# Quaternary glucocorticoid receptor structure highlights allosteric interdomain communication

Sandra Postel, Lisa Wissler, Carina Johansson, Anders Gunnarsson, Euan Gordon, Barry Collins, Marie Castaldo, Christian Köhler, David Öling, Patrik Johansson, et al.

► To cite this version:

Sandra Postel, Lisa Wissler, Carina Johansson, Anders Gunnarsson, Euan Gordon, et al. Quaternary glucocorticoid receptor structure highlights allosteric interdomain communication. Nature Structural and Molecular Biology, 2023, 30 (3), pp.286-295. 10.1038/s41594-022-00914-4. hal-04281562

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1 **Quaternary GR structure highlights allosteric interdomain communication**

2

3 Sandra Postel¹, Lisa Wissler¹, Carina A. Johansson¹, Anders Gunnarsson¹, Euan Gordon²,

4 Barry Collins³, Marie Castaldo², Christian Köhler³, David Öling², Patrik Johansson¹, Linda

5 Fröderberg Roth², Brice Beinsteiner⁴,⁵,⁶,⁷, Ian Dainty³, Stephen Delaney³, Bruno P.

6 Klaholz⁴,⁵,⁶,⁷, Isabelle M.L. Billas⁴,⁵,⁶,⁷, Karl Edman¹

7

8 ¹Mechanistic and Structural Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg,

9 Sweden

10 ²Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden

11 ³Research and Early Development, Respiratory and Immunology, BioPharmaceuticals R&D,

12 AstraZeneca, Gothenburg, Sweden

13 ⁴IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Illkirch, France

14 ⁵Université de Strasbourg, Illkirch, France

15 ⁶Institut National de la Santé et de la Recherche Médicale (INSERM) U1258, Illkirch, France

16 ⁷Centre National de la Recherche Scientifique (CNRS) UMR 7104, Illkirch, France

17

18 **Abstract**

19 The glucocorticoid receptor (GR) is a ligand activated transcription factor that binds DNA and
20 assembles context dependent coregulator complexes to regulate gene transcription. GR
21 agonists are widely prescribed to patients with inflammatory and autoimmune diseases. Here,
22 we determine the first high resolution, multi-domain structures of GR in complex with ligand,
23 DNA and a coregulator peptide. The structures reveal how the receptor forms an asymmetric
24 dimer on the DNA and provide a detailed view of the domain interactions within and across
25 the two monomers. Hydrogen deuterium exchange and DNA binding experiments
demonstrate that ligand dependent structural changes are communicated across the different domains in the full-length receptor. Based on the structural analysis, we design mutant receptor constructs and functional assays to validate the signaling pathways identified in the structures. This study demonstrates how GR forms a distinct architecture on DNA and how signal transmission can be modulated by the ligand pharmacophore. The results provide a platform where we can build a new level of understanding for how receptor modifications can drive disease progression and offer key insight for future drug design.

33

## Introduction

The glucocorticoid receptor (GR) is a ubiquitously expressed nuclear hormone receptor. Together with the androgen, progesterone and mineralocorticoid receptors, GR forms the keto-steroid receptor subfamily which evolved from an estrogen receptor (ER) like ancestor in vertebrates¹. GR is activated through endocrine signaling by the glucocorticoid hormone cortisol, and is of fundamental importance for development, skeletal growth, behavior, glucose homeostasis and inflammation.

41

GR comprises a large, disordered N-terminal domain, a DNA-binding domain (DBD) and a ligand-binding domain (LBD). In the apo state, GR primarily resides in a chaperone complex in the cytoplasm. Ligand activation triggers nuclear translocation and receptor binding to specific DNA sequences. On the DNA, GR recruits co-regulators and other transcription factors in a context dependent manner to ultimately repress or activate transcription of a large set of target genes². As such, GR provides a plastic scaffold which integrates signaling input from the ligand pharmacophore, the sequence of the DNA and post-translational
49 modifications and the outcome of the signaling event is governed by the identity of the assembled co-regulators.
50

51
52 Signaling efficiency is dependent on comprehensive allosteric networks that enable communication in between distinct functional sites across the receptor domains. GR typically binds as a dimer to the canonical GR binding sequence (GBS), which is an imperfect palindromic hexameric DNA sequence repeat, separated by a 3-nucleotide spacer³. However, GR can also bind as a monomer to a canonical half-site⁴ or as discrete monomers on opposite sides of the DNA to an inverted repeat sequence⁵. This adds a layer of combinatorial flexibility and contributes to signaling diversity, but also suggests that the protein interfaces must be able to adapt to different boundary conditions.
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61 To date, structural studies have focused on the individual domains within the receptor. Studies of the DBD in complex with DNA have demonstrated how the nucleotide sequence drives structural rearrangement within the DBD dimer⁶. The structures of the LBD in complex with a wide array of ligands have highlighted the plasticity of the ligand binding pocket⁷. However, a full understanding of the allosteric transmission in between the ligand and DNA binding events and effects on co-regulator recruitment requires structural information from a multi-domain construct.
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69 Here we present the crystal structures of a GR construct (residues 385-777, encompassing the DBD and LDB) in complex with the agonists velsecorat⁸ and fluticasone furoate⁹, a natural 70
71 GBS and a co-regulator peptide. These are the first high-resolution multidomain structures of 72 a steroid receptor and reveal how the receptor forms a unique architecture on the DNA.
73

## 74 Results

To determine the multidomain structure of GR, we expressed and purified several GR constructs (Fig. 1a) in the presence of the strong agonists velsecorat (Vel), fluticasone furoate (FF), or dexamethasone (Dex) (Fig. 1b). All three ligands exhibit strong anti-inflammatory responses, inhibiting lipopolysaccharide (LPS) induced tumor necrosis factor α (TNFα) secretion in human whole blood with an IC₅₀ of 10.74 nM, 3.63 nM and 34.70 nM, respectively (Fig. 1c), and have demonstrated clinical efficacy in a range of disease indications¹⁰⁻¹².

<figure><img src="image_2.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

82

**Figure 1** | GR protein constructs and agonists used in this study. **a**, GR protein constructs used in this study. The GR<sub>ΔN</sub> construct includes five stabilizing mutants highlighted in the schematic. **b**, The chemical structure of the GR agonists velsecorat, fluticasone furoate and dexamethasone. **c**, Velsecorat, fluticasone furoate and dexamethasone inhibit LPS induced

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87 TNFα production in human whole blood. Data points are mean ± s.e.m. of six replicates from
88 three donors.

89
90 The purified receptors were then mixed with dsDNA and coregulator peptide to obtain
91 quaternary complexes (Extended Data Fig. 1). The GR<sub>ΔN</sub> contains 5 stabilizing mutants (Fig.
92 1a) and yielded diffracting crystals with both velsecorat and fluticasone furoate. The highest
93 resolution structure (2.5 Å, Extended Data Table 1) was obtained from the GR<sub>ΔN</sub> construct in
94 complex with velsecorat [GR<sub>ΔN</sub>(Vel)], a GBS from the serum and glucocorticoid-regulated
95 kinase-1 (SGK-1) promoter, and a peptide (residues 134-154) derived from the co-regulator
96 peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α).

97
98 The structure reveals that the LBD dimer is asymmetrically placed on top of the center of the
99 DBD dimer resulting in a shifted tetrahedral architecture on the DNA (Fig. 2). In the crystal
100 lattice, there are two alternative LBD dimer interfaces; one consisting of two separate regions
101 spanning the N-terminal end of H10/11 and H6-H7 (Fig. 2a, b, d), and one primarily mediated
102 by H1 (Fig. 2c). We could not detect any electron density for the linker residues (residues
103 489-525) in between the LBD and DBD in any of our structures, which is likely due to the
104 flexible nature of this region¹³. However, the H10/11 interface partially overlaps with the
105 canonical ER dimer interface¹⁴ and exhibits a larger total buried surface area of 904.4 Å²
106 (compared to 710.2 for the H1 dimer interface). We propose that this is the primary LBD
107 dimer for GR activation on the SGK-1 GBS. The LBD dimer is tilted relative to the DBD
108 dimer (Fig. 2a, b), which positions the LBD N-termini in proximity of the DBD C-termini.
109 Based on distances, number of missing residues and space constraints between the C-terminus
110 of the DBD domains and the N-terminus of LBD domains, we suggest that DBD1 is
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connected to LBD1 and DBD2 to LBD2, but the alternative connectivity cannot be fully excluded.

