evidence for dbd-lbd communications in the androgen receptor
TRANSCRIPT
1
Evidence for DBD-LBD communications in the androgen receptor 1
2
Christine Helsen1, Vanessa Dubois1, Annelien Verfaillie1, Jacques Young2, Mieke 3
Trekels3, Renée Vancraenenbroeck3, Marc De Maeyer3 and Frank Claessens1# 4
5
Laboratory of Molecular Endocrinology, Department of Cellular and Molecular Medicine, 6
KU Leuven, Herestraat 49, 3000 Leuven, Belgium1 7
Hôpital de Bicêtre, Service d’Endocrinologie et Maladies de la Reproduction, Rue du 8
Général Leclerc 78, 94275 Le Kremlin Bicêtre, France2 9
Division of Biochemistry, Molecular and Structural Biology, Department of Chemistry, KU 10
Leuven Celestijnenlaan 200g, 3001 Heverlee, Belgium3 11
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Running title: Communication between DBD and LBD of the AR 13
14
Corresponding author: Frank Claessens, PhD, Katholieke Universiteit Leuven, 15
Department of Cellular and Molecular Medicine, Molecular Endocrinology Laboratory, 16
Herestraat 49, B-3000 Leuven, Belgium. Tel.: +3216330253; Fax: +32163430735; 17
19
Word count : Abstract: 189 words, Materials and methods: 885 words; Introduction, 20
Results and Discussion: 3004 words, Conclusion: 127 words, Figure legends: 677 21
words. 22
Abbreviations: AIS, androgen insensitivity syndrome; PrCa, prostate cancer. 23
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.00151-12 MCB Accepts, published online ahead of print on 29 May 2012
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ABSTRACT 24
DNA binding as well as ligand binding by nuclear receptors has been studied 25
extensively. Both binding functions are attributed to isolated domains of which the 26
structure is known. The crystal structure of a complete receptor in complex with its 27
ligand and DNA-response element, however, has only been solved for the PPARγ-RXRα 28
heterodimer. This structure provided the first indication of direct interactions between 29
DNA- and ligand-binding domains. In this study, we investigated whether there is a 30
similar interface between the DNA- and ligand-binding domains for the androgen 31
receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a 32
combination of in silico modeling and docking pointed out a putative interface between 33
AR-DBD and LBD. The surfaces were subjected to a point mutation analysis, which was 34
inspired by known AR mutations described in androgen insensitivity syndromes and 35
prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S and P766A 36
induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-37
residing mutations K590A, K592A and E621A lowered the ligand binding affinity but not 38
DNA binding. We therefore propose that these residues are involved in allosteric 39
communications between the AR-DBD and LBD. 40
41
42
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INTRODUCTION 43
Nuclear receptors (NR) are involved in many physiological processes, diseases and 44
therapeutic applications. They are transcription factors that contain a DNA-binding 45
domain (DBD) composed of 2 zinc-fingers (39) and a ligand-binding domain (LBD) 46
formed by twelve α-helices (60). The structures of the separate DNA-binding and ligand-47
binding domain of many receptors have already revealed a large amount of information. 48
Especially the structure of the LBD has led to a more focused search for new agonists 49
and antagonists for many therapeutic indications where NRs are involved. The exact 50
structure of full-size NRs bound to DNA and ligand will help us in understanding the 51
basic mechanism of nuclear receptor signaling but will also provide new targets for 52
therapeutic strategies. The coordinates of the PPARγ-RXRα co-crystal bound as a 53
heterodimer to the DNA has been reported (11) and revealed a more intimate contact 54
between PPARγ and RXRα than expected. The existence of the previously unknown 55
interface between PPARγ-LBD and RXRα-DBD was corroborated with a mutation 56
analysis. Since this has not yet been confirmed by other techniques like SAXS and 57
SANS (49), it is debated whether such a communication exists. There is, however, 58
strong evidence for allosteric communications e.g. between the DBD and LBD in nuclear 59
receptors. A most remarkable observation was made for the glucocorticoid receptor 60
(GR) when slightly different response elements were tested in gene reporter assays: 61
small changes in the DNA sequence had an important impact on receptor activity (44, 62
58). FRAP-experiments with GFP-tagged NRs show that binding of a ligand affects the 63
receptor-mobility in the nucleus, indicating an influence of the LBD on DNA binding (24, 64
