T. & Wilson, E. M. (2004). Mol. Cell, 16, 425–438. [DOI] [PubMed]
  19. Hou, X., Yan, H., Wang, A., Liu, C., Zhou, Q., Ma, L., Chen, J., Ren, Z. & Tong, Y. (2022). bioRxiv, 2022.06.29.498191.
  20. Hsu, C.-L., Liu, J.-S., Wu, P.-L., Guan, H.-H., Chen, Y.-L., Lin, A.-C., Ting, H.-J., Pang, S.-T., Yeh, S.-D., Ma, W.-L., Chen, C.-J., Wu, W.-G. & Chang, C. (2014). Mol. Oncol. 8, 1575–1587. [DOI] [PMC free article] [PubMed]
  21. Hur, E., Pfaff, S. J., Payne, E. S., Grøn, H., Buehrer, B. M. & Fletterick, R. J. (2004). PLoS Biol. 2, e274. [DOI] [PMC free article] [PubMed]
  22. Joseph, J. D., Lu, N., Qian, J., Sensintaffar, J., Shao, G., Brigham, D., Moon, M., Maneval, E. C., Chen, I., Darimont, B. & Hager, J. H. (2013). Cancer Discov. 3, 1020–1029. [DOI] [PubMed]
  23. Kabsch, W. (2010). Acta Cryst. D66, 133–144. [DOI] [PMC free article] [PubMed]
  24. Kocak, A. & Yildiz, M. (2022). J. Mol. Graph. Model. 111, 108081. [DOI] [PubMed]
  25. Korpal, M., Korn, J. M., Gao, X., Rakiec, D. P., Ruddy, D. A., Doshi, S., Yuan, J., Kovats, S. G., Kim, S., Cooke, V. G., Monahan, J. E., Stegmeier, F., Roberts, T. M., Sellers, W. R., Zhou, W. & Zhu, P. (2013). Cancer Discov. 3, 1030–1043. [DOI] [PubMed]
  26. Lack, N. A., Axerio-Cilies, P., Tavassoli, P., Han, F. Q., Chan, K. H., Feau, C., LeBlanc, E., Guns, E. T., Guy, R. K., Rennie, P. S. & Cherkasov, A. (2011). J. Med. Chem. 54, 8563–8573. [DOI] [PMC free article] [PubMed]
  27. Lallous, N., Snow, O., Sanchez, C., Parra Nuñez, A. K., Sun, B., Hussain, A., Lee, J., Morin, H., Leblanc, E., Gleave, M. E. & Cherkasov, A. (2021). Cancers, 13, 2939. [DOI] [PMC free article] [PubMed]
  28. Ledet, E. M., Lilly, M. B., Sonpavde, G., Lin, E., Nussenzveig, R. H., Barata, P. C., Yandell, M., Nagy, R. J., Kiedrowski, L., Agarwal, N. & Sartor, O. (2020). Oncologist, 25, 327–333. [DOI] [PMC free article] [PubMed]
  29. Liebschner, D., Afonine, P. V., Baker, M. L., Bunkóczi, G., Chen, V. B., Croll, T. I., Hintze, B., Hung, L.-W., Jain, S., McCoy, A. J., Moriarty, N. W., Oeffner, R. D., Poon, B. K., Prisant, M. G., Read, R. J., Richardson, J. S., Richardson, D. C., Sammito, M. D., Sobolev, O. V., Stockwell, D. H., Terwilliger, T. C., Urzhumtsev, A. G., Videau, L. L., Williams, C. J. & Adams, P. D. (2019). Acta Cryst. D75, 861–877.
  30. Matias, P. M., Carrondo, M. A., Coelho, R., Thomaz, M., Zhao, X.-Y., Wegg, A., Crusius, K., Egner, U. & Donner, P. (2002). J. Med. Chem. 45, 1439–1446. [DOI] [PubMed]
  31. Matias, P. M., Donner, P., Coelho, R., Thomaz, M., Peixoto, C., Macedo, S., Otto, N., Joschko, S., Scholz, P., Wegg, A., Bäsler, S., Schäfer, M., Egner, U. & Carrondo, M. A. (2000). J. Biol. Chem. 275, 26164–26171. [DOI] [PubMed]
  32. Nadal, M., Prekovic, S., Gallastegui, N., Helsen, C., Abella, M., Zielinska, K., Gay, M., Vilaseca, M., Taulès, M., Houtsmuller, A. B., van Royen, M. E., Claessens, F., Fuentes-Prior, P. & Estébanez-Perpiñá, E. (2017). Nat. Commun. 8, 14388. [DOI] [PMC free article] [PubMed]
  33. Nique, F., Hebbe, S., Peixoto, C., Annoot, D., Lefrançois, J.-M., Duval, E., Michoux, L., Triballeau, N., Lemoullec, J.-M., Mollat, P., Thauvin, M., Prangé, T., Minet, D., Clément-Lacroix, P., Robin-Jagerschmidt, C., Fleury, D., Guédin, D. & Deprez, P. (2012). J. Med. Chem. 55, 8225–8235. [DOI] [PubMed]
  34. Pereira de Jésus-Tran, K., Côté, P. L., Cantin, L., Blanchet, J., Labrie, F. & Breton, R. (2006). Protein Sci. 15, 987–999. [DOI] [PMC free article] [PubMed]
