A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

The box C/D snoRNP assembly factor Bcd1 interacts with the histone chaperone Rtt106 and controls its transcription dependent activity.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.

Structural studies of RNase M5 reveal two-metal-ion supported two-step dsRNA cleavage for 5S rRNA maturation.

All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus, the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3'- and 5'-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3'-before-5' order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.

Mechanistic insights into the synergistic activation of the RXR-PXR heterodimer by endocrine disruptor mixtures.

Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR-PXR and potentially several other nuclear receptor heterodimers.

Structural basis for DNA recognition and allosteric control of the retinoic acid receptors RAR-RXR.

Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR-RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR-RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR-RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR-RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR-RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR-RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR-RAR heterodimers.

Molecular determinants of MED1 interaction with the DNA bound VDR-RXR heterodimer.

The MED1 subunit of the Mediator complex is an essential coactivator of nuclear receptor-mediated transcriptional activation. While structural requirements for ligand-dependent binding of classical coactivator motifs of MED1 to numerous nuclear receptor ligand-binding domains have been fully elucidated, the recognition of the full-length or truncated coactivator by full nuclear receptor complexes remain unknown. Here we present structural details of the interaction between a large part of MED1 comprising its structured N-terminal and the flexible receptor-interacting domains and the mutual heterodimer of the vitamin D receptor (VDR) and the retinoid X receptor (RXR) bound to their cognate DNA response element. Using a combination of structural and biophysical methods we show that the ligand-dependent interaction between VDR and the second coactivator motif of MED1 is crucial for complex formation and we identify additional, previously unseen, interaction details. In particular, we identified RXR regions involved in the interaction with the structured N-terminal domain of MED1, as well as VDR regions outside the classical coactivator binding cleft affected by coactivator recruitment. These findings highlight important roles of each receptor within the heterodimer in selective recognition of MED1 and contribute to our understanding of the nuclear receptor-coregulator complexes.

Structures of B. subtilis Maturation RNases Captured on 50S Ribosome with Pre-rRNAs.

The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.

Recurrent activating mutations of PPARγ associated with luminal bladder tumors.

The upregulation of PPARγ/RXRα transcriptional activity has emerged as a key event in luminal bladder tumors. It renders tumor cell growth PPARγ-dependent and modulates the tumor microenvironment to favor escape from immuno-surveillance. The activation of the pathway has been linked to PPARG gains/amplifications resulting in PPARγ overexpression and to recurrent activating point mutations of RXRα. Here, we report recurrent mutations of PPARγ that also activate the PPARγ/RXRα pathway, conferring PPARγ-dependency and supporting a crucial role of PPARγ in luminal bladder cancer. These mutations are found throughout the protein-including N-terminal, DNA-binding and ligand-binding domains-and most of them enhance protein activity. Structure-function studies of PPARγ variants with mutations in the ligand-binding domain allow identifying structural elements that underpin their gain-of-function. Our study reveals genomic alterations of PPARG that lead to pro-tumorigenic PPARγ/RXRα pathway activation in luminal bladder tumors and may open the way towards alternative options for treatment.

Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex.

RPAP3 and PIH1D1 are part of the HSP90 co-chaperone R2TP complex involved in the assembly process of many molecular machines. In this study, we performed a deep structural investigation of the HSP binding abilities of the two TPR domains of RPAP3. We combined 3D NMR, non-denaturing MS, and ITC techniques with Y2H, IP-LUMIER, FRET, and ATPase activity assays and explain the fundamental role played by the second TPR domain of RPAP3 in the specific recruitment of HSP90. We also established the 3D structure of an RPAP3:PIH1D1 sub-complex demonstrating the need for a 34-residue insertion, specific of RPAP3 isoform 1, for the tight binding of PIH1D1. We also confirm the existence of a complex lacking PIH1D1 in human cells (R2T), which shows differential binding to certain clients. These results highlight similarities and differences between the yeast and human R2TP complexes, and document the diversification of this family of co-chaperone complexes in human.

The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.

Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches.

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.

Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor.

Most nuclear receptors (NRs) bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs) involves specific protein-DNA and protein-protein interactions. The estrogen-related receptor (ERR) belongs to the steroid hormone nuclear receptor (SHR) family and shares strong similarity in its DNA-binding domain (DBD) with that of the estrogen receptor (ER). In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3), but in vivo, it preferentially binds to single half-site REs extended at the 5'-end by 3 bp [estrogen-related response element (ERREs)], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS), non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3), such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3), where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site is followed by a weaker one with degenerated sequence.