43). By means of Isothermal Titration Calorimetry, the thyroid receptor was 65
demonstrated to display DNA-dependent DBD-LBD interactions that were bi-directional. 66
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TRE-binding by the DBD appears to decrease its affinity for the LBD and thereby 67
facilitates recruitment of SRC-1 to the LBD (47). Moreover for the VDR-RXR 68
heterodimer, HDX studies provided structural evidence for DNA-dependent allosteric 69
communication between DBD and LBD of one receptor and also between the binding 70
partners (62). Also for the AR, it was demonstrated that some receptor mutations 71
outside the DBD affect the activity of the receptor in a DNA sequence-dependent way (8, 72
9). 73
The androgen receptor (AR) is a NR involved in the early development of the male 74
sexual organs, in the establishment and maintenance of the secondary male 75
characteristics and fertility in puberty and in adult live. The AR is activated by 76
testosterone and dihydrotestosterone (DHT) and regulates the expression of androgen-77
responsive genes by binding to a nearby regulatory sequence in the DNA, also known 78
as the androgen-response element (ARE). In addition, the AR is an important survival 79
factor in primary as well as castration-resistant forms of prostate cancer (PrCa) (2). 80
Despite considerable differences in primary amino acid sequences, the structure of the 81
AR-DBD and LBD closely resemble that of other NRs. The DBD is the most conserved 82
NR domain and consists of 2 α-helices that are coordinated by zinc molecules thus 83
forming two zinc-finger modules. The first zinc-finger is responsible for the recognition of 84
AREs, while the second zinc-finger is involved in DNA-dependent dimerization (31, 39, 85
51). 86
The LBD is the second most conserved domain and consists of 11 α-helices that form a 87
ligand-binding pocket which is delineated by helix 3, 4, 5, 7, 11 and 12 and the β-sheet 88
preceding helix 6 (41). The carboxy-terminal helix, named helix 12, functions as an 89
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intramolecular switch (52). Binding of an agonist results in the repositioning of helix 12 90
and creates a hydrophobic groove formed by helix 3, 4, 5 and 12. This site serves as a 91
binding site for LxxLL-containing coactivators as well as for the amino-terminal domain 92
of the AR (30, 38, 56). 93
94
Regarding the AR, dimerization occurs primarily at the level of the DBD via the so-called 95
D-box residues in the second zinc-finger (51) as well as between the amino-terminal 96
domain (NTD) and the LBD (21, 30). The latter interaction takes place between the 97
conserved 23FQNLF27 motif and the ligand-induced hydrophobic groove at the surface of 98
the LBD (29). The extent to which the N/C-interaction is impaired by mutating residues in 99
the AR-LBD seems to correlate with the severity of the corresponding AIS-phenotype 100
(26, 36). However, the exact role of the N/C interactions in transcription control still 101
remains obscure since it is largely lost once the receptor binds to the DNA (50, 57). 102
Dimerization via the LBD has been described for many receptor-homodimers, such as 103
GR, PR, ER and RXR as well as for heterodimeric receptor complexes, such as PPAR 104
and RXR (4, 6, 11, 55), but the existence of such LBD-dimerization surface for the AR-105
homodimer remains controversial (45). Furthermore, it is unknown whether DBD-LBD 106
interactions as described for the PPARγ-RXRα heterodimer can occur within or between 107
AR monomers. To investigate this, we have taken advantage of the many AR mutations 108
that have been described to correlate with pathologies. Indeed AR mutations described 109
in patients suffering from the androgen insensitivity syndrome (AIS) can provide valuable 110
information on the importance of the mutated residues in AR-functioning. 111
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In this report, we investigate the existence of a DBD-LBD interface in the AR-112
homodimer. By means of in silico approaches potential interaction surfaces between 113
DBD and LBD were predicted and analyzed. 114
115
MATERIALS AND METHODS 116
Plasmid constructs 117
The cDNA of the human AR was present in a flag-tagged pSG5-expression vector. 118
Mutations in the LBD of the AR were introduced by two-step PCR site-directed 119
mutagenesis using two internal primers and two external primers. The resulting mutated 120
human AR-LBD cDNA fragments were re-cloned as PsyI/AsuII fragments into the pSG5-121
flag-wtAR construct. Mutations in the DBD were made by site-directed mutagenesis of 122