112

<figure><img src="image_7.png" /></figure>

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<caption>**Figure 2** | Structure of GR<sub>AN</sub>(Vel) in complex with SGK and PGC1α<sub>134-154</sub>.</caption>

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**a**, **b**, High resolution crystal structure of GR<sub>AN</sub>(Vel) in complex with dsDNA SGK (red) and the coactivator peptide PGC1α<sub>134-154</sub> (yellow). Velsecorat is shown as a stick model (magenta) and Zn atoms as spheres (gray). GR LBD helix numbering is annotated within the circles. The DBD dimerization loops and lever arms are marked D and L, respectively. The nucleotide sequence of the SGK GBS is highlighted with the two half-sites in red. **c**, The alternative LBD dimer (LBD1: LBD2<sub>H1</sub>) in the crystallographic lattice in which the LBD-LBD interface is mediated by H1 interactions. **d**, Top view of the head-to-tail LBD dimer interface as shown in **a** and **b**.
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The asymmetric placement of the LBD dimer on the DBD dimerization loops (D-loops) results in LBD1 interacting with the D-loops of both DBD1 and DBD2 with a combined buried surface area of 731 Å², while LBD2 only contacts the lever arm of DBD1 with a buried surface area of 145.2 Å² (Fig. 2a, b). This arrangement of the LBD dimer on the DBD dimer agrees with NMR studies of DBD complexes with multiple GBSs which, combined with functional cell assay data, suggested that both the D-loop and the lever arm could be involved in inter-domain communication³,⁶. In addition, NMR studies of WT GR LBD revealed an allosteric pathway from the ligand binding pocket to the N-terminal end of helix 1¹⁵. In our structure the LBD1 H1 is in direct contact with the DBD D-loops, and likely forms a communication network that links the DNA sequence to the ligand pharmacophore.

In the keto-steroid receptors, a conserved C-terminal extension (residues 767-777) prevents formation of the stable head-to-head LBD dimer observed for ER¹⁶. In contrast, the GRΔN(Vel) LBDs form a unique head-to-tail dimer (Fig. 2c), which has not been observed in any GR LBD structure to date¹⁷. The LBD construct alone remains predominantly monomeric in solution¹⁸ and formation of a relevant LBD dimer likely requires DNA binding (Extended Data Fig. 1).

To study the degree of sequence conservation of the LBD interface residues, we collected 34 GR and 34 reference ER sequences from vertebrates with a similar degree of sequence diversity (see Materials and Methods and Extended Data Fig. 3 and 4). We then mapped the mean pairwise column identity of each residue onto the corresponding LBD structure surface (ER PDB: 3ERD) with the dimerization interface outlined in red (Fig. 3a, b). The ER LBD forms a stable dimer in solution with a total buried surface area of 1581.5 Å². The ER dimer
148 interface and the conservation pattern are strikingly different from the ones observed in GR.

149 However, when superimposing the ER LBD dimer onto the GR LBD dimer (Fig. 3c), the GR
150 dimer interface has merely shifted to an overlapping position due to the GR LBD1 C-terminus
151 shielding the original ER interface. In addition, both interfaces exhibit a similar degree of
152 total conservation, with an average mean pairwise column identity of 0.70 and 0.72 for GR
153 and ER, respectively. A detailed investigation of the GR LBD dimerization interface reveals a
154 cluster of conserved polar residues forming a network of interactions across the two domains
155 (Fig. 3d). Analysis of other key interfaces suggests that the LBD regions facing the DBD
156 dimer (Extended Data Fig. 5a) and the coregulator binding site (AF-2, Extended Data Fig. 5b)
157 are highly conserved.

158
<figure><img src="image_8.png" /><figcaption>**Figure 3 a,** Degree of conservation of GR and **b,** ER. LBD residues colored according to column identity between 0.4 (white) and 1.0 (blue) with LBD dimerization interfaces outlined in red (using a 5Å distance cutoff). **c,** The ER LBD dimer (in black and gray) superposed on the GR LBD dimer presented in this study (in blue and green). The structures have been overlaid using GR LBD1 as reference. **d,** Highly conserved polar residues in the GR LBD dimer interface.</figcaption></figure>

The dimer we observe is distinct from the ones reported in published structures of the GR LBD alone. The dimer interface proposed in the first GR LBD publication includes the loop
169 in between H1 and H3, the antiparallel β-sheet region and the C-terminal end of H5¹⁹. In our 170 structure, this area is directed towards the DBD-DNA for both LBD1 and LBD2, preventing 171 formation of this interface in the crystal lattice. A recent evaluation of all GR LBD structures 172 to date¹⁷ highlighted that H9 forms significant crystal contacts either in a parallel or anti- 173 parallel fashion in many of the LBD structures. In the quaternary structure we present here, 174 this interaction is obstructed by dsDNA from crystallographic neighbors in the lattice. The 175 most prevalent potential dimer interaction in the GR LBD structures, however, is the option 176 mediated by H1¹⁷. This interface is also present in our lattice (Fig. 2c). It has been proposed 177 that GR may form transient higher-order oligomeric states²⁰,²¹. It is plausible that the H1 178 interaction (or even H9 interactions) may mediate formation of tetramers or even higher-order 179 states (Extended Data Fig. 6), bringing distal GBS together in a folded assembly. 180

181 The best characterized co-regulator interaction surface is the activation function 2 (AF-2), at 182 the intersect of LBD helices 3, 4 and 12 (H3, H4 and H12), where the canonical co-activator 183 LxxLL peptide sequence motif binds upon agonist activation. The LBD arrangement 184 presented here, aligns both AF-2 surfaces on the outside of the tetrahedron, accessible for co- 185 regulator binding. However, while both LBDs have H12 arranged in an active position, only 186 the LBD1 AF-2 site is occupied by the PGC1α peptide (Fig. 2a, b). Crystal packing analysis 187 suggests that the LBD2 AF-2 is in close proximity to DBD1 of a crystallographic neighbor 188 which likely precludes peptide binding. 189

190 The DBDs form a head-to-head dimer arrangement in the multidomain structure, which 191 recapitulates the structure of the isolated GR DBDs on the same GBS (PDB: 3G9O) with a Cα 192 root mean square deviation (r.m.s.d.) of 0.876 (138 atoms) (Extended Data Fig. 2a). To 193 validate the LBD structures in the complex, we determined the structure of the isolated,
monomeric wild type (WT) GR LBD in complex with velsecorat [GR<sub>LBD</sub>(Vel)] and PGC1α<sub>134-154</sub> (Extended Data Table 1). The GR<sub>ΔN</sub>(Vel) LBD1 and LBD2 both exhibit the typical 3 layered α-helical sandwich fold and overlay on the GR<sub>LBD</sub>(Vel) structure with a Cα r.m.s.d. of 0.480 and 0.549 for LBD1 (248 atoms) and LBD2 (248 atoms), respectively (Extended Data Fig. 2b, c), confirming that the LBD V571M, F602S and C628D mutants of the GR<sub>ΔN</sub> construct have minimal impact on the GR LBD conformation. The additional S404A and N517D mutants of the GR<sub>ΔN</sub> construct are located in unstructured regions adjacent to the DBDs and are unlikely to affect the structure.

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In contrast to the electron microscopy structures of multi-domain nuclear hormone receptors<sup>22,23</sup>, the X-ray structures<sup>24-27</sup> generally display a more compact arrangement with significant contacts in between the LBDs and DBDs. However, the domain arrangement among the X-ray structures varies dramatically and the structural overlay reveals that the GR organization is unique (Extended Data Fig. 7). The previous X-ray structures all bind to direct repeat DNA response elements with the DBDs arranged in a head-to-tail fashion in the quaternary complex. As the nucleotide linker length in between the binding sequences vary, the DBDs are placed on different sides along the DNA duplex. In addition, as for the other keto-steroid receptors, the isolated GR LBD is monomeric in solution, while the receptors with known multi-domain structures form stable head-to-head LBD-LBD dimers that are conserved in the multi-domain structures. Together, this presents distinct boundary conditions for positioning the LBDs in the quaternary complex. However, despite the differences in the domain arrangement, it is interesting to note that as observed for the previous structures<sup>24-27</sup>, the LBD1 H9-H10 loop is part of the interface with the DBD on the 5' DNA binding sequence (DBD1).

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The receptor transcriptional regulation is highly ligand dependent²⁸,²⁹. To investigate potential ligand impact on the domain arrangement, we determined the structure of GR<sub>ΔN</sub> in complex with the steroid fluticasone furoate, the GBS from SGK-1 and the PGC1α coregulator peptide [(GR<sub>ΔN</sub>(FF)-SGK-PGC1α), Extended Data Table S1]. GR<sub>ΔN</sub>(FF)-SGK-PGC1α crystallizes in the same space group as GR<sub>ΔN</sub>(Vel)-SGK-PGC1α, and the structures of the individual domains are very similar with a Cα r.m.s.d. of 1.004 and 0.852 for LBD1 and LBD2 (248 atoms), respectively, and 0.340 for the DBDs (142 atoms, Fig. 4a).

