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PPARγ is a key regulator of glucose homeostasis and insulin sensitization. PPARγ must heterodimerize with its dimeric partner, the retinoid X receptor (RXR), to bind DNA and associated coactivators such as p160 family members or PGC-1α to regulate gene networks. To understand how coactivators are recognized by the functional heterodimer PPARγ/RXRα and to determine the topological organization of the complexes, we performed a structural study using small angle X-ray scattering of PPARγ/RXRα in complex with DNA from regulated gene and the TIF2 receptor interacting domain (RID). The solution structures reveal an asymmetry of the overall structure due to the crucial role of the DNA in positioning the heterodimer and indicate asymmetrical binding of TIF2 to the heterodimer.

Control of transcription allows the regulation of cell activity in response to external stimuli and research in the field has greatly benefited from efforts in structural biology. In this review, based on specific examples from the European SPINE2-COMPLEXES initiative, we illustrate the impact of structural proteomics on our understanding of the molecular basis of gene expression. While most atomic structures were obtained by X-ray crystallography, the impact of solution NMR and cryo-electron microscopy is far from being negligible. Here, we summarize some highlights and illustrate the importance of specific technologies on the structural biology of protein-protein or protein/DNA transcription complexes: structure/function analysis of components the eukaryotic basal and activated transcription machinery with focus on the TFIID and TFIIH multi-subunit complexes as well as transcription regulators such as members of the nuclear hormone receptor families. We also discuss molecular aspects of promoter recognition and epigenetic control of gene expression. © 2011 Elsevier Inc., This work forms part of SPINE2-complexes Contract No. LSHG-CT-2006-031220, funded by the European Commission under the Integrated Programme ‘Quality of Life and Management of Living Resources’. This work was also supported by grants from the EC FP7 IS project P-CUBE (to I.B.), from the Spanish ‘Ministerio de Ciencia e Innovación’ (Grants BFU2008-02372/BMC and CSD2006-00023) and the ‘Generalitat de Catalunya’ (Grant 2009SGR-1309) (to A.B. and M.C.), from the Netherlands Organisation for Scientific Research, Division of Chemical Sciences, NWO-CW (to R.B.) and from the Agence Nationale de la Recherche, the Association de la Recherche sur le Cancer and the Institut National du Cancer (to J.C., P.S, D.M. and A.P.).

International audience; In insects, the nuclear receptor family comprises 19 transcription factors, among which a majority play a crucial role in ecdysteroid signaling, notably the receptor of ecdysone itself (ECR). A small network of regulators that control the ecdysone cascade during the onset of metamorphosis underwent an increase of evolutionary rate in a large group of insects, Mecopterida (1/3 of insect species), which includes Diptera and Lepidoptera. This network contains at least five nuclear receptors that probably coevolved together: ECR, USP, HR3, E75 and HR4. By comparing sequences and structures between Drosophila and a non-Mecopterida species, the flour beetle Tribolium castaneum, we try to understand the coevolution between ECR and USP. The crystal structure of the Tribolium USP has revealed a novel type of ligand-independent USP-RXR, with no ligandbinding pocket. The ECR-USP heterodimerisation surface is symmetric and 30%larger in Mecopterida. This innovation may have been associated with a loss of homodimerisation for both USP and ECR and a reinforcement of their heterodimerisation. These results indicate that the coevolution between ECR and USP occurred, at least in part, through intra-molecular epistasis and adaptation of the heterodimerisation surface. In order to test the hypothesis that selection has shaped the USP ligand-binding domain during the evolution of insects, we analysed the evolutionary rate at different time scales. We also tested the possibility that GC-biased gene conversion played a role in the increase of nucleotide substitution rate observed for USP. Our results suggest that adaptation, drift and molecular drive shaped the structure of the nuclear receptor USP in insects. The structural evolutionary plasticity of nuclear receptors is a determinant factor for the maintenance of hormonal regulations of insect development, growth, reproduction and physiology.

International audience; Understanding how the variability of protein structure arises during evolution and leads to new structurefunction relationships ultimately promoting evolutionary novelties is a major goal of molecular evolution and is critical for interpreting genome sequences. We addressed this issue using the ecdysone receptor (ECR), a major developmental factor that controls development and reproduction of arthropods. The functional ECR is a heterodimer of two nuclear receptors: ECR, which binds ecdysteroids, and its obligatory partner ultraspirade (USP), which is orthologous to the retinoid X receptor of vertebrates. Both genes underwent a dramatic increase of evolutionary rate in Mecopterida, the major insect terminal group containing Dipteras and Lepidopteras. We therefore questioned the implication of this event in terms of coevolution of their dimerization interface. A structural comparison revealed a 30% larger ligand-binding domain (LBD) heterodimerization surface in the Lepidoptera Heliothis when compared with basal insects, associated with a symmetrization of the interface, which is exceptional for nuclear receptors. Reconstruction of ancestral sequences and homology modeling of the ancestral Mecopterida ECRUSP reveal that this enlarged dimerization surface is a synapomorphy for Mecopterida. Furthermore, we show that the residues implicated in the new dimerization surface underwent specific evolutionary constraints in Mecopterida indicative of their new and conserved role in the dimerization interface. Most of all, the novel surface originates from a 15° torsion of a subdomain of USP LBD toward its partner ECR, which is a long-range consequence of the peculiar position of a Mecopterida-specific insertion in loop L1-3, located outside of the interaction surface, in a less crucial domain of the partner protein. These results indicate that the coevolution between ECR and USP occurred through a novel mechanism of intramolecular epistasis that will undoubtedly be generalized for other molecules because it uses flexibility of a less-constrained region of a protein to modify the structure of another, critical part of the molecule.

International audience; Retinoid X receptor (RXR) and Ultraspiracle (USP) play a central role as ubiquitous heterodimerization partners of many nuclear receptors. While it has long been accepted that a broad range of ligands can activate vertebrate/mollusc RXRs, the existence and necessity of specific endogenous ligands activating RXR-USP in vivo is still matter of intense debate. Here we report the existence of a novel type of RXR-USP with a ligand-independent functional conformation. Our studies involved Tribolium USP (TcUSP) as representative of most arthropod RXR-USPs with high sequence homology to vertebrate/mollusc RXRs. The crystal structure of the ligand-binding domain of TcUSP was solved in the context of the functional heterodimer with the ecdysone receptor (EcR). While EcR exhibits a canonical ligand-bound conformation, USP adopts an original apo structure. Our functional data demonstrate that TcUSP is a constitutively silent partner of EcR and that none of the RXR ligands can bind and activate TcUSP. These findings together with a phylogenetical analysis suggest that RXR-USPs have undergone remarkable functional shifts during evolution and give insight into receptor-ligand binding evolution and dynamics.