the wild-type AR-DBD-encoding cDNA fragment inserted in pGEMT (Promega) using 123
two primers containing the mutation enabling amplification of the complete vector. The 124
mutated AR-DBD cDNA was subsequently cloned as a HindIII/PsyI fragment into the the 125
pSG5-flag-wtAR construct. The pCMV-β-gal expression vector was obtained from 126
Stratagene (La Jolla, CA). The classical ARE-based luciferase reporter plasmid was 127
described before (19). 128
129
Cell culture, transfections and nuclear extracts 130
COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA) 131
and were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% Fetal 132
Calf Serum. For assessing the transactivation capacity of the mutant ARs, cells were 133
seeded at a density of 10 000 cells/well in a 96-well plate. In each well 10 ng expression 134
vector of the receptor, 100 ng of reporter plasmid and 10 ng of the pCMV-β-gal 135
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expression vector were transfected. The next day, the medium in the wells was replaced 136
by medium containing DHT (Sigma-Aldrich) or R1881 (Perkin Elmer). Cells were 137
harvested in Passive Lysis Buffer (Promega) and luciferase-activity and β-138
Galactosidase-activity were measured as described before (27). 139
Nuclear extracts of COS-7 cells expressing wild-type or mutant ARs were prepared as 140
described earlier (19). 141
142
Electrophoretic mobility shift assay (EMSA) 143
EMSAs were performed using nuclear extracts of COS-7 cells expressing wild-type AR 144
or a mutant AR. The binding of these receptors to [α-32P]dCTP-labeled ARE-sequences 145
was determined as described earlier. TAT-GRE with sequence 5’-AGAACAtccTGTACA-146
3’ and SLP-MUT (SLP-HRE2 mutated (-4, T-A)) with sequence 5’-147
AGAACTgccTGTCCA-3’ were used as ARE (19). 148
149
Whole cell competition assay (WCCA) 150
COS-7 cells were seeded in a 48-well plate at a density of 30 000 cells per well. The 151
next day, the cells were transfected with 375 ng expression vector for either wild-type or 152
a mutant AR and 75 ng of pCMV-β-gal expression vector (per well). After approximately 153
48 h, the cells were incubated with [3H]-labeled Mibolerone (³H-Mib, 72.2 Ci/mmol) to 154
investigate the binding capacity in the transfected cells. Both total and aspecific binding 155
was determined, from which the specific binding of [3H]-labeled Mibolerone to the 156
receptor was derived. A dilution series of [³H]-Mib (300, 100, 30, 10, 3, 1 and 0.3 nM 157
[³H]-Mib) was made to assess total binding. An excess of DHT (10 µM) was added to 158
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the dilution series to determine aspecific binding of the cells. After incubating the cells 159
for 90 minutes at 37°C, the cells were washed with ice-cold PBS and lysed in 100 µl of 160
Passive Lysis Buffer (Promega). To 75 µl cell lysate, 2 ml Lumasafe Plus (Perkin Elmer) 161
was added and the radioactivity (as counts per minute, cpm) was detected in a Wallac 162
1409 liquid scintillation counter from Perkin Elmer. Receptor expression was compared 163
by Western blot analysis and protein concentrations were used to convert cpm into 164
fmol/µg protein. Via nonlinear regression analyses in GraphPad Prism 5 (GraphPad 165
Software), the KD and the Bmax were calculated from at least 2 independent experiments. 166
The Bmax of AR wt was set at 100%, the Bmax of every other receptor is given relative to 167
the Bmax of AR wt. One-way ANOVA was performed using Graphpad Prism 5. 168
169
In silico 3D-alignment and docking analysis 170
Using the coordinates from the model of the PPARγ-RXRα heterodimer (PDB ID: 3DZY), 171
the rat AR-DBD (PDB ID: 1R4I) was aligned onto the RXRα-DBD and the human AR-172
LBD (PDB ID: 1XQ3) was aligned onto the PPARγ-LBD using the ‘CEAlign’ tool in 173
PyMOL (17). 174
The crystal structure of human AR-LBD (PDB: 2AM9) and rat AR-DBD (PDB: 1R4I) 175
were used to perform protein-protein docking with HADDOCK2.0 (20). Since at the 176
moment of the docking experiments, mutagenesis data on the contact between AR-LBD 177
and AR-DBD were still absent, we chose not to define Ambiguous Interaction Restraints 178
since they might bias the docking results. Two patches on the surface of AR-LBD were 179
used as semi-flexible segments in two individual docking experiments. During the first 180
docking, residues 720-730, 732-733, 737, 741-746, 767, 830-831, and 834 (patch 1) 181
were indicated as semi-flexible, while in the second experiment residues 681-684, 686-182
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688, 710-712, 748-752, 754-759, and 762-768 (patch 2) formed the semi-flexible 183