<figure><img src="image_9.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_12.png" /></figure>

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<caption>**Figure 4** | Ligand specific structural rearrangements.</caption>
229 **a**, Overlay of the structures of GR<sub>ΔN</sub>-SGK-PGC1α complexes in the presence of velsecorat (in color) and FF (in white). The structures have been overlaid using the 5' GBS binding motif as reference. GR LBD helix numbering is annotated within the circles. **b**, Ligand binding site with velsecorat (magenta) and FF (dark gray). |Fo|-|Fc| densities (in green) were calculated before models were refined with ligands and are contoured at 3σ. **c**, HDX difference plots showing deuterium uptake difference [complex A] – [complex B] for all peptides captured from the full-length receptor. Negative values indicate that peptides are protected in A relative to B and vice versa. **d**, Competition binding experiments monitoring exchange of 6-FAM-SGK dsDNA in complex with GR(ligand) with unlabeled SGK dsDNA using fluorescence polarization. Data points are mean ± s.e.m. of three replicates from one experiment.

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240 Velsecorat and fluticasone furoate both bind in the same central ligand binding pocket
241 forming an interaction with N564, which is important for stabilizing H12 in the active
242 position (Fig. 4b)³⁰. As for GR<sub>ΔN</sub>(Vel), H12 is positioned in the active conformation in both
243 GR<sub>ΔN</sub>(FF) LBDs, but the PGC1α peptide is only observed at the AF-2 site for LBD1.

244 Opposite N564, both compounds form an interaction with Q642, but the position of the side-chain is different due to the less bulky, non-steroidal scaffold, of velsecorat. The most notable distinction, however, is beyond the intersect in between H3 and H5. While the 3-keto moiety of the steroid interacts with Q572 and R611, velsecorat rearranges these residues and extends into a novel pocket beneath W577, at the intersect in between H1, H3 and H5.

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250 While the domain arrangement is conserved in the quaternary complexes, the position of
251 LBD2 is slightly shifted in the GR<sub>ΔN</sub>(FF)-SGK-PGC1α structure (Fig. 4a). This is likely
252 because fluticasone furoate has a bulkier substituent at the 17α position and pushes on the H6-
253 H7 region in LBD1 (Extended Data Fig. 8). This region is part of the LBD-LBD dimer
254 interface and consequently shifts the position of the H10/11 in the other monomer. The H6-
255 H7 region is known to be flexible and may be part of the ligand entry mechanism³¹. While
256 fluticasone furoate also makes an analogous push on the H6-H7 region in LBD2, LBD1 is less
257 likely to move since the H10/11 region interacts with DBD1. As a consequence, the
258 GR<sub>ΔN</sub>(FF)-SGK-PGC1α LBD2 position is shifted, and the total buried surface areas are
259 reduced to 704.5 Å² at the LBD dimer interface and to 16.6 Å² in between LBD2 and DBD1.

260

261 To investigate how these structural changes translate to receptor modulation in solution, we
262 expressed the full-length receptor with velsecorat, fluticasone furoate or dexamethasone and
263 performed hydrogen/deuterium exchange mass spectrometry (HDX-MS). The deuterium
264 uptake over time correlates with solvent accessibility and protein dynamics³². Comparing
265 GR(Vel) to GR(FF) (Fig. 4c, top panel [GR(Vel)]-[GR(FF)], and Extended Data Fig. 9), the
266 data confirms that velsecorat protects the protein near helix 1 where the ligand extends into
267 the novel pocket (residues 536-565) and the region near H6 and H7 where we observed the
268 rearrangements in the GR<sub>ΔN</sub>(FF) structure (residues 621-647). Interestingly, the data revealed
269 that velsecorat and fluticasone furoate also impact the deuterium uptake level in a region of
270 the DBD differently, suggesting ligand specific communication across the two domains, even
271 in absence of DNA.

272

273 When analyzing the complex of GR with SGK GBS and the PGC1α peptide with HDX-MS,
274 we obtained a lower peptide coverage than for the GR protein alone (Extended Data Fig. 10).
275 A large section of the DBD (residues 424-467), including the N-terminal α-helix 1, the
276 dimerization loop (D-loop) and the lever arm, is protected from HDX in the GR(Vel)-SGK-
277 PGC1α and GR(FF)-SGK-PGC1α complexes compared to the isolated GR(Vel) and GR(FF)
278 samples, confirming DNA binding (Fig. 4c, second and third panel from top). However, when
279 comparing the HDX-MS data from the GR(Vel)-SGK-PGC1α and the GR(FF)-SGK-PGC1α complexes, we did not detect differences in the DBD domain (Fig. 4c, bottom panel). This 280 may be due to DNA binding itself being such a dominant stabilization process engaging many 281 residues at the interface that it obscures potential ligand specific rearrangements across the 282 two domains. The HDX difference data for GR(Dex) supports that dexamethasone is of the 283 same steroid chemotype as fluticasone furoate with no significant signal in the LBD 284 (Extended Data Fig. 11, top panel). The GR(Dex) data also confirms ligand specific 285 communication across the LBD-DBD domains in absence of DNA. 286

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288 To further explore how the ligand pharmacophore impacts the DNA binding event, we used 289 fluorescence polarization (FP) to analyze the stability of the GR(ligand) in complex with 290 fluorescently labelled 6-FAM-SGK dsDNA, by adding increasing concentrations of 291 competing unlabeled SGK and determined the IC₅₀. As the cytokine release inhibition assay 292 highlighted (Fig. 1c), velsecorat and fluticasone furoate are the stronger GR agonists and they 293 also lead to a 2-fold increase in the stability of the GR–SGK complex in comparison to 294 dexamethasone, reflected by the higher concentration of unlabeled SGK required to exchange 295 the labelled DNA (Fig. 3d). Altogether, the HDX-MS and FP data provide biophysical 296 support for inter-domain communication that could form the basis for ligand driven functional 297 differentiation. 298
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<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

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<figure><img src="image_15.png" /></figure>

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<figure><img src="image_16.png" /></figure>

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<caption>**Figure 5** | Domain interfaces in the GR<sub>AN</sub>(Vel) complex. **a**, MMTV reporter assay using</caption>

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GR<sub>cryst</sub>(S404A, N517D, V571M, F602S, C638D), GR<sub>WT</sub>, GR(A458T), GR(R614A),

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GR(K720D), GR(D641K) and GR(Y640S). Data points are mean ± s.e.m. of eight replicates

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from four experiments. The corresponding western blots showing GR and actin protein

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expression levels in the bottom panel. **b**, Interface of LBD1 with DBD1 and DBD2 and **c**,

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LBD1-LBD2 interface in GR(Vel)-SGK-PGC1α. The residues mutated in the transcriptional

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activation studies (a) are highlighted in bold.

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To explore the relevance of the receptor interfaces for the signaling complex, we designed FL
GR constructs with mutants located in the domain junctions. We transfected the GR
constructs and a luciferase reporter under the control of the mouse mammary tumor virus
(MMTV) promoter into COS7 cells, which lack endogenous GR, to monitor receptor
activation in response to increasing concentrations of dexamethasone. All GR protein
constructs express well (Fig. 5a, bottom panels). WT GR activates the luciferase reporter in
response to dexamethasone with an EC₅₀ of 257 pM (Fig. 5a, top graph). The GR construct
containing the crystallization mutations, GRcryst, exhibits a similar EC₅₀ of 252 pM confirming
that mutants in the GRAN construct have limited effect on signaling potency. However, the
maximal signaling efficacy is reduced. The crystallization mutant construct is designed to
stabilize the receptor in a specific state. It is conceivable that the signaling event requires
structural dynamics that the mutations in GRcryst interfere with. The control mutant A458T,
which is located in the DBD D-loops (Fig. 5b) causing a clash in the DBD-DBD interface³³,
lowers the EC₅₀ to 555 pM accompanied by a reduction of the maximal signaling efficacy. In
LBD1, R614 engages in H-bonds with residues in the D-loops of both DBD1 (G459, A458)
and DBD2 (D462, R469) and exhibits hydrophobic contacts with I464 of DBD2 and R460 of
DBD1 (Fig. 5b). In keeping with this central position, the R614A mutant shifts the signaling
EC₅₀ to 533 pM. At the LBD1-LBD2 interface, we designed the Y640S, D641K and K720D
mutant constructs (Fig. 5c), yielding a change in the signaling potency to an EC₅₀ of 1.50 nM,
1.79 nM and 762 pM, respectively. In humans, the D641V mutant has been reported to be
associated with glucocorticoid resistance³⁴. Altogether, the functional data presented here
confirm that the strategically placed mutants impair receptor signaling. However, a full
characterization of the receptor interfaces requires a comprehensive functional study with
mutants distributed across all receptor surfaces.
333 70 years ago Phillip Hench administered the hormone cortisone to patients with rheumatoid
arthritis with a transformative outcome³⁵. After the isolation of human and rat receptors³⁶,³⁷
334 and identification of the GR cDNA³⁸,³⁹, GR remains one of the most intensely studied nuclear
335 receptors. GR is distinct among the steroid receptors in that it binds a wider range of DNA
336 elements, either as a dimer or as a monomer⁴⁰. As such, the individual receptor domains are
337 required to adapt to different oligomeric arrangements. The structures of GR<sub>ΔN</sub> binding to the
338 canonical GBS reveal a distinct domain arrangement. The keto-steroid receptor specific C-
339 terminal extension prevents the formation of the strong LBD dimer observed in ER¹⁶. Instead,
340 the GR LBD dimerization surface is shifted to an adjacent overlapping position, and the LBDs
341 form a unique head-to-tail dimer with a relatively small interface. We propose that this may
342 be a consequence of the requirement to signal in different contexts, as a strong LBD dimer
343 would potentially shift the signaling balance. The detailed quaternary arrangement presented
344 here provides a structural context for disease mutations and the information on the domain
345 interfaces will provide critical insights for future drug design. Future studies of multidomain
346 GR constructs in complex with DNA half-sites, inverted repeat binding elements and different
347 coregulators will further build the understanding of how the signaling flexibility correlates
348 with different structural arrangements.
349

