Allosteric interactions prime androgen receptor dimerization and activation

“Individual domains of the AR LBD and DBD were manually docked into respective EM maps and subject to rigid body refinement in Chimera (Pettersen et al., 2004) using the PDB coordinates for the AR LBD (1XOW) (He et al.”

“S11L, M) (Edgar, 2004), we queried whether ERG could modulate other 3K-SRs, including the mineralocorticoid receptor (MR) and GR, as the latter can bypass AR blockade to drive ENZ resistance in CRPC (Arora et al.”

“Frames were motion-corrected using MotionCor2 (Zheng et al., 2017) and the contrast transfer function estimated using CTFFIND4 (Rohou and Grigorieff, 2015).”

“Driven by the biology of ETS transcription factor translocations in prostate cancer (Cancer Genome Atlas Research, 2015; Chen et al., 2013; Yu et al., 2010), we introduced the oncoprotein ERG into this system and demonstrated ERG is a bonafide AR cofactor, endowed with a LXXLL-like AR interacting motif (AIM) that can…”

“—The ARE repeat reporter (4X-ARE, firefly luciferase) has been previously described (Tran et al., 2009).”

“Consistent with our model that partially primed AR is more vulnerable to ERG modulation, we noted organoid establishment and gene expression were more pronounced under conditions favoring low AR activity, either through ENZ treatment or DHT withdrawal (Fig.”

“S11L, M) (Edgar, 2004), we queried whether ERG could modulate other 3K-SRs, including the mineralocorticoid receptor (MR) and GR, as the latter can bypass AR blockade to drive ENZ resistance in CRPC (Arora et al., 2013).”

“Driven by the biology of ETS transcription factor translocations in prostate cancer (Cancer Genome Atlas Research, 2015; Chen et al., 2013; Yu et al., 2010), we introduced the oncoprotein ERG into this system and demonstrated ERG is a bonafide AR cofactor, endowed with a LXXLL-like AR interacting motif (AIM) that can…”

“Indeed, the few reported dimeric structures of 3K-SRs exhibit variability around their dimerization interfaces, likely indicative of a low affinity interaction (Bledsoe et al., 2002; Nadal et al., 2017; Williams and Sigler, 1998).”

“This scenario is in contrast to that of the AnSRs that exist as obligate dimers, exhibit less flexibility, and maintain binding to repeat consensus sequences in vivo (70%), even in the presence of oncoprotein cofactors (Arruabarrena-Aristorena et al., 2020; Chandra et al., 2008; Chandra et al., 2013; Chandra et al., 2…”

“Mouse prostate organoids were isolated and cultured in Matrigel (Corning) using standard methods (Karthaus et al., 2014).”

“However, AR’s highly degenerate cistrome is inconsistent with a model whereby dimerization relies on canonical ARE repeats (Massie et al., 2007; Wang et al., 2007; Wilson et al., 2016).”

“, 2004) could be due in part to flexibility conferred by the Hinge and the conserved five residue “lever arm,” a loop in the DBD previously identified in GR that permits degeneracy within the DNA consensus motif without altering affinity (Meijsing et al., 2009).”

“Indeed, the few reported dimeric structures of 3K-SRs exhibit variability around their dimerization interfaces, likely indicative of a low affinity interaction (Bledsoe et al., 2002; Nadal et al., 2017; Williams and Sigler, 1998).”

“FL ERG repressed activity of an AR variant lacking the N-terminus, but exerted no effect on AR V7, a splice isoform lacking the LBD that is expressed in CRPC patients and associated with anti-androgen resistance (Watson et al., 2010) (Fig.”

“That these disorders present with a spectrum of physical and molecular phenotypes (Cancer Genome Atlas Research, 2015; Jeske et al., 2007; La Spada et al., 1991; Lee et al., 2019; McPhaul et al., 1992; Robinson et al., 2015) suggests AR can exist in fully and partly primed states.”

“Nuclear AR binds both palindromic and direct repeats of DNA hexamers known as androgen response elements (AREs) to activate its gene expression program, and is further regulated through association with numerous protein cofactors that bind the NTD or LBD through LXXLL and related motifs (Brooke et al., 2008; Weikum et…”

“This scenario is in contrast to that of the AnSRs that exist as obligate dimers, exhibit less flexibility, and maintain binding to repeat consensus sequences in vivo (70%), even in the presence of oncoprotein cofactors (Arruabarrena-Aristorena et al., 2020; Chandra et al., 2008; Chandra et al., 2013; Chandra et al., 2…”

“Whether allosteric surfaces within the 3K-SR LBD contribute to DNA binding remains unclear as structural information has been limited to individual domains (He et al., 2004; Shaffer et al., 2004) or lacks structural features to unambiguously assign DNA or individual domains (Yu et al.”