segments. For AR-DBD, we defined the residues opposite the DNA-binding surface as 184
semi-flexible segments in both experiments. These included residues 569-584, 596-597, 185
599-600, and 602-611 (586-601, 613-614, 616-617, and 619-628 in human). In a first 186
step, 1000 structures were generated using randomization and rigid body energy 187
minimization, followed by semi-flexible simulated annealing in torsion-angle space on 188
the best 200 structures and final refinement in an explicit solvent layer (8 Å radius) 189
containing water molecules. The structures were sorted according to a weighted sum 190
(HADDOCK score) of, among other factors, buried surface area, van der Waals and 191
electrostatic energy. The solutions were, for both experiments, divided into 13 clusters 192
(16) containing at least 5 complexes with an interface backbone root mean square 193
deviation of 5 Å from which the best scoring one was further analyzed. For these 194
structures, the total non-bonded energy and total interface area were calculated using 195
Brugel (18). 196
197
RESULTS 198
3D-alignment of the AR on the PPAR-RXR crystal structure 199
To test the hypothesis of DBD-LBD communications in the AR, we based our first AR 200
DBD-LBD model on the crystal structure of PPARγ-RXRα (PDB ID: 3DZY) (11). Via 201
CEAlign, a plugin for Pymol (17), the main-chain coordinates of the AR-LBD were fitted 202
onto those of the PPARγ-LBD and the coordinates of the AR-DBD were fitted onto those 203
of the RXRα-DBD (Fig. 1A). The position of the LBD in relation to the DBD in the 3D-204
alignment of the AR has to be interpreted with caution since the PPARγ-RXRα 205
heterodimer was bound to a DR1 (direct repeat with 1-nucleotide spacer), while the AR-206
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homodimer binds to response elements with a 3-nucleotide spacer. Spacing influences 207
the position of the DBDs on the DNA-helix and perhaps also the positioning of the LBDs 208
towards each other. Secondly, the structural elements involved in the interaction surface 209
on the PPAR-LBD are not well conserved in the AR. In the PPARγ-RXRα heterodimer, 210
the residues in helix 5 are not situated on the surface due to the presence of helix 2a, an 211
additional β-strand and helix 2b. In the AR, this region corresponds to a linker with little 212
secondary structure resulting in a gap between the AR-LBD and the AR-DBD in the 213
model. However, in this alignment model, a first potential interaction surface on the DBD 214
is formed by residues 587-599, 612-614 and 617-626 which interact with the regions 215
682-695 and 754-774 in the LBD (Fig. 1A). To test the functional relevance of this 216
hypothetical LBD-DBD contact, these regions were subjected to mutational analysis. 217
Target residue selection was based on data in ‘The Androgen Receptor Gene Mutations 218
Database’ on AR mutations in AIS-patients and PrCa-biopsies (42). 219
220
Effect of alignment-based mutations on transactivation 221
The transactivation capacity of the mutant receptors mentioned in Table 1 were 222
assessed and compared to the wild-type AR (Fig. 2). Two mutant ARs (AR R617P and 223
AR L768A) did not show any activity in transfection assays, even at higher agonist 224
levels. Three mutations (D695N, F754S and P766A) reduced transactivation at 225
physiological ligand concentrations, but at higher levels, their transactivation capacity 226
was comparable to that of wild-type AR. Two other mutations (N758T and E772A) did 227
not significantly affect transactivation at low ligand concentration, while AR R760S 228
showed a surprising increase in transactivation capacity at R1881-concentrations of 1 229
nM or higher. 230
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Docking of the AR-DBD onto the AR-LBD 232
In an in silico docking experiment, the most energy-favorable position of AR-DBD and 233
AR-LBD in relation to each other was calculated with HADDOCK2.0 (20). For the in 234
silico docking of the AR-DBD onto the AR-LBD, we selected the DBD-surface opposite 235
of the DNA-binding surface and for the LBD we focused on two mutational hotspots. The 236
hotspots on the LBD were named patch 1 and patch 2 and are defined in the Materials 237
and Methods section. Because the docking experiment using patch 1 as semi-flexible 238
segment resulted in lower scoring complexes than when patch 2 was used, we selected 239
three clusters generated by docking with patch 2. For each of these clusters the most 240
energy-favorable structure was chosen as a representative. Each of these DBD-LBD 241