350

351
352 References

353

354 1 Eick, G. N. & Thornton, J. W. Evolution of steroid receptors from an estrogen-sensitive ancestral receptor. *Molecular and Cellular Endocrinology* **334**, 31-38, doi:10.1016/j.mce.2010.09.003 (2011).

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360 3 Meijsing, S. H. et al. DNA Binding Site Sequence Directs Glucocorticoid Receptor Structure and Activity. *Science* **324**, 407-410, doi:10.1126/science.1164265 (2009).

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448
449 33 Heck, S. et al. A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-1. *The EMBO Journal* **13**, 4087-4095, doi:10.1002/j.1460-2075.1994.tb06726.x (1994).

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469 40 Hudson, W. H. et al. Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space. *Proceedings of the National Academy of Sciences* **113**, 326-331, doi:10.1073/pnas.1518960113 (2016).

473
474
a

<figure><img src="image_17.png" /><figcaption>2 - GR<sub>ΔN</sub>(Vel)-SGK-PGC1α</figcaption></figure>

<figure><img src="image_18.png" /></figure>

c

<figure><img src="image_19.png" /><figcaption>3 - GR<sub>ΔN</sub>(FF)</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>4 - GR<sub>ΔN</sub>(FF)-SGK-PGC1α</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>3 4</figcaption></figure>

<figure><img src="image_22.png" /></figure>

b

<table><thead><tr><th>Sample No</th><th>Sample</th><th>Protein</th><th>Peptide</th><th>dsDNA</th><th>Ligand</th><th>MALS MW (kDa)</th><th>calculated MW (kDa)</th></tr></thead><tbody><tr><td>1</td><td>GR<sub>ΔN</sub>(Vel)</td><td>GR<sub>385-777</sub>[S404A, N517D, V571M, F602S, C638D]</td><td></td><td></td><td>velsecorat</td><td>43.14</td><td>44.5</td></tr><tr><td>2</td><td>GR<sub>ΔN</sub>(Vel)-SGK-PGC1α</td><td>GR<sub>385-777</sub>[S404A, N517D, V571M, F602S, C638D]</td><td>PGC1α</td><td>SGK23blunt</td><td>velsecorat</td><td>98.14</td><td>107.5</td></tr><tr><td>3</td><td>GR<sub>ΔN</sub>(FF)</td><td>GR<sub>385-777</sub>[S404A, N517D, V571M, F602S, C638D]</td><td></td><td></td><td>fluticasone furoate</td><td>45.15</td><td>44.5</td></tr><tr><td>4</td><td>GR<sub>ΔN</sub>(FF)-SGK-PGC1α</td><td>GR<sub>385-777</sub>[S404A, N517D, V571M, F602S, C638D]</td><td>PGC1α</td><td>SGK23overhang</td><td>fluticasone furoate</td><td>98.47</td><td>107.5</td></tr></tbody></table>

475

476

477

Extended Data Figure 1 | SEC-MALS of GR<sub>ΔN</sub>(Vel), GR<sub>ΔN</sub>(FF), GR<sub>ΔN</sub>(Vel)-SGK-PGC1α

478

and GR<sub>ΔN</sub>(FF)-SGK-PGC1α. a, The monomeric GR proteins and dimeric GR complexes

479

eluted as single peaks. b, The experimentally determined and expected molecular weights. c,

480

GR proteins and GR complexes separated on a native PAGE.

481

482

483

484
<table><caption>**Extended Data Table 1 | Crystallographic data collection and refinement statistics.**</caption><thead><tr><th></th><th>GR<sub>AN</sub>(Vel)-SGK-PGC1α</th><th>GR<sub>LBD</sub>(Vel)-PGC1α</th><th>GR<sub>AN</sub>(FF)-SGK-PGC1α</th></tr></thead><tbody><tr><td><strong>Name</strong></td><td>GRα<sub>385-777</sub> [S404A, N517D, V571M, F602S, C638D]</td><td>GRα<sub>529-777</sub> [WT]</td><td>GRα<sub>385-777</sub> [S404A, N517D, V571M, F602S, C638D]</td></tr><tr><td><strong>GR construct</strong></td><td></td><td></td><td></td></tr><tr><td><strong>Ligand</strong></td><td>Velsecorat</td><td>Velsecorat</td><td>fluticasone furoate</td></tr><tr><td><strong>peptide</strong></td><td>PGC1α<sub>134-154</sub></td><td>PGC1α<sub>134-154</sub></td><td>PGC1α<sub>134-154</sub></td></tr><tr><td><strong>DNA</strong></td><td>SGK23blunt</td><td>N/A</td><td>SGK23overhang</td></tr><tr><td><strong>Data collection</strong></td><td></td><td></td><td></td></tr><tr><td>Space group</td><td>P212121</td><td>P21</td><td>P212121</td></tr><tr><td>α, b, c (Å)</td><td>80.21, 122.72, 130.45</td><td>42.34, 73.43, 43.93</td><td>79.67, 119.72, 135.53</td></tr><tr><td>α, β, γ (°)</td><td>90.0, 90.0, 90.0</td><td>90.0, 105.86, 90.0</td><td>90.0, 90.0, 90.0</td></tr><tr><td>Wavelength (Å)</td><td>0.96550</td><td>0.97242</td><td>0.96545</td></tr><tr><td>Resolution (Å)</td><td>89.39-2.5 (2.87-2.5)</td><td>73.43-2.2 (2.27-2.2)</td><td>68.7-2.7 (3.0-2.7)</td></tr><tr><td>Rmerge</td><td>0.082 (0.792)</td><td>0.124 (0.928)</td><td>0.067 (1.035)</td></tr><tr><td>Rpim</td><td>0.036 (0.373)</td><td>0.081 (0.466)</td><td>0.029 (0.425)</td></tr><tr><td>I/σ(I)</td><td>12.6 (1.9)</td><td>6.6 (1.7)</td><td>16.3 (1.6)</td></tr><tr><td>CC1/2 (%)</td><td>0.999 (0.744)</td><td>0.995 (0.709)</td><td>0.999 (0.706)</td></tr><tr><td>Completeness spherical (%)</td><td>51.0 (7.6)</td><td></td><td>70.2 (13.8)</td></tr><tr><td>Completeness ellipsoidal (%)</td><td>93.4 (76.1)</td><td></td><td>93.9 (63.5)</td></tr><tr><td>Completeness</td><td></td><td>99.0 (88.1)</td><td></td></tr><tr><td>Multiplicity (%)</td><td>6.3 (5.4)</td><td>3.3 (3.1)</td><td>6.5 (6.9)</td></tr><tr><td><strong>Refinement</strong></td><td></td><td></td><td></td></tr><tr><td>Resolution (Å)</td><td>89.39-2.5</td><td>42.26-2.2</td><td>68.7-2.7</td></tr><tr><td>Total reflections</td><td>45176</td><td>42980</td><td>36333</td></tr><tr><td>Unique reflections</td><td>23070</td><td>13075</td><td>24963</td></tr><tr><td>Rwork/Rfree (%)</td><td>0.202/0.263</td><td>0.226/0.281</td><td>0.216/0.254</td></tr><tr><td>No. of non-hydrogen atoms</td><td></td><td></td><td></td></tr><tr><td>Protein</td><td>5299</td><td>2168</td><td>5262</td></tr><tr><td>Nucleic Acid</td><td>939</td><td>-</td><td>896</td></tr><tr><td>Ligands</td><td>116</td><td>44</td><td>90</td></tr><tr><td>Water</td><td>64</td><td>46</td><td>30</td></tr><tr><td>Average B-factor (Å<sup>2</sup>)</td><td>88.0</td><td>50.0</td><td>103.0</td></tr><tr><td>Wilson B-factor (Å<sup>2</sup>)</td><td>66.0</td><td>27.7</td><td>76.1</td></tr><tr><td>RMS deviations</td><td></td><td></td><td></td></tr><tr><td>Bond lengths (Å)</td><td>0.008</td><td>0.008</td><td>0.008</td></tr><tr><td>Bond angles (°)</td><td>0.92</td><td>0.97</td><td>0.92</td></tr><tr><td>Ramachandran</td><td></td><td></td><td></td></tr><tr><td>Most favored (%)</td><td>94</td><td>98</td><td>94</td></tr><tr><td>Outliers (%)</td><td>0</td><td>0</td><td>1</td></tr><tr><td>Clashscore</td><td>5</td><td>2</td><td>6</td></tr><tr><td>PDB code</td><td>7PRW</td><td>7PRX</td><td>7PRV</td></tr></tbody></table>