  35. Qu, F., Gu, Y., Wang, Q., He, M., Zhou, F., Sun, J., Wang, G. & Peng, Y. (2020). Invest. New Drugs, 38, 1292–1302. [DOI] [PubMed]
  36. Radaeva, M., Li, H., LeBlanc, E., Dalal, K., Ban, F., Ciesielski, F., Chow, B., Morin, H., Awrey, S., Singh, K., Rennie, P. S., Lallous, N. & Cherkasov, A. (2022). Cells, 11, 2785. [DOI] [PMC free article] [PubMed]
  37. Sack, J. S., Kish, K. F., Wang, C., Attar, R. M., Kiefer, S. E., An, Y., Wu, G. Y., Scheffler, J. E., Salvati, M. E., Krystek, S. R. Jr, Weinmann, R. & Einspahr, H. M. (2001). Proc. Natl Acad. Sci. USA, 98, 4904–4909. [DOI] [PMC free article] [PubMed]
  38. Saeed, A., Vaught, G. M., Gavardinas, K., Matthews, D., Green, J. E., Losada, P. G., Bullock, H. A., Calvert, N. A., Patel, N. J., Sweetana, S. A., Krishnan, V., Henck, J. W., Luz, J. G., Wang, Y. & Jadhav, P. (2016). J. Med. Chem. 59, 750–755. [DOI] [PubMed]
  39. Sumiyoshi, T., Mizuno, K., Yamasaki, T., Miyazaki, Y., Makino, Y., Okasho, K., Li, X., Utsunomiya, N., Goto, T., Kobayashi, T., Terada, N., Inoue, T., Kamba, T., Fujimoto, A., Ogawa, O. & Akamatsu, S. (2019). Sci. Rep. 9, 4030. [DOI] [PMC free article] [PubMed]
  40. Tong, Y., Chen, C. D., Wu, J., Yang, J., Zhang, H., Wu, X., Duan, Y., Gao, W., Qian, W., Niu, X., Mi, L. & Guo, C. (2014). Cancer Res. 74, 614.
  41. Tran, C., Ouk, S., Clegg, N. J., Chen, Y., Watson, P. A., Arora, V., Wongvipat, J., Smith-Jones, P. M., Yoo, D., Kwon, A., Wasielewska, T., Welsbie, D., Chen, C. D., Higano, C. S., Beer, T. M., Hung, D. T., Scher, H. I., Jung, M. E. & Sawyers, C. L. (2009). Science, 324, 787–790. [DOI] [PMC free article] [PubMed]
  42. Unwalla, R., Mousseau, J. J., Fadeyi, O. O., Choi, C., Parris, K., Hu, B., Kenney, T., Chippari, S., McNally, C., Vishwanathan, K., Kilbourne, E., Thompson, C., Nagpal, S., Wrobel, J., Yudt, M., Morris, C. A., Powell, D., Gilbert, A. M. & Chekler, E. L. P. (2017). J. Med. Chem. 60, 6451–6457. [DOI] [PubMed]
  43. Wang, F., Liu, X., Li, H., Liang, K., Miner, J. N., Hong, M., Kallel, E. A., van Oeveren, A., Zhi, L. & Jiang, T. (2006). Acta Cryst. F62, 1067–1071. [DOI] [PMC free article] [PubMed]
  44. Wasmuth, E. V., Broeck, A. V., LaClair, J. R., Hoover, E. A., Lawrence, K. E., Paknejad, N., Pappas, K., Matthies, D., Wang, B., Feng, W., Watson, P. A., Zinder, J. C., Karthaus, W. R., de la Cruz, M. J., Hite, R. K., Manova-Todorova, K., Yu, Z., Weintraub, S. T., Klinge, S. & Sawyers, C. L. (2022). Mol. Cell, 82, 2021–2031. [DOI] [PMC free article] [PubMed]
  45. Watson, P. A., Arora, V. K. & Sawyers, C. L. (2015). Nat. Rev. Cancer, 15, 701–711. [DOI] [PMC free article] [PubMed]
  46. Zhou, T., Xu, W., Zhang, W., Sun, Y., Yan, H., Gao, X., Wang, F., Zhou, Q., Hou, J., Ren, S., Yang, Q., Yang, B., Xu, C., Zhou, Q., Wang, M., Chen, C. & Sun, Y. (2020). Eur. J. Cancer, 134, 29–40. [DOI] [PubMed]
  47. Zhou, X. E., Suino-Powell, K. M., Li, J., He, Y., Mackeigan, J. P., Melcher, K., Yong, E.-L. & Xu, H. E. (2010). J. Biol. Chem. 285, 9161–9171. [DOI] [PMC free article] [PubMed]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

PDB reference: human androgen receptor ligand-binding domain, L702H/H875Y/F877L/T878A mutations, 8fgy

PDB reference: L702H/H875Y/F877L mutations, 8fgz

PDB reference: H875Y/F877L/T878A mutations, 8fh0

PDB reference: F877L/T878A mutations, 8fh1

PDB reference: L702H/H875Y mutations, 8fh2

Articles from Acta Crystallographica. Section F, Structural Biology Communications are provided here courtesy of International Union of Crystallography

RESOURCES