positions provided us with a well-defined hypothetical interface and the contribution of 242
specific residues to the interaction (Fig. 1B and Table S1 and S2). Residues that 243
contributed most to the stabilization of the interaction by lowering the non-bonded 244
interaction energy were mutated. When possible, mutations described to occur in AIS or 245
PrCa were introduced. If not indicated otherwise, the residues were changed to alanine. 246
247
Effect of docking-based mutations on transactivation 248
Four DBD mutations (K590A, K592A, Y620A and E621A) were tested for their effect on 249
the transcriptional activity of the AR. K590A, K592A and E621A slightly but significantly 250
reduced transactivation of the receptor, while Y620A had no apparent effect (Fig. 3). Of 251
the seven LBD mutations (E678A, N691A, E706A, R710A, H714A, K717A and Q798E), 252
only AR R710A showed a clear reduction in activity compared to AR wt at low, but not at 253
higher ligand concentration (Fig. 4). No significant difference with AR wt was seen for 254
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AR E678A, AR N691A, AR H714A and AR Q798E. Surprisingly, two mutations (E706A 255
and K717A) increased the potency of the AR significantly at higher ligand 256
concentrations. 257
258
DNA- and ligand-binding capacities of AR mutants 259
Mutated receptors with reduced transactivation capacity or lower ligand sensitivity were 260
subjected to EMSA and WCCA to assess their ligand- and DNA-binding capacity. DNA 261
binding as determined with EMSA was not affected by the DBD-mutations K590A, 262
K592A, Y620A and E621A (Fig. 5A). For several LBD-mutants, namely AR D695N, AR 263
R710A, AR F754S, AR N758T and AR P766A the DNA binding was impaired when 264
compared to AR wt (Fig. 5B). Importantly, the mutations did not affect the expression 265
level of the receptor as shown by Western blot analysis (Fig. 5). 266
In return, the affinity for ligand of the DBD-mutants AR K590A, AR K592A was reduced 267
(Table 2). Their KD for mibolerone was 3.57 nM and 3.83 nM respectively, which is a 2-268
fold increase compared to the KD of AR wt (1.69 nM). This increase was, however, not 269
significant. The DBD-mutants also display aberrant maximal ligand-binding capacities 270
(Bmax) of 74.5%, 158.1%, 46.3% and 60.8% for AR K590A, AR K592A, AR Y620A and 271
AR E621A, respectively. These are all significantly different compared to the Bmax of AR 272
wt (105.1%) (Table 2). Clearly, some of the LBD-mutations (D695N, R710A, F754S, 273
N758T and P766A) did not affect the ligand-binding capacity since KD and Bmax were in 274
the normal range (Table 2). 275
The DBD-mutant AR R617P was transcriptionally inactive due to abrogated DNA binding 276
(Fig. S1) while the LBD mutant AR L768A was inactive due to its inability to bind ligand 277
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(data not shown). All data on transactivation or DNA and ligand binding of the mutated 278
ARs and the correlated patient phenotypes (when available) are summarized in Table 1. 279
280
DISCUSSION 281
A lot of experimental data hint towards allosteric interferences between the LBD and 282
DBD of NR (7-9, 24, 43, 44, 47), but only the report by Chandra et al. in 2008 has 283
provided strong evidence for a physical interaction between the DBD and LBD of the 284
PPARγ-RXRα heterodimer. In this study, we aimed to identify residues at the surface of 285
the AR-DBD and AR-LBD that might be involved in such DBD-LBD communications. 286
Candidate residues were identified with the help of in silico models and information from 287
‘The Androgen Receptor Gene Mutation Database’ (42). Both the 3D-alignment of AR 288
with the PPARγ-RXRα coordinates as well as the AR-docking provided potential 289
interaction surfaces for the AR. The functional relevance of the predicted interfaces was 290
scrutinized by mutational analysis. The strength of this study of the AR is that some of 291
these mutations have already been described in AIS and/or PrCa, which provides direct 292
indications on the in vivo relevance of the mutated residues. 293
Superposition of the AR on the PPARγ-RXRα crystal coordinates resulted in the 294
selection of one DBD-mutation and seven LBD-mutations found in MAIS, PAIS and/or 295
CAIS that are present in the thus defined DBD-LBD interface. A further four DBD and 296
seven LBD residues were selected from the surfaces generated by AR-docking 297
(summarized in Table 1). 298
299
Mutational analysis of the DBD- and LBD-surface 300
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Two mutations (R617P and L768A) affected the primary function of the receptor domain 301