Values in parentheses are for the highest resolution shell.
a

<figure><img src="image_23.png" /></figure>

b

<figure><img src="image_24.png" /></figure>

c

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<caption>Extended Data Figure 2 | GR<sub>AN</sub>(Vel)-SGK-PGC1α domains overlaid on structures of the isolated domains. a, DBD1 (purple), DBD2 (green) and dsDNA (red) overlaid on the structure of the DBD dimer alone on the same GBS (PDB: 3G9O, all in white). Zn atoms are denoted as grey and white spheres, respectively. b, LBD1 (blue) with velsecorat (magenta) and coactivator peptide PGC1α<sub>134-154</sub> (yellow) overlaid on the structure of GR<sub>LBD</sub> (white) in complex with velsecorat (black) and coactivator peptide PGC1α<sub>134-154</sub> (black). c, LBD2 (green) with velsecorat (magenta) overlayed on the structure of GR<sub>LBD</sub> (white) in complex with velsecorat (black) and coactivator peptide PGC1α<sub>134-154</sub> (black).</caption>
<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

503

504

**Extended Data Figure 3 | Sequence conservation of the GR LBD.** Alignment of a set of diverse GR related vertebrate sequences with a pairwise identity of 37-84%. The mean pairwise column identity of each residue as calculated by Geneious Prime is shown as bars in the top graph. The residues involved in the LBD:LBD and LBD:DBD interfaces are indicated by orange and green boxes, respectively.

505

506

507

508

509

510
<figure><img src="image_29.png" /><figcaption>**Extended Data Figure 4 | Sequence conservation of the ER LBD. a, Alignment of a set of diverse ER related vertebrate sequences with a pairwise identity of 38-90%. The mean pairwise column identity of each residue as calculated by Geneious Prime is shown as bars in the top graph. LBD dimer interface residues are highlighted with orange boxes.**</figcaption></figure>

511

512

513

514

515

516
517

a

<figure><img src="image_30.png" /></figure>

b

<figure><img src="image_31.png" /></figure>

518

519

**Extended Data Fig. 5** | Sequence conservation of key interfaces in the GR<sub>AN</sub>(Vel)-SGK-PGC1α complex. Conservation of GR LBD1 residues colored according to column identity between 0.4 (white) and 1.0 (blue) and shown as cartoon highlighting the LBD1 interface with **a**, the DBD and DNA and **b**, the PGC1α peptide (yellow).

520

521

522

523

524
<figure><img src="image_32.png" /><figcaption>Extended Data Fig. 6 | Structure of GR<sub>ΔN</sub>(Vel) in complex with SGK and PGC1α<sub>134-154</sub>.</figcaption></figure>

The structure of GR<sub>ΔN</sub>(Vel)-SGK-PGC1α and a crystallographic neighbor highlighting putative tetramer formation through the H1 interface.
<figure><img src="image_33.png" /><figcaption>Extended Data Figure 7 | The domain organization of the multidomain nuclear receptor X-ray structures on DNA. a, GR<sub>ΔN</sub>(Vel)-SGK-PGC1α. b, LXRβ-RXRα c, RARβ-RXRα. d, PPARγ-RXRα. e, HNF-4α. The sequence of the DNA binding motif is indicated below the DNA duplex with x denoting nucleotide spacer. The structures have been overlaid using the 5' DNA binding sequence as reference (highlighted in red) and are all shown from the same angle. The different coregulator peptides are shown in yellow and the ligands in magenta.</figcaption></figure>
a

<figure><img src="image_34.png" /></figure>

b

<figure><img src="image_35.png" /></figure>

Extended Data Figure 8 | Fluticasone furoate rearranges the region where H6 and H7

**meet. a,** Overlay of the GR<sub>ΔN</sub>(FF)-SGK-PGC1α LBD1 (white) with PGC1α peptide in black on the GR<sub>ΔN</sub>(Vel)-SGK-PGC1α LBD1 (blue) with PGC1α peptide in yellow. Fluticasone furoate and velsecorat are shown in black and magenta, respectively. **b,** Fluticasone furoate repositions Q642 and pushes on D638 and M639, rearranging the H6-H7 loop. Helix numbering is annotated within the circles.
a

<figure><img src="image_36.png" /></figure>

b

<figure><img src="image_37.png" /></figure>

c

<figure><img src="image_38.png" /><figcaption>Extended Data Figure 9 | Relative deuteration of specific peptides in GR(Vel)-SGK-PGC1α, GR(FF)-SGK-PGC1α, GR(Dex)-SGK-PGC1α, GR(Vel), GR(FF) and GR(Dex) complexes. a, peptide 425-436. b, peptide 536-544. c, peptide 628-636.</figcaption></figure>

551

552

553

554

555

556

557

558

559

560

561

562

563

564
<figure><img src="image_39.png" /></figure>

Total: 144 Peptides, 89.2% Coverage, 2.49 Redundancy

Total: 92 Peptides, 61.8% Coverage, 1.95 Redundancy

<caption>Extended Data Figure 10 | Protein coverage of GR and GR-SGK-PGC1α in HDX-MS. a, peptides used for HDX-MS analysis of GR. A coverage of 89.2% of the sequence was achieved. b, peptides used for HDX-MS analysis of GR-SGK-PGC1α complexes. A coverage of 61.8% of the sequence was achieved.</caption>

<figure><img src="image_40.png" /><figcaption>Extended data table 2.docx</figcaption></figure>

<caption>Extended Data Table 3 | Relative Uptake and Relative Uptake Error as identified with the DynamX 3.0 software (Waters) for all peptides and used for HDX analysis of GR(Dex), GR(FF), GR(Vel) and the complexes GR(Vel)-SGK-PGC1α, GR(FF)-SGK-</caption>
582 PGC1α and GR(Dex)-SGK-PGC1α. Vel=velsecorat, FF=fluticasone furoate,

583 Dex=dexamethasone.

<figure><img src="image_41.png" /><figcaption>Extended data table
3.docx</figcaption></figure>

584

585

586 Extended Data Table 4 | Relative Uptake Difference as identified with the DynamX 3.0

587 software (Waters) and p-values for all peptides used for HDX analysis of [GR(Vel)]

588 minus [GR(FF)]. Significant p-values are highlighted in grey. Vel=velsecorat,

589 FF=fluticasone furoate.

<figure><img src="image_42.png" /><figcaption>Extended data table
4.docx</figcaption></figure>

590

591

592 Extended Data Table 5 | Relative Uptake Difference as identified with the DynamX 3.0

593 software (Waters) and p-values for all peptides used for HDX analysis of [GR(Vel)-SGK-

594 PGC1α] minus [GR(FF)-SGK-PGC1α], [GR(Vel)-SGK-PGC1α] minus [GR(Vel)] and

595 [GR(FF)-SGK-PGC1α] minus [GR(FF)]. Significant p-values are highlighted in grey.

596 Vel=velsecorat, FF=fluticasone furoate.

<figure><img src="image_43.png" /><figcaption>Extended data table
5.docx</figcaption></figure>

597

598

599
Difference in deuterium uptake (Da)

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>

C-terminal peptide residue

<figure><img src="image_49.png" /></figure>

601

602

<caption>Extended Data Figure 11 | HDX difference plots showing deuterium uptake difference</caption>

603

between [complex A] – [complex B] for all peptides. Negative value indicate that peptides

604

are protected in A relative to B and vice versa.

605
606 Extended Data Table 6 | Relative Uptake Difference as identified with the DynamX 3.0
607 software (Waters) and p-values for all peptides used for HDX analysis of [GR(Vel)]
608 minus [GR(Dex)] and [GR(FF)] minus [GR(Dex)]. Significant p-values are highlighted in
609 grey. Vel=velsecorat, FF=fluticasone furoate, Dex=dexamethasone.

