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Rev-erbs are members of the nuclear receptor (NR) transcription factor superfamily and are widely expressed, but are most prevalent in liver, adipose tissue, skeletal muscle and brain. Rev-erbs are key regulators of the circadian rhythm and are expressed in a circadian manner. The discovery that Rev-erbs are ligand-regulated receptors, whose repressive activity is regulated by the endogenous porphyrin ligand, heme, as well as the recent report of the first synthetic Rev-erb ligand, GSK4112/SR6452, suggests that pharmacological modulation through Rev-erb may provide new routes to treat metabolic diseases. Here, we review the work leading to the discovery that Rev-erbs are indeed ligand-regulated and the role that both natural and synthetic Rev-erb ligands have on adipogenesis.

Several nuclear receptors (NRs) are still character-ized as orphan receptors because ligands have not yet been identified for these proteins. The retinoic acid receptor-related receptors (RORs) have no well-defined physiological ligands. Here, we describe the identification of a selective RORα synthetic ligand, SR3335 (ML-176). SR3335 directly binds to RORα, but not other RORs, and functions as a selective partial inverse agonist of RORα in cell-based assays. Furthermore, SR3335 suppresses the expression of endogenous RORα target genes in HepG2 involved in hepatic gluconeogenesis including glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Pharmacokinetic studies indicate that SR3335 displays reasonable exposure following an ip injection into mice. We assess the ability of SR3335 to suppress gluconeogenesis in vivo using a diet-induced obesity (DIO) mouse model where the mice where treated with 15 mg/kg b.i.d., ip for 6 days followed by a pyruvate tolerance test. SR3335-treated mice displayed lower plasma glucose levels following the pyruvate challenge consistent with suppression of gluconeogenesis. Thus, we have identified the first selective synthetic RORα inverse agonist, and this compound can be utilized as a chemical tool to probe the function of this receptor both in vitro and in vivo. Additionally, our data suggests that RORα inverse agonists may hold utility for suppression of elevated hepatic glucose production in type 2 diabetics.

Summary Peroxisome proliferator-activated receptors (PPARs) are pharmacological targets for the treatment of metabolic disorders. Previously, we demonstrated the anti-diabetic effects of SR1664, a PPARγ modulator lacking classical transcriptional agonism, despite its poor pharmacokinetic properties. Here, we report identification of the antagonist SR11023 as a potent insulin sensitizer with significant plasma exposure following oral administration. To determine the structural mechanism of ligand-dependent antagonism of PPARγ, we employed an integrated approach combining solution-phase biophysical techniques to monitor activation helix (helix 12) conformational dynamics. While informative on receptor dynamics, hydrogen/deuterium exchange mass spectrometry and nuclear magnetic resonance data provide limited information regarding the specific orientations