they are situated in (DBD and LBD, respectively) thus explaining their inactivating effect 302
on the receptor’s function in the transactivation assay. A possible implication of these 303
residues in DBD-LBD communications can therefore not be commented on. 304
Conversely, many of the tested mutations left the receptor unaffected (Y620A, E678A, 305
N691A, H714A and Q798E) or even increased its ability to transactivate (E706A, K717A 306
and R760S). For R760S, E772A and Q798E the absence of any effect in our assays is 307
in surprising contradiction with the AIS-phenotypes of the patients in which they were 308
described (3, 37, 53). In particular Q798E was expected to have functional implications 309
because mutations of this residue have been found in a number of AIS as well as PrCa 310
cases. However, its mutation did not affect AR activity in our transactivation assays. 311
These data are in accordance with Bevan et al. who reported normal ligand-binding 312
capacity for this mutant (3). This still leaves the molecular effects of these AIS and PrCa 313
mutations unexplained. 314
315
Evidence for DBD-LBD communications in the AR 316
Four mutations in the AR-LBD (D695N, R710A, F754S and P766A) do not affect ligand 317
binding but reduce DNA binding and transactivation. Three mutations in the AR-DBD 318
(K590A, K592A and E621A) do not affect DNA binding, but reduce ligand-binding and 319
transactivation of the AR. We propose that these mutations affect direct communications 320
between the DBD and the LBD. 321
322
A surface on the LBD affects DNA binding 323
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The four LBD mutations are located at the surface of the LBD (Fig. 6). This surface 324
consists of residues located in the linker corresponding to helix 2 (D695), in helix 3 325
(R710), in helix 5 (F754) and the β-turn (P766). The functional importance of these four 326
residues in AR-functioning is underlined by their involvement in several cases of AIS 327
(Table 1). Moreover, two of the residues (D695 and P766) are conserved in PR, MR and 328
GR, and because of their surface exposures in the LBD crystal structures might be 329
involved in DBD-LBD communications. 330
331
A surface on the DBD affects DNA binding 332
Three mutations that affect AR ligand-binding are located at the surface of the DBD 333
(K590A, K592A and E621A; Fig. 6), again strongly suggesting DBD-LBD 334
communications. Residues K590 and K592 are located in the linker region between the 335
two zinc-fingers, which corresponds to the lever arm in the GR. For the GR, the 336
conformation of this lever arm is influenced by the sequence of the DNA response 337
element. Since the activity of the GR depends greatly on the DNA sequence it is binding 338
to, it was suggested that the lever arm transmits this DNA signal allosterically to other 339
domains of the receptor (44). Our results are in agreement with the possibility of a 340
similar allosteric influence of the lever arm on other domains of the AR. 341
In summary, we identified seven residues that are implicated in inter-domain 342
communications between the DBD and LBD of the AR. This opens up new questions: 343
344
Are the interactions direct or indirect? 345
It should be noted here that double hybrid assays between AR-DBD and LBD did not 346
reveal protein-protein interactions (data not shown). However, the expected interface is 347
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rather small and the affinity between the two surfaces does not need to be high, since 348
they are both present in close vicinity either within the same or in two dimerizing 349
proteins. Indeed, whether these interactions are intra- or intermolecular remains to be 350
determined. 351
SAXS analyses and cryo-EM structures have demonstrated NR dimer conformations 352
with extended hinge regions and minimal interactions between the DBD and LBDs (46, 353
49). Our results seem to be in favor of direct interactions, rather than to match the more 354
elongated common architecture for NRs (49), unless the DBD- and LBD-surfaces we 355
identified are interacting with bridging factors. 356
Obviously, structural analyses, such as a crystal structure of the complete AR-357
homodimer bound to ligand, DNA and possibly coactivator or N-terminal peptides would 358
provide a better picture of the exact DBD-LBD interactions. For the AR, any structural 359
data surpassing the isolated DBD and LBD are missing. However, the positions of the 360
residues with trans-domain effects are in accordance with a direct interface as observed 361
for the PPAR-RXR dimer (11). 362