<figure><img src="image_50.png" /><figcaption>Extended data table
6.docx</figcaption></figure>

610

611

612 Extended Data Table 7 | Relative Uptake Difference as identified with the DynamX 3.0
613 software (Waters) and p-values for all peptides used for HDX analysis of [GR(Dex)-
614 SGK-PGC1α] minus [GR(Dex)], [GR(Vel)-SGK-PGC1α] minus [GR(Dex)-SGK-PGC1α]
615 and [GR(FF)-SGK-PGC1α] minus [GR(Dex)-SGK-PGC1α]. Significant p-values are
616 highlighted in grey. Vel=velsecorat, FF=fluticasone furoate, Dex=dexamethasone.

614

615

616

<figure><img src="image_51.png" /><figcaption>Extended data table
7.docx</figcaption></figure>

617
618 **Materials and Methods**

619

620 **Protein expression and purification**

621 Optimized DNA sequences of GR (human GRα₁-777 [WT]) and GRΔN (human GRα₃₈₅-777

622 [S404A, N517D, V571M, F602S, C638D]) with N-terminal 6xHN-tag followed by a TEV

623 site were cloned into pFastBac™1 and recombinant baculoviruses generated using the Bac-to-

624 Bac® system (invitrogen). S404 is a known phosphorylation site¹ and the mutations N517D,

625 V571M, F602S and C638D stabilize the GR LBD²⁻⁴. *Spodoptera frugiperda* insect cells

626 (SF21) were cultured at 27°C in Gibco™ Sf-900™ II SFM medium and infected with

627 baculovirus at a density of 2.5-3.0 x 10⁶ mL⁻¹. 24 h after transfection 10 µM ligand in DMSO

628 (velsecorat, fluticasone furoate or dexamethasone) was added to the cultures and 48 h after

629 transfection the cells were harvested. The cells were resuspended in 20 mM Hepes pH 7.0, 10

630 % (v/v) glycerol, 1 mM TCEP, 10 µM ligand, cOmplete™ EDTA-free protease inhibitor

631 cocktail (Roche, 1 tablet per 50 ml of solution) and flash frozen in liquid nitrogen. After

632 thawing the NaCl concentration was adjusted to 0.5 M by adding adequate amounts of a stock

633 solution containing 4.5 M NaCl, 20 mM Hepes pH 7.0, 10 % (v/v) glycerol, 1 mM TCEP, 10

634 µM ligand, the solution incubated for 15 Min on ice and the supernatant cleared by

635 centrifuging at 30.000 g for 45 Min. The supernatant was incubated with WorkBeads™ 40

636 Ni-NTA (Bio-Works) for 2 hours at 4°C, the beads were washed by gravity flow with buffer

637 containing 20 mM HEPES pH 7.0, 500 mM NaCl, 10 % glycerol, 1 mM TCEP, 10 µM

638 ligand, 25 mM Imidazole and 5 mM ATP and the protein eluted with increasing Imidazole

639 concentrations. The elution buffer was exchanged on a desalting column to 20 mM HEPES

640 pH 7.0, 500 mM NaCl, 10 % glycerol, 1 mM TCEP, 10 µM ligand before the HN-tag was

641 cleaved off by TEV digestion overnight at 4°C. The proteins were further purified by size
642

exclusion and the peak fractions pooled, concentrated to 1-3 mg/mL, flash frozen in liquid N₂
and stored at -80°C.

643

644

The wildtype GR<sub>LBD</sub>(Vel) (human GRα<sub>529-777</sub> [WT] with velsecorat) was cloned into the pET24a vector (Novagen) featuring an N-terminal His6-tag and a TEV protease cleavage site.

645

646

The expression vector was transformed into *E. coli* BL21(DE3) STAR, followed by expression in PASM-5052 autoinduction medium. 50 µM velsecorat was added after the cell culture reached an OD of 0.6 followed by expression over 48 hours at 16 °C. All purification buffers were degassed and contained 2 mM TCEP and 20 µM velsecorat. The harvested cells were resuspended in lysis buffer (50 mM Tris pH 8, 10% glycerol, 1% CHAPS) supplemented by protease inhibitors (Complete, Roche) and DNAse. Cells were lysed by sonication. The cleared lysate was applied to a nickel affinity column equilibrated with wash buffer (50 mM Tris pH 8.0, 10% glycerol, 1% CHAPS, 60 mM NaCl) and eluted by a 300 mM imidazole gradient. The purification tag was removed by TEV protease cleavage while dialyzing against a 50 mM Tris buffer at pH 9.0 overnight followed by a second Nickel affinity step to collect the cleaved target protein in the flowthrough. Remaining impurities were removed by size exclusion using 50 mM Tris buffer at pH 9.0, 2 mM TCEP, 20 µM velsecorat as running buffer. The purified protein in 50 mM Tris buffer pH 9.0, 2 mM TCEP, 10 µM velsecorat was flash frozen in liquid N₂ and stored at -80°C.

647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

## GRα complex formation with DNA and peptide

662

Peptides of human peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α<sub>134-154</sub>: PPQEAEEPSLLKKLLLLAPANT) dissolved in water and dsDNA (see below) from the GR DNA response element from the serum and glucocorticoid-regulated kinase-1 (SGK-1) were used for complex formation with GRα. The purified ligand bound GRα proteins were mixed with dsDNA (1 : 0.6 ratio of LBD monomer to ds DNA) and peptide (1 : 1.3 ratio of

663

664

665

666
LBD monomer to peptide) and dialyzed overnight into 20 mM MOPS pH 7.0, 150 mM NaCl, 3mM MgCl₂, 0.75 % glycerol, 1 mM TCEP, 10 µM ligand for crystallization or SEC-MALS.

DNA oligonucleotides (Sigma Aldrich) used for SGK dsDNA formation:

<table><thead><tr><th>SGK dsDNA</th><th>Oligo 1</th><th>Oligo 2</th></tr></thead><tbody><tr><td>SGK23overhang</td><td>SGK23oF:<br>5'-TACAGAACATTTTGTCCGTCGAC-3'</td><td>SGK23oR:<br>5'-TCGACGGACAAAATGTTCTGTAC-3'</td></tr><tr><td>SGK23blunt</td><td>SGK23bF:<br>5'-GTACAGAACATTTTGTCCGTCGA-3'</td><td>SGK23bR:<br>5'-TCGACGGACAAAATGTTCTGTAC-3'</td></tr><tr><td>SGK24blunt</td><td>SGK24F:<br>5'-GTACAGAACATTTTGTCCGTCGAC-3'</td><td>SGK24R:<br>5'-GTCGACGGACAAAATGTTCTGTAC-3'</td></tr><tr><td>FAM-SGK24</td><td>6-FAM-SGK24F:<br>5'-(6-FAM)-GTACAGAACATTTTGTCCGTCGAC-3'</td><td>SGK24R:<br>5'-GTCGACGGACAAAATGTTCTGTAC-3'</td></tr></tbody></table>

## Crystallization and crystal structure determination

The complex GR<sub>ΔN</sub>(FF)-SGK-PGC1α (human GRα<sub>385-777</sub> [S404A, N517D, V571M, F602S, C638D] with the agonist fluticasone furoate, the dsDNA SGK23overhang and the peptide PGC1α<sub>134-154</sub>) was concentrated to approx. 10 mg/mL and crystallization optimized in hanging drops using multiple rounds of seeding. Drops were set up with a 1 : 1 ratio of protein complex solution to well solution (8 % PEG3350, 0.1 M Bis-Tris-Propane pH 6.5, 8 % 2,2,2-Trifluoroethanol, 0.1 M Guanidine HCl, 0.3 M Hexanediol). The complex GR<sub>ΔN</sub>(Vel)-SGK-PGC1α with the agonist velsecorat, SGK23blunt and the peptide PGC1α was concentrated to approx. 10 mg/mL and crystallization optimized in hanging drops using multiple rounds of seeding. Drops were set up with a 1 : 1 ratio of protein complex to well solution (8.6 % PEG3350, 0.1 M Bis-Tris-Propane pH 6.5, 2 % 2,2,2-Trifluoroethanol, 0.1 M Guanidine HCl,
683 0.3M 1,6-Hexanediol). 30 % ethylene glycol or 30 % glycerol in well solution was used as
684 cryoprotectant and crystals were flash frozen in liquid nitrogen for data collection. Data was
685 collected with a Pilatus3 x 2M detector at the automated beamline ID30A-1/MASSIF-1 at the
686 European Synchrotron Radiation Facility (ESRF).