“Segmentation of individual domains of AR was performed using Segger (Pintilie et al., 2010) in ChimeraX.”

“Crosslinked lysines were depicted in 2D using xiNET cross-link viewer (Combe et al., 2015) and rendered in 3D as solid-colored spheres mapped onto the Entrenched model using PyMol.”

“Current structural studies suggest that 3K-SRs may have acquired a mechanism of activation distinct from other NRs, as their LBDs most often crystallize as monomers (He et al., 2004), in contrast to ER (Greschik et al.”

“Notably, germline mutations within the dimer (N759 and R761) and DBD interfaces (R846 and R854) have been detected in individuals presenting with PAIS, underscoring the physiological importance of these surfaces (Boehmer et al., 2001; Jeske et al., 2007; McPhaul et al., 1992).”

“Driven by the biology of ETS transcription factor translocations in prostate cancer (Cancer Genome Atlas Research, 2015; Chen et al., 2013; Yu et al., 2010), we introduced the oncoprotein ERG into this system and demonstrated ERG is a bonafide AR cofactor, endowed with a LXXLL-like AR interacting motif (AIM) that can…”

“However, AR’s highly degenerate cistrome is inconsistent with a model whereby dimerization relies on canonical ARE repeats (Massie et al., 2007; Wang et al., 2007; Wilson et al., 2016).”

“To enrich for and identify productively bound AR complexes, we performed gentle cross-linking during ultracentrifugation (Stark, 2010) followed by a combination of AFM, negative stain and single particle cryo-EM to screen individual fractions (Fig.”

“progression by redistributing AR binding from higher affinity ARE repeats to lower affinity half sites or degenerate sequences (Chen et al., 2013; Jin et al., 2014; Mao et al., 2019; Norris et al., 2009).”

“S10C) and measured their ability to activate reporters with either degenerate (half-site) or palindromic ARE sequences (Huang et al., 1999; Zhang et al., 2000).”

“Regulation of AR is multimodal, with androgen binding, relief of NTD-mediated autoinhibition, and NTD-cofactor interactions all contributing to AR activation (He et al., 2001; He et al., 2004; Schaufele et al., 2005; Wasmuth et al., 2020; Yu et al., 2020).”

“This scenario is in contrast to that of the AnSRs that exist as obligate dimers, exhibit less flexibility, and maintain binding to repeat consensus sequences in vivo (70%), even in the presence of oncoprotein cofactors (Arruabarrena-Aristorena et al., 2020; Chandra et al., 2008; Chandra et al., 2013; Chandra et al., 2…”

“While all three models share this common dimerization interface, the Entrenched state is the most compact, exhibiting the best fit for the X-ray structure of a LBD dimer (Nadal et al., 2017), whereas the extended conformation in the Divorced state deviates the most (Fig.”

“While many of these sites are not normally associated with active transcription, the overexpression of AR cofactors in prostate cancer is thought to activate expression of pro-proliferative genes at these lower affinity degenerate sites (Chen et al., 2013; Jin et al., 2014; Liu et al., 2017; Mao et al., 2019; Wasmuth…”

“S10C) and measured their ability to activate reporters with either degenerate (half-site) or palindromic ARE sequences (Huang et al., 1999; Zhang et al., 2000).”

“For AR Hinge variants, residues of unknown function proximal to the LBD (652-671) were excised, as this region is distal from the bipartite nuclear localization sequence previously implicated in DNA binding and acetylation (Haelens et al., 2007).”

“Notably, germline mutations within the dimer (N759 and R761) and DBD interfaces (R846 and R854) have been detected in individuals presenting with PAIS, underscoring the physiological importance of these surfaces (Boehmer et al., 2001; Jeske et al., 2007; McPhaul et al., 1992).”

“Regulation of AR is multimodal, with androgen binding, relief of NTD-mediated autoinhibition, and NTD-cofactor interactions all contributing to AR activation (He et al., 2001; He et al., 2004; Schaufele et al., 2005; Wasmuth et al., 2020; Yu et al., 2020).”

“The fact that AR exists as a non-obligate dimer when bound to DNA is in stark contrast to AnSRs and type II NRs, whose LBDs are sufficient for dimerization at a distinct yet conserved interface (see below) (Hochberg et al., 2020; Weikum et al., 2018).”