Most importantly, it should be clear from the work presented here, that the domains in 363
the AR do not act independently but that optimal activation of transcription by the AR 364
requires allosteric communications between different domains. 365
366
Can the DBD-LBD interactions be modulated? 367
It was recently reported that phosphorylation by CDK5 of serine 273 on the PPARγ-LBD 368
interface with the RXRα-DBD, controls the transactivation by PPARγ (13). This opens up 369
the possibility that receptor activities may be controlled via modulations of the DBD-LBD 370
interactions. Although for the AR, there are no data to indicate such a control, the 371
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presence of acetylation and phosphorylation sites in the hinge region that connects the 372
DBD and LBD are intriguing (15). Another question is how the DBD-LBD interactions 373
relate to the N/C-interactions within the AR and to the coactivator-interactions via LBD or 374
via NTD. Since the LBD interacts with coactivators or with the NTD via a known 375
hydrophobic groove which is situated on the opposite site of the proposed interface with 376
the DBD (7, 14), we propose that the effect of the mutations described here only 377
specifically disrupt the intra- or intermolecular DBD-LBD communications of the AR. 378
Estebanez-Perpina et al. described an alternative antagonist binding site at the surface 379
of the AR-LBD (22). This surface too does not overlap with the DBD-contacting patch 380
and hence antagonists binding to this alternative site are not believed to directly affect 381
the DBD-LBD interactions. 382
383
CONCLUSIONS 384
We discovered seven residues in the AR which we propose to be involved in a DBD-385
LBD interaction: D695, R710, F754 and P766 in the LBD and K590, K592 and E621 in 386
the DBD (Fig. 6). Mutation of these residues affected the transactivation capacity of the 387
full-size receptor without affecting the primary function of the domain they are situated 388
in, but by affecting the function of the adjacent domain. Importantly, since all seven 389
residues are situated at the surface, we conclude that these residues are most likely 390
involved in direct intra- or intermolecular communications between the DBD and LBD of 391
the AR. The functional relevance of these communications is further indicated by the 392
description of several cases of androgen insensitivity that correlate with mutations of 393
these residues. 394
395
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ACKNOWLEDGEMENTS 396
We thank R. Bollen and H. De Bruyn for their excellent technical assistance. C.Helsen 397
was holder of a Belgian l’Oréal-Unesco fellowship for Young Women in Life Sciences 398
and a fellowship of the ‘Fonds voor Wetenschappelijk onderzoek’ of Flanders. The work 399
was supported by FWO grants G.0369.02 and G.0858.11. 400
401
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nuclear receptors. Nat Struct Biol 3:87-94. 614
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androgen receptor are a frequent cause of receptor-binding positive androgen 625
resistance. Mol Endocrinol 6:409-15. 626
627
628
629
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FIGURE LEGENDS 630
Fig. 1: In silico models applied to investigate DBD-LBD communication and 631
position of mutated residues. (A) Front- and side-view of the 3D-alignment of AR-DBD 632
(1R4I) and AR-LBD (1XQ3) onto the heterodimeric complex of PPARγ-RXRα (3DZY). 633
The interaction surfaces on the AR-DBD and AR-LBD are depicted in purple. The DNA-634
helix is shown in black. (B) Docking of AR-DBD (1R4I) onto AR-LBD (2AM9). Three 635
potential positions of the AR-DBD (Yellow, Red and Blue) are given in respect to the AR-636
LBD (White). Each position corresponds to a cluster and represents an energy-favorable 637
DBD-LBD position. (C) Front-and side-view of the position of the residues in LBD and 638
DBD that have been tested for their role in DBD-LBD communication in the AR. They 639
are K590, K592, R617, Y620 and E621 in the DBD and E678, N691, D695, E706, R710, 640
H714, K717, F754, N758, R760, P766, L768, E772 and Q798 in the LBD. Residues that 641
affect the function of the adjacent domain are indicated in orange, other residues are in 642
green. 643
644
Fig. 2: The transactivation of AR is affected by mutation of residues selected from 645
the 3D-alignment. 646
Transactivation capacity of the mutant ARs was investigated by transient transfection in 647
COS-7 cells. The ability of the mutant receptors to initiate transcription of an androgen-648
regulated luciferase-reporter gene was compared to the activity of AR wt. R1881 or DHT 649