687 A twofold molar excess of PGC1$\alpha_{134-154}$ was added to GR$_{LBD}$(Vel) (human GR$\alpha_{529-777}$ [WT],
688 co-expressed with velsecorat) and the protein concentrated to 10 mg/mL. Crystals were grown
689 at 20 °C in sitting drops using a 1 : 1 (100 nL + 100 nL) ratio of protein and well solution (18
690 % PEG8000, 2 % 2-Propanol, 0.1 M Sodium acetate, 0.1 M HEPES pH7.5). Crystals were
691 cryo-protected in well solution supplemented with 20 % glycerol and flash frozen in liquid
692 nitrogen. Data was collected with a Pilatus 6M detector at the beamline ID23-1 at the ESRF.

693 All datasets were integrated and scaled with autoPROC (Global Phasing)⁵ and initial models
694 obtained performing molecular replacement with the known structures 4P6W and 3G9O using
695 PHASER⁶. The models were improved by iterative rounds of refinement using BUSTER
696 (Global Phasing) ⁷ and manual model building in COOT⁸. The PyMOL Molecular Graphics
697 System, Version 2.1.0, Schrödinger, LLC, was used to generate figures.

698 Detailed statistics of data collection and refinement can be found in Supplementary Table 1.
699 All coordinates and structure factors have been deposited in the Protein Data Bank (PDB)
700 under the accession codes 7PRV, 7PRW and 7PRX.

701 PDBePISA⁹ has been used for interface analysis and Cα root mean square deviation (r.m.s.d.)
702 values calculated with lsqkab¹⁰.

704 Analysis of the sequence conservation of GR

705 A set of GR-like vertebrate sequences were gathered through iterative Blast searches ¹¹,
706 starting with human GR (NP_000167.1). Sequences with an identity above 50% to the query
707 were aligned with Geneious Prime v. 2021.1.1 using MUSCLE ¹². All entries with an identity
708 >90% to any other entry in the set were purged, while the least similar sequence to the query
709 was used for subsequent searches. This resulted in a diverse alignment of the GR family with
710 a pairwise sequence identity of 37-84% between all sequences (Extended Data Fig. 3). A set
711 of ER-like vertebrate sequences were gathered by a similar approach, starting with human ER
712 (NP_000116.2) (Extended Data Fig. 4) and purged to match the diversity of the GR set. The
713 mean pairwise column identity of each residue was calculated by Geneious, plotted by
714 Spotfire Analyst 11.4 (Extended Data Figure 3 and 4) and subsequently color mapped onto
715 the GR and ER LBD structures (Fig. 2d, e and Extended Data Fig. 5)

716

717 Binding assay to dsDNA in competition mode

718 The affinity of GR (human WT GRα₁-777) in the presence of different ligands to fluorescein
719 (6-FAM) labelled dsDNA FAM-SGK24 was analyzed using fluorescence polarization in
720 competition mode.

721 150 nM GR complexed with the ligands dexamethasone [GR(Dex)], velsecorat [GR(Vel)] or
722 fluticasone furoate [GR(FF)] was mixed with 10 nM FAM-SGK24 in HBSP buffer (10 mM
723 HEPES pH 7.4, 150 mM NaCl, 0.005 % Tween-20®). This preformed complex was mixed
724 with a dilution series (1.5 µ, 750 nM, 375 nM, 187.5 nM, 93.75 nM, 46.88 nM, 23.44 nM,
725 11.7 nM, 5.9 nM and 0 nM) of competing unlabeled dsDNA SGK24blunt. Fluorescence
726 polarization was measured using a PHERAStar microplate reader (BMG LABTECH) with the
727 FP Module 485-520/520. IC50 value was determined by simultaneous fitting all curve data
728 (three curve replicates from one experiment) using a four parameter non-linear curve fit in
729 GraphPad Prism 9.

730

731 Hydrogen/deuterium exchange mass spectrometry (HDX-MS)
The HDX experiments were carried out using a HDX Manager (Waters) equipped with a CTC
PAL sample handling robot (LEAP Technologies). GR (human GRα₁-777 [WT]) in the
presence of the ligand dexamethasone [GR(Dex)], velsecorat [GR(Vel)] or fluticasone furoate
[GR(FF)] and also the complexes GR(Dex)-SGK-PGC1α, GR(FF)-SGK-PGC1α and
GR(Vel)-SGK-PGC1α (GR with the dsDNA SGK24blunt and PGC1α₁₃₄-1₅₄) were dialyzed
into the HDX compatible buffer 20 mM HEPES pH 7.0, 200 mM NaCl, 2.5 % glycerol, 1
mM TCEP, 20 µM ligand and the exchange reactions performed with a CTC PAL sample
handling robot (LEAP Technologies). The samples were labelled for 0 s, 30 s, 60 s, 180 s, 600
s and 1800 s by incubating 3 µl protein (GR at 1 mg/mL or GR complexes at 0.6 mg/mL) with
57 µl of D₂O buffer (20 mM HEPES pD 7.4, 200 mM NaCl, 2.5 % glycerol, 1 mM TCEP) at
22 °C. To stop the exchange reaction Quench (3M Urea, 0.1 % TFA, pH2.5) was added 1 : 1
at 4 °C and the mixture was injected onto an online pepsin digestion system with a Waters
Enzymate™ BEH Pepsin Column (2.1 x 30 mm, 5 µm) in 0.1 % formic acid in water at 150
uL/min for 3 min at 0.1 °C. The peptides were trapped/desalted for 3 min on a Waters
ACQUITY UPLC BEH C18 VanGuard Pre-Column (2.1 × 5 mm, 1.7 µm) and separated on a
using a C18 reverse phase column (ACQUITY UPLC BEH C18 Column, 1.7 µm,
2.1 × 100 mm, Waters) by running a linear gradient from 5 - 40 % solvent B (solvent A: 0.1 %
formic acid in water; solvent B: 0.1 % formic acid in acetonitrile) over 9 min. The peptides
were analyzed on a Waters Synapt G2-Si mass spectrometer, identified using MSE
fragmentation. Mass spectrometry experiments acquired over a mass range from 50 to
2000 m/z using an electrospray ionization source operated at a temperature of 200 °C and a
spray voltage of 4.5 kV. All reactions were carried out in triplicates and no correction was
made for back exchange, therefore all results are reported as relative deuterium exchange
levels.
756

Peptides were identified using the ProteinLynx Global Server 3.0.2 (PLGS, Waters) and

757

deuterium incorporation was analyzed in DynamX 3.0 (Waters). Tables including all peptides

758

with sequences, monoisotopic mass, retention time and mean deuterium uptake is supplied as

759

Extended Data Tables 2 and 3 following guidelines by¹³. Relative deuterium levels were

760

calculated for each peptide by subtracting the average mass of the deuterium-labeled GR with

761

ligand 1 from that of the deuterium-labeled GR with ligand 2. Statistical significance was

762

determined in t tests as p values < 0.05 and listed in Extended Data Tables 4, 5, 6 and 7.

763

Visualization was done using GraphPad Prism 9.

764

765

**Size exclusion chromatography multi angle light scattering (SEC-MALS)**

766

A Cytiva Superdex® 200 Increase 10/300 GL column on an OMNISEC RESOLVE/REVEAL

767

System (Malvern Panalytical) was used to analyze the absolute molecular weight (MW) of

768

GR<sub>ΔN</sub>(FF) and GR<sub>ΔN</sub>(Vel) (human GRα<sub>385-777</sub> [S404A, N517D, V571M, F602S, C638D] with

769

the agonist fluticasone furoate or velsecorat) in the buffer 20 mM HEPES pH 7.0, 250 mM

770

NaCl, 2.5 % glycerol, 1 mM TCEP, 10 µM ligand at a flow rate of 0.4 mL/min.

771

A Cytiva Superose® 6 Increase 10/300 GL column on an OMNISEC RESOLVE/REVEAL

772

System (Malvern Panalytical) was used to analyze the absolute molecular weight (MW) of the

773

complexes GR<sub>ΔN</sub>(FF)-SGK-PGC1α and GR<sub>ΔN</sub>(Vel)-SGK-PGC1α (human GRα<sub>385-777</sub> [S404A,

774

N517D, V571M, F602S, C638D] with the agonist fluticasone furoate or velsecorat, the

775

dsDNA SGK23overhang or SGK23blunt respectively and PGC1α<sub>134-154</sub>) in the buffer 20 mM

776

MOPS pH 7.0, 150 mM NaCl, 3 mM MgCl₂, 0.75 % glycerol, 1 mM TCEP, 10 µM ligand at

777

a flow rate of 0.4 mL/min.

778

The software OmniSEC5.12 was used to analyze the data with the 3 detector method and to

779

determine the molecular weights of the proteins and protein-DNA-peptide complexes.
780 Analyzed proteins and protein complexes were also separated on a native PAGE
(NuPAGE™ 3 to 8%, Tris-Acetate, Invitrogen).

782
783 Inhibition of cytokine (TNF-α) release in whole blood

784 Whole blood from human with sodium heparin as anticoagulant was used as an anti-
785 inflammatory model to test for steroid like activity (TNF-α inhibition) of GR agonists. A
786 dilution series (1 µM, 0.33 µM, 0.11 µM, 0.037 µM, 0.012 µM, 0.0041 µM, 0.0013 µM,
787 0.00045 µM) of velsecorat, fluticasone furoate or dexamethasone in DMSO was analyzed. 1
788 µl of GR agonist was mixed with 190 µL of whole blood and incubated for 45 min at 37°C in
789 96-well plates. 10 µl LPS, Lipopolysaccharide from E.coli serotype O127:B8 2mg/ml in PBS
790 (SigmaAldrich), was added per well and the plates were incubated overnight at 37°C. The
791 cells were spun down at 1800 rpm for 10 min at 4°C and the culture supernatants stored at -
792 80°C until analysis. The culture supernatants were diluted and added to ELISA plates (R&D
793 Systems) and TNF-α was measured according to the manufacturer's instructions. The raw
794 data in pg/ml was normalized to % inhibition. The IC50 value was determined by
795 simultaneous fitting all curve data (six curve replicates from three donors) using a four
796 parameter non-linear curve fit in GraphPad Prism 9.

797
798 Transactivation assay

799 A reporter vector for GR activity was constructed by cloning the glucocorticoid receptor
800 responsive MMTV promoter upstream of a codon optimized (GeneArt, ThermoFisher
801 Scientific) NanoLuc® Luciferase reporter to replace the CMV promoter in a pcDNA3.1
802 backbone. A plasmid containing a Firefly luciferase driven by the PGK promoter (#E5011,
803 Promega) was co-transfected and used for normalization.
804 GR<sub>WT</sub> (human GRα<sub>1-777</sub> [WT]) and 6 different GR mutants (GR<sub>cryst</sub>(S404A, N517D, V571M, F602S, C638D), GR(A458T), GR(R614A), GR(K720D), GR(D641K) and GR(Y640S) were synthesized, codon optimized (GeneArt, ThermoFisher Scientific) and cloned under the control of a CMV promoter into pcDNA3.1.

808 For transactivation assays COS-7 cells (ECACC 87021302, <sup>14</sup>) were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions and seeded into 384-well OptiPlates (PerkinElmer) at 5000 cells / well and seeded into 6 well plates (620,000 cells /well) for subsequent immunoblotting. Following 24 hours of incubation the 384-well plates were treated with dexamethasone at the indicated concentrations. Reporter gene activation of NanoLuc® and Firefly Luciferase was read out 24 hours later using Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions. GraphPad Prism 9 was used for data analysis and graph generation. The EC50 value was determined by simultaneous fitting all curve data (eight curve replicates from four experiments) using a four parameter non-linear curve fit in GraphPad Prism 9.

818 To verify the expression of the different GR mutants in COS-7 cells, immunoblots of the transfected cells were carried out using a rabbit anti-GR-antibody (Cell Signaling, #3660) and a mouse anti-actin antibody (Invitrogen, # MA5-11869). Detection was performed using corresponding secondary antibodies (LI-COR #926-68071; #926-32210). Densitometry quantification was performed using Image Studio Software (LI-COR)(LICOR).

823

824

825 References

826

827 1 Galliher-Beckley, A. J., Williams, J. G., Collins, J. B. & Cidlowski, J. A. Glycogen

828 synthase kinase 3beta-mediated serine phosphorylation of the human glucocorticoid
829 receptor redirects gene expression profiles. *Molecular and cellular biology* **28**, 7309-
830 7322, doi:10.1128/MCB.00808-08 (2008).

831 2 Bledsoe, R. K. et al. Crystal Structure of the Glucocorticoid Receptor Ligand Binding Domain Reveals a Novel Mode of Receptor Dimerization and Coactivator Recognition. *Cell* **110**, 93-105, doi:10.1016/S0092-8674(02)00817-6 (2002).

834 3 Carlsson, P., Koehler, K. F. & Nilsson, L. Glucocorticoid Receptor Point Mutation V571M Facilitates Coactivator and Ligand Binding by Structural Rearrangement and Stabilization. *Molecular Endocrinology* **19**, 1960-1977, doi:10.1210/me.2004-0203 (2005).

838 4 Kauppi, B. et al. The Three-dimensional Structures of Antagonistic and Agonistic Forms of the Glucocorticoid Receptor Ligand-binding Domain: RU-486 INDUCES A TRANSCONFORMATION THAT LEADS TO ACTIVE ANTAGONISM. *Journal of Biological Chemistry* **278**, 22748-22754, doi:10.1074/jbc.M212711200 (2003).

842 5 Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. *Acta Crystallographica Section D* **67**, 293-302, doi:10.1107/S0907444911007773 (2011).

844 6 McCoy, A. J. et al. Phaser crystallographic software. *Journal of Applied Crystallography* **40**, 658-674, doi:10.1107/S0021889807021206 (2007).

846 7 Bricogne, G. et al. BUSTER version *Cambridge, United Kingdom: Global Phasing Ltd.* (2017).

848 8 Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. *Acta Crystallographica Section D* **66**, 486-501, doi:10.1107/S0907444910007493 (2010).

851 9 Krissinel, E. & Henrick, K. Inference of Macromolecular Assemblies from Crystalline State. *Journal of Molecular Biology* **372**, 774-797, doi:10.1016/j.jmb.2007.05.022 (2007).
854 10 Kabsch, W. A solution for the best rotation to relate two sets of vectors. *Acta Crystallographica Section A* **32**, 922-923, doi:10.1107/S0567739476001873 (1976).

856 11 Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. *Nucleic acids research* **25**, 3389-3402, doi:10.1093/nar/25.17.3389 (1997).

859 12 Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. *Nucleic Acids Research* **32**, 1792-1797, doi:10.1093/nar/gkh340 (2004).

861 13 Masson, G. R. et al. Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. *Nature Methods* **16**, 595-602, doi:10.1038/s41592-019-0459-y (2019).

864 14 Gluzman, Y. SV40-transformed simian cells support the replication of early SV40 mutants. *Cell* **23**, 175-182, doi:10.1016/0092-8674(81)90282-8 (1981).

867 Acknowledgments

868 We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and we would like to thank the beamline staff for assistance in using beamline ID30A-1/MASSIF-1 and ID23-1. S.P. and C.K. were supported by the AstraZeneca postdoctoral program. We thank John Steele, Rose Maciewicz, Nils-Olov Hermansson, Matti Lepistö and Richard Neutze for scientific discussions. This work was supported by CNRS, Inserm, Institut National du Cancer (INCa_16099), Fondation pour la Recherche Médicale (FRM), Agence Nationale pour la Recherche (ANR) and the French Infrastructure for Integrated Structural Biology FRISBI ANR-10-INSB-05-01, Instruct-ERIC, and the French Proteomic Infrastructure ProFI ANR-10-INBS-08-03.

879 Author contributions
880 S.P. purified the proteins and crystallized the GR<sub>ΔN</sub> complexes. C.K. purified the wildtype

881 GR<sub>LBD</sub> protein. L.W. crystallized the GR<sub>LBD</sub>. S.P. and K.E. solved the structures and wrote the

882 manuscript with input from all authors. S.P. and C.J. performed HDX experiments and

883 analyzed the data. S.P. and A.G. performed fluorescence polarization assays. S.P., M.C. and

884 E.G. performed SEC-MALS and analyzed the data. P.J. analyzed the sequence conservation

885 of GR and ER. B.C., D.Ö., L.F.R., S.P., K.E., I.D., S.D designed and cloned constructs and

886 performed cell assays. B.B., B.P.K. and I.B.M. helped to conceive and conceptualize the

887 study and interpret structural data.

888

889 **Competing interests**

890 Sandra Postel, Lisa Wissler, Carina A. Johansson, Anders Gunnarsson, Euan Gordon, Barry

891 Collins, Christian Köhler, Marie Castaldo, David Öling, Patrik Johansson, Linda Fröderberg

892 Roth, Ian Dainty, Stephen Delaney and Karl Edman were employed by AstraZeneca at the

893 time of the study. The authors declare no competing financial interest.

894

895 **Data availability**

896 The X-ray data and coordinates for the GR<sub>ΔN</sub>(Vel)-SGK-PGC1α, GR<sub>LBD</sub>(Vel)-PGC1α and

897 GR<sub>ΔN</sub>(FF)-SGK-PGC1α structures are deposited in the PDB (7PRW, 7PRX and 7PRV,

898 respectively). The data will be released upon publication.

899

900 **Corresponding Author**

901 Karl.Edman@astrazeneca.com