Asymmetric dimerization in a transcription factor superfamily is promoted by allosteric interactions with DNA.

Transcription factors, such as nuclear receptors achieve precise transcriptional regulation by means of a tight and reciprocal communication with DNA, where cooperativity gained by receptor dimerization is added to binding site sequence specificity to expand the range of DNA target gene sequences. To unravel the evolutionary steps in the emergence of DNA selection by steroid receptors (SRs) from monomeric to dimeric palindromic binding sites, we carried out crystallographic, biophysical and phylogenetic studies, focusing on the estrogen-related receptors (ERRs, NR3B) that represent closest relatives of SRs. Our results, showing the structure of the ERR DNA-binding domain bound to a palindromic response element (RE), unveil the molecular mechanisms of ERR dimerization which are imprinted in the protein itself with DNA acting as an allosteric driver by allowing the formation of a novel extended asymmetric dimerization region (KR-box). Phylogenetic analyses suggest that this dimerization asymmetry is an ancestral feature necessary for establishing a strong overall dimerization interface, which was progressively modified in other SRs in the course of evolution.

Crystal Structure of Staphopain C from Staphylococcus aureus.

Staphylococcus aureus is a common opportunistic pathogen of humans and livestock that causes a wide variety of infections. The success of S. aureus as a pathogen depends on the production of an array of virulence factors including cysteine proteases (staphopains)-major secreted proteases of certain strains of the bacterium. Here, we report the three-dimensional structure of staphopain C (ScpA2) of S. aureus, which shows the typical papain-like fold and uncovers a detailed molecular description of the active site. Because the protein is involved in the pathogenesis of a chicken disease, our work provides the foundation for inhibitor design and potential antimicrobial strategies against this pathogen.

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.

A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

A structural signature motif enlightens the origin and diversification of nuclear receptors.

Nuclear receptors are ligand-activated transcription factors that modulate gene regulatory networks from embryonic development to adult physiology and thus represent major targets for clinical interventions in many diseases. Most nuclear receptors function either as homodimers or as heterodimers. The dimerization is crucial for gene regulation by nuclear receptors, by extending the repertoire of binding sites in the promoters or the enhancers of target genes via combinatorial interactions. Here, we focused our attention on an unusual structural variation of the α-helix, called π-turn that is present in helix H7 of the ligand-binding domain of RXR and HNF4. By tracing back the complex evolutionary history of the π-turn, we demonstrate that it was present ancestrally and then independently lost in several nuclear receptor lineages. Importantly, the evolutionary history of the π-turn motif is parallel to the evolutionary diversification of the nuclear receptor dimerization ability from ancestral homodimers to derived heterodimers. We then carried out structural and biophysical analyses, in particular through point mutation studies of key RXR signature residues and showed that this motif plays a critical role in the network of interactions stabilizing homodimers. We further showed that the π-turn was instrumental in allowing a flexible heterodimeric interface of RXR in order to accommodate multiple interfaces with numerous partners and critical for the emergence of high affinity receptors. Altogether, our work allows to identify a functional role for the π-turn in oligomerization of nuclear receptors and reveals how this motif is linked to the emergence of a critical biological function. We conclude that the π-turn can be viewed as a structural exaptation that has contributed to enlarging the functional repertoire of nuclear receptors.

Nonsteroidal ecdysone receptor agonists use a water channel for binding to the ecdysone receptor complex EcR/USP.

The ecdysone receptor (EcR) possesses the remarkable capacity to adapt structurally to different types of ligands. EcR binds ecdysteroids, including 20-hydroxyecdysone (20E), as well as nonsteroidal synthetic agonists such as insecticidal dibenzoylhydrazines (DBHs). Here, we report the crystal structures of the ligand-binding domains of Heliothis virescens EcR/USP bound to the DBH agonist BYI09181 and to the imidazole-type compound BYI08346. The region delineated by helices H7 and H10 opens up to tightly fit a phenyl ring of the ligands to an extent that depends on the bulkiness of ring substituent. In the structure of 20E-bound EcR, this part of the ligand-binding pocket (LBP) contains a channel filled by water molecules that form an intricate hydrogen bond network between 20E and LBP. The water channel present in the nuclear receptor bound to its natural hormone acts as a critical molecular adaptation spring used to accommodate synthetic agonists inside its binding cavity.

Acyl Transfer Catalytic Activity in De Novo Designed Protein with N-Terminus of α-Helix As Oxyanion-Binding Site.