“This scenario is in contrast to that of the AnSRs that exist as obligate dimers, exhibit less flexibility, and maintain binding to repeat consensus sequences in vivo (70%), even in the presence of oncoprotein cofactors (Arruabarrena-Aristorena et al., 2020; Chandra et al., 2008; Chandra et al., 2013; Chandra et al., 2…”

“However, AR’s highly degenerate cistrome is inconsistent with a model whereby dimerization relies on canonical ARE repeats (Massie et al., 2007; Wang et al., 2007; Wilson et al., 2016).”

“2F), suggesting that when AR is partly primed through limiting ligand or compromised allosteric interactions, DNA consensus repeats can directly promote 3K-SR dimerization (Hochberg et al., 2020; McKeown et al., 2014).”

“To model the shortest (HNF-4α, a type II NR) and longest (MR) known NR hinges, we altered AR’s 42 residue Hinge by 20 amino acids in regions of uncharacterized function (Clinckemalie et al., 2012) (Fig.”

“This scenario is in contrast to that of the AnSRs that exist as obligate dimers, exhibit less flexibility, and maintain binding to repeat consensus sequences in vivo (70%), even in the presence of oncoprotein cofactors (Arruabarrena-Aristorena et al., 2020; Chandra et al., 2008; Chandra et al., 2013; Chandra et al., 2…”

“Nuclear AR binds both palindromic and direct repeats of DNA hexamers known as androgen response elements (AREs) to activate its gene expression program, and is further regulated through association with numerous protein cofactors that bind the NTD or LBD through LXXLL and related motifs (Brooke et al., 2008; Weikum et…”

“While many of these sites are not normally associated with active transcription, the overexpression of AR cofactors in prostate cancer is thought to activate expression of pro-proliferative genes at these lower affinity degenerate sites (Chen et al., 2013; Jin et al., 2014; Liu et al., 2017; Mao et al., 2019; Wasmuth…”

“S11C, D) (Mackereth et al., 2004; Regan et al., 2013), consistent with the ERG-AR interface mapped by XL-MS (Fig.”

“This scenario is in contrast to that of the AnSRs that exist as obligate dimers, exhibit less flexibility, and maintain binding to repeat consensus sequences in vivo (70%), even in the presence of oncoprotein cofactors (Arruabarrena-Aristorena et al., 2020; Chandra et al., 2008; Chandra et al., 2013; Chandra et al., 2…”

“S11C, D) (Mackereth et al., 2004; Regan et al., 2013), consistent with the ERG-AR interface mapped by XL-MS (Fig.”

“intrinsically disordered, is not necessary for ERG association and cooperative stimulation, and contributes to N-C autoinhibition in the absence of NTD-cofactor association (He et al., 2001; Schaufele et al., 2005; van Royen et al., 2012; Wasmuth et al., 2020).”

“AR can tolerate higher levels of sequence degeneracy within its ARE, an important feature required for normal development, and a unique feature among steroid receptors (Adler et al., 1993; Sahu et al., 2014), with 70% of its cistrome comprised of half-sites and up to 99% exhibiting some level of degeneracy (Massie et…”

“That these disorders present with a spectrum of physical and molecular phenotypes (Cancer Genome Atlas Research, 2015; Jeske et al., 2007; La Spada et al., 1991; Lee et al., 2019; McPhaul et al., 1992; Robinson et al., 2015) suggests AR can exist in fully and partly primed states.”

“Pten−/−;sgERG organoids previously generated by electroporation of Cas9-sgRNA ribonucleoprotein complexes against ERG (Feng et al., 2021) were transduced with an empty vector control, or an allelic series of WT and mutant ERG variants and selected with 2 μg/mL puromycin for 1 week.”

“While many of these sites are not normally associated with active transcription, the overexpression of AR cofactors in prostate cancer is thought to activate expression of pro-proliferative genes at these lower affinity degenerate sites (Chen et al., 2013; Jin et al., 2014; Liu et al., 2017; Mao et al., 2019; Wasmuth…”

“Notably, germline mutations within the dimer (N759 and R761) and DBD interfaces (R846 and R854) have been detected in individuals presenting with PAIS, underscoring the physiological importance of these surfaces (Boehmer et al., 2001; Jeske et al., 2007; McPhaul et al., 1992).”

“Driven by the biology of ETS transcription factor translocations in prostate cancer (Cancer Genome Atlas Research, 2015; Chen et al., 2013; Yu et al., 2010), we introduced the oncoprotein ERG into this system and demonstrated ERG is a bonafide AR cofactor, endowed with a LXXLL-like AR interacting motif (AIM) that can…”

“The direct interaction between the AR and ERG oncogene has recently been confirmed by a report of the cryo-EM structure elucidating this interaction (52).”