were added in a concentration gradient ranging from 1 pM to 10 µM. Activities that are 650
significantly different compared to AR wt are signalized with a * (p<0.05; student’s t-651
test). The mean and standard deviation of at least 3 independent experiments are given. 652
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(A) AR wt, AR R617P, AR D695N, AR N758T, AR R760S, AR P766A, AR L768A and 653
AR E772A. (B) AR wt and AR F754S. 654
655
Fig. 3: The transactivation of AR is affected by mutation of residues in the DBD 656
selected from the docking. 657
Transactivation capacity of the mutant AR (indicated on top of each panel) was 658
investigated by transient transfection in COS-7 cells. The ability of the mutant receptors 659
to initiate transcription of an androgen-regulated luciferase-reporter gene was compared 660
to the activity of AR wt. DHT was added in a concentration gradient ranging from 0.1 pM 661
to 1 µM. Activities that are significantly different compared to AR wt are signalized with a 662
* (p<0.05; student’s t-test). The mean and standard deviation of at least 3 independent 663
experiments are given. 664
665
Fig. 4: The transactivation of AR is affected by mutation of residues in the LBD 666
selected from the docking. 667
Transactivation capacity of wild type and mutant ARs was investigated by transient 668
transfection in COS-7 cells. The ability of the mutant receptors to initiate transcription of 669
an androgen-regulated luciferase-reporter gene was compared to the activity of AR wt. 670
DHT was added in a concentration gradient ranging from 0.1 pM to 1 µM. Activities that 671
are significantly different compared to AR wt are signalized with a * (p<0.05; student’s t-672
test). The mean and standard deviation of at least 3 independent experiments are given. 673
674
Fig. 5: DNA-binding ability of mutant ARs. 675
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By means of EMSA, the DNA-binding capacity of several DBD-mutants (A) and several 676
LBD-mutants (B) were investigated. Radio-actively labeled TAT-GRE and SLP-MUT 677
ARE were used for the DBD-mutants and LBD-mutants, respectively. Binding of the 678
receptor to the ARE will give a complex at the position of the arrow. Addition of an AR-679
antibody will cause a supershift, indicated by SS. Loading of equal amounts of receptor 680
was confirmed by Western blot analysis. The free probe which is present at the bottom 681
of the gel is not included in this picture. The DNA-binding capacity and the expression 682
level of each mutant receptor were quantified using ImageJ (NIH) resulting in a relative 683
DNA-binding capacity given as % of the binding of AR wt. 684
685
Fig. 6: Proposed position of the DBD relative to the LBD. 686
The position of DBD and LBD was derived from the 3D-alignment of the AR on the 687
PPARγ-RXRα model. The residues that were shown to be involved in DBD-LBD 688
communication are depicted in orange. Helix 1, 3 and 5 are indicated (H1, H3 and H5). 689
690
691
692
693
694
695
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Table 1: Overview of the selected mutations; their location in the AR; the obtained 696
results on transactivation, DNA binding and ligand binding and their occurrence in AIS 697
and PrCa. 698
699
Position Mutation
Trans-activation
DNA binding
Ligand binding
Phenotype Reference
DBD
Lever arm K590A ↓ Normal KD↑a Bmax↓
K592A ↓ Normal KD↑a Bmax↑
2nd
α-helix
R617P Inactive Inactive Normal PAIS, CAIS (40, 63)
Y620A Normal Normal Bmax↓
E621A ↓ Normal Bmax↓
LBD
Helix 1 E678A Normal
‘Helix 2’ N691A Normal
D695N ↓ ↓ Normal MAIS,
PAIS, CAIS (1, 12, 25, 28,
34, 48)
Helix 3
E706A ↑ at high conc.
R710A ↓ at low conc. ↓ Normal
H714A Normal
K717A ↑ at high conc.
End of Helix 5
F754S ↓ ↓ Normal PAIS (54)
N758T Normal ↓ Normal PAIS (61)
R760S ↑ PAIS (37)
β-turn
P766A ↓ at low conc. ↓ Normal CAIS (5)
L768A Inactive ↓ Inactive
E772A Normal
PAIS (53)
End of Helix 7
Q798E Normal
MAIS, PAIS, PrCa
(3, 10, 23, 25, 32, 33, 35, 59)
a 2-fold increase compared to AR wt, not significant 700
701
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Table 2: Ligand-binding ability of mutant ARs. 702
KD (nM)
SE of K
D
Bmax
(% fmol/µg of AR wt)
SE of Bmax
AR wt 1,69 0,63 105,1 8,3
DBD
AR K590A 3,57 1,00 74,5* 5,0
AR K592A 3,83 1,31 158,1* 13,1
AR Y620A 1,83 0,39 46,3* 2,2
AR E621A 2,33 2,06 60,8* 12,0
LBD
AR D695N 2,43 0,49 96,3 4,4
AR R710A 2,73 0,48 97,1 3,9
AR F754S 2,11 0,61 109,9 7,0
AR N758T 2,15 0,49 89,8 4,6
AR P766A 2,83 0,83 119,6 8,1
* Significant difference compared to AR wt (p<0.05, One-way ANOVA). 703
704
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