The design of catalytic proteins with functional sites capable of specific chemistry is gaining momentum and a number of artificial enzymes have recently been reported, including hydrolases, oxidoreductases, retro-aldolases, and others. Our goal is to develop a peptide ligase for robust catalysis of amide bond formation that possesses no stringent restrictions to the amino acid composition at the ligation junction. We report here the successful completion of the first step in this long-term project by building a completely de novo protein with predefined acyl transfer catalytic activity. We applied a minimalist approach to rationally design an oxyanion hole within a small cavity that contains an adjacent thiol nucleophile. The N-terminus of the α-helix with unpaired hydrogen-bond donors was exploited as a structural motif to stabilize negatively charged tetrahedral intermediates in nucleophilic addition-elimination reactions at the acyl group. Cysteine acting as a principal catalytic residue was introduced at the second residue position of the α-helix N-terminus in a designed three-α-helix protein based on structural informatics prediction. We showed that this minimal set of functional elements is sufficient for the emergence of catalytic activity in a de novo protein. Using peptide-αthioesters as acyl-donors, we demonstrated their catalyzed amidation concomitant with hydrolysis and proved that the environment at the catalytic site critically influences the reaction outcome. These results represent a promising starting point for the development of efficient catalysts for protein labeling, conjugation, and peptide ligation.

FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts.

Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP-A, VAP-B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP-A, VAP-B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non-conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non-conventional FFAT motifs (named Phospho-FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho-FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho-FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER-endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter-organelle contacts.

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.

Aminobenzosuberone derivatives as PfA-M1 inhibitors: Molecular recognition and antiplasmodial evaluation.

Aminobenzosuberone-based PfA-M1 inhibitors were explored as novel antimalarial agents against two different Plasmodium falciparum strains. The 4-phenyl derivative 7c exhibited the most encouraging growth inhibitory activity with IC50 values of 6.5-11.2 µM. X-ray crystal structures and early assessment of DMPK/ADME-Tox parameters allowed us to initiate structure-based drug design approach and understand the liabilities (such as potential metabolic and aqueous solubility issues) as well as identify the opportunities for improvement of this aminobenzosuberone series. It also suggested that compound 7c should be regarded as an attractive chemical tool to investigate the different biological roles of this multifunctional PfA-M1 protein.

Structural Basis for DNA Gyrase Interaction with Coumermycin A1.

Coumermycin A1 is a natural aminocoumarin that inhibits bacterial DNA gyrase, a member of the GHKL proteins superfamily. We report here the first cocrystal structures of gyrase B bound to coumermycin A1, revealing that one coumermycin A1 molecule traps simultaneously two ATP-binding sites. The inhibited dimers from different species adopt distinct sequence-dependent conformations, alternative to the ATP-bound form. These structures provide a basis for the rational development of coumermycin A1 derivatives for antibiotherapy and biotechnology applications.

A revisited version of the apo structure of the ligand-binding domain of the human nuclear receptor retinoic X receptor α.

The retinoic X receptor (RXR) plays a crucial role in the superfamily of nuclear receptors (NRs) by acting as an obligatory partner of several nuclear receptors; its role as a transcription factor is thus critical in many signalling pathways, such as metabolism, cell development, differentiation and cellular death. The first published structure of the apo ligand-binding domain (LBD) of RXRα, which is still used as a reference today, contained inaccuracies. In the present work, these inaccuracies were corrected using modern crystallographic tools. The most important correction concerns the presence of a π-bulge in helix H7, which was originally built as a regular α-helix. The presence of several CHAPS molecules, which are visible for the first time in the electron-density map and which stabilize the H1-H3 loop, which contains helix H2, are also revealed. The apo RXR structure has played an essential role in deciphering the molecular mode of action of NR ligands and is still used in numerous biophysical studies. This refined structure should be used preferentially in the future in interpreting experiments as well as for modelling and structural dynamics studies of the apo RXRα LBD.

Modulation of RXR-DNA complex assembly by DNA context.

Retinoid X Receptors (RXRs) act as dimer partners for several nuclear receptors including itself, binding to genomic DNA response elements and regulating gene transcription with cell and gene specificity. As homodimers, RXRs bind direct repeats of the half-site (A/G)G(G/T)TCA separated by 1 nucleotide (DR1) and little variability of this consensus site is observed for natural DR1s. However, these variations are responsible of the modulation of RXR receptors function through differential binding affinity and conformational changes. To further our understanding of the molecular mechanisms underlying RXR-DNA interactions, we examined how RXR DBDs bind to different DR1s using thermodynamics, X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. We show that the half-site sequences modulate the binding cooperativity that results from the protein-protein contacts between the two DBDs. Chemical shifts perturbation NMR experiments revealed that sequence variations in half-sites induce changes that propagate from the protein-DNA interface to the dimerization interface throughout the DBD fold.

Insight into the remarkable affinity and selectivity of the aminobenzosuberone scaffold for the M1 aminopeptidases family based on structure analysis.

Aminopeptidases are ubiquitous hydrolases that cleave the N-terminal residues of proteins and oligopeptides. They are broadly distributed throughout all kingdoms of life and have been implicated in a wide variety of physiological processes, including viral infection, parasite metabolism, protein processing, regulation of peptide hormones, and cancer cell proliferation. Members of the M1 family, also termed gluzincins, are defined by two highly conserved motifs in the catalytic domain: a zinc-binding motif, HEXXH-(X18)-E; and an exopeptidase motif, GXMEN. We report the high-resolution X-ray structures of E. coli aminopeptidase N (PepN) in complex with three aminobenzosuberone scaffolds that display various Ki values (50, 0.33, and 0.034 µM) and provide a compelling view of the outstanding selectivity of these chemical entities for the M1 aminopeptidases. This series of inhibitors interacts as transition state mimics with highly conserved residues of the catalytic machinery and substrate recognition sites. Structural comparisons and model-building studies allowed a deep interpretation of the SAR observed for bacterial, as well as mammalian enzymes. Proteins 2017; 85:1413-1421. © 2017 Wiley Periodicals, Inc.

The crystal structure of the 5΄ functional domain of the transcription riboregulator 7SK.

In vertebrates, the 7SK RNA forms the scaffold of a complex, which regulates transcription pausing of RNA-polymerase II. By binding to the HEXIM protein, the complex comprising proteins LARP7 and MePCE captures the positive transcription elongation factor P-TEFb and prevents phosphorylation of pausing factors. The HEXIM-binding site embedded in the 5΄-hairpin of 7SK (HP1) encompasses a short signature sequence, a GAUC repeat framed by single-stranded uridines. The present crystal structure of HP1 shows a remarkably straight helical stack involving several unexpected triples formed at a central region. Surprisingly, two uridines of the signature sequence make triple interactions in the major groove of the (GAUC)2. The third uridine is turned outwards or inward, wedging between the other uridines, thus filling the major groove. A molecular dynamics simulation indicates that these two conformations of the signature sequence represent stable alternatives. Analyses of the interaction with the HEXIM protein confirm the importance of the triple interactions at the signature sequence. Altogether, the present structural analysis of 7SK HP1 highlights an original mechanism of swapping bases, which could represent a possible '7SK signature' and provides new insight into the functional importance of the plasticity of RNA.

Structural basis of natural promoter recognition by the retinoid X nuclear receptor.

Retinoid X receptors (RXRs) act as homodimers or heterodimerisation partners of class II nuclear receptors. RXR homo- and heterodimers bind direct repeats of the half-site (A/G)G(G/T)TCA separated by 1 nucleotide (DR1). We present a structural characterization of RXR-DNA binding domain (DBD) homodimers on several natural DR1s and an idealized symmetric DR1. Homodimers displayed asymmetric binding, with critical high-affinity interactions accounting for the 3' positioning of RXR in heterodimers on DR1s. Differing half-site and spacer DNA sequence induce changes in RXR-DBD homodimer conformation notably in the dimerization interface such that natural DR1s are bound with higher affinity than an idealized symmetric DR1. Subtle changes in the consensus DR1 DNA sequence therefore specify binding affinity through altered RXR-DBD-DNA contacts and changes in DBD conformation suggesting a general model whereby preferential half-site recognition determines polarity of heterodimer binding to response elements.

Structure of the Elongator cofactor complex Kti11/Kti13 provides insight into the role of Kti13 in Elongator-dependent tRNA modification.

UNLABELLED: Modification of wobble uridines of many eukaryotic tRNAs requires the Elongator complex, a highly conserved six-subunit eukaryotic protein assembly, as well as the Killer toxin-insensitive (Kti) proteins 11-14. Kti11 was additionally shown to be implicated in the biosynthesis of diphthamide, a post-translationally modified histidine of translation elongation factor 2. Recent data indicate that iron-bearing Kti11 functions as an electron donor to the [4Fe-4S] cluster of radical S-Adenosylmethionine enzymes, triggering the subsequent radical reaction. We show here that recombinant yeast Kti11 forms a stable 1 : 1 complex with Kti13. To obtain insights into the function of this heterodimer, the Kti11/Kti13 complex was purified to homogeneity, crystallized, and its structure determined at 1.45 Å resolution. The importance of several residues mediating complex formation was confirmed by mutagenesis. Kti13 adopts a fold characteristic of RCC1-like proteins. The seven-bladed ß-propeller consists of a unique mixture of four- and three-stranded blades. In the complex, Kti13 orients Kti11 and restricts access to its electron-carrying iron atom, constraining the electron transfer capacity of Kti11. Based on these findings, we propose a role for Kti13, and discuss the possible functional implications of complex formation. DATABASE: Structural data have been submitted to the Protein Data Bank under accession number 4X33.

TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration.

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration.

Structural basis for hijacking of cellular LxxLL motifs by papillomavirus E6 oncoproteins.

E6 viral oncoproteins are key players in epithelial tumors induced by papillomaviruses in vertebrates, including cervical cancer in humans. E6 proteins target many host proteins by specifically interacting with acidic LxxLL motifs. We solved the crystal structures of bovine (BPV1) and human (HPV16) papillomavirus E6 proteins bound to LxxLL peptides from the focal adhesion protein paxillin and the ubiquitin ligase E6AP, respectively. In both E6 proteins, two zinc domains and a linker helix form a basic-hydrophobic pocket, which captures helical LxxLL motifs in a way compatible with other interaction modes. Mutational inactivation of the LxxLL binding pocket disrupts the oncogenic activities of both E6 proteins. This work reveals the structural basis of both the multifunctionality and the oncogenicity of E6 proteins.

On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase.

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40) hydrocarbon substrate.