“The 3D structures of several NRs truncated by their NTDs and in complex with DNA have now been obtained by X-ray crystallography or cryoEM for PPAR–RXR [61], HNF4 [62], LXR–RXR [63], RAR–RXR [64], EcR–USP [65] and AR [66].”

“76 The LBD is located at the receptor ’ s C-terminus and contains 220--250 AAs.”

“There is also now clear evidence now in both the AR homodimer DNA complex and the ER homodimer DNA complex for DBD–LBD interactions (Huang et al. 2018, Wasmuth et al. 2022).”

“Amplification of the AR gene, mutation of AR’s ligand-binding domain (LBD) and/or AR cofactordirected mechanisms can lead to a hyperactive AR signaling during prostate tumorigenesis (5,6); in addition, the AR gene undergoes alternative splicing with inclusion of cryptic exons in CRPC, giving rise to the truncated, hor…”

“…co-crystal structures (PPAR g -RXR a heterodimer, HNF4 a homodimer and RXR a - RAR b heterodimer) by X-ray crystallography, and subsequent NR complex structure determinations consistently fi nd domain-domain contacts and physical interactions within all multi-domain complexes (30, 37, 39, 40, 71).”

“…for a refinement of our understanding of NR structure–function relationships, including quaternary structure and interdomain communication, 8 9 hence improving the search for novel ligands/drugs targeting these NRs. 10 However, no ligands have so far been identified for some NRs, which…”

“Moreover, using a range of DHT concentrations that partially to fully prime the AR for activity, partially saturated or antagonist-bound AR were most susceptible to regulation by ERG on AR half sites [97].”

“AR is a nuclear receptor that regulates gene expression programs required for prostate development and male phenotype maintenance [41].”

“Structural models of PTGES3 and AR chaperone and DNA-bound complexes were generated using a combination of experimentally derived structural models (PDB: 1XOW; PDB: 1R4I; PDB: 7KRJ) 43,50,67,68 and Alphafold 3 69 .”

“Accumulating evidence has indicated that progression to advanced drug-resistant CRPC involves multiple molecular mechanisms correlated with irregular AR signaling [4, 5].”

“Given that AR binds to response element DNA as a homodimer (Nadal et al., 2017; Wasmuth et al., 2022), ADP-ribose reading by the oligomeric DTX3L-PARP9 complex should be an efficient mechanism for concentrating ubiquitin E3 ligase activity on AR target genes.”

“CryoEM structures of full-size AR are revealing how AR dimers interact and recruit coregulators in greater detail, although the resolution of these structures doesn ’ t reach the atomic resolution of our crystal-based structures and no effects of varying DNA sequences has been reported [83,84,85].”

“Thanks to a multitude of crystal and solution structures of the DBD and the LBD(Wasmuth et al., 2022) in combination with the cryoEM solution structure of the full AR in complex with SRC-3 and p300 (Yu et al., 2020), the unique head-to-head and tail-to-tail dimerization of the AR has been…”

“Notably, a recent study using cryo-EM combined with cross-linking mass spectrometry found that an adjacent lysine (K913) was in proximity to the DNA binding domain, suggesting that ubiquitylation at K911 could disrupt DNA binding 28 .”

“Furthermore, not only can 3 K-SR protein allostery be mediated by the degree of ligand affinity but also by the degree of DNA affinity; with contributions from both elements ultimately determining the activity of receptor [127].”

“Furthermore, AlphaFold 17 models of nuclear receptors frequently show cloud-like AF-1/NTD structural depictions that are thought to be an artifact of the computational method to avoid steric clashes with structured domains 18 .”

“More recently, it has become clear that ERG expression has marked global effects on gene expression in PCa cells and, in particular, on the AR cistrome and transcriptome, where it directly interacts with AR and functions to maintain or expand AR signaling and luminal epithelial lineage (16–23).”

“22 27 However, in vivo assessments of AR activation in animal models have not been thoroughly examined.”

“conformational flexibility that corresponds to a range of AR activity.(116) This is facilitated in part by a plastic dimerization interface within the LBD that is likely conserved in related 3‐ketosteroid receptors (such as the glucocorticoid receptor (GR)(117)) but “inside out” relative to other nuclear receptors, in…”

“Within the nucleus, AR dimerizes and binds to androgen response elements (ARE) in target gene promoters, orchestrating transcriptional regulation through dynamic interactions with coregulatory proteins [27, 28].”

“Allosteric surfaces involved in AR activation and cooperative interaction with ERG, and which contribute to resistance to AR-directed inhibitor treatment, have been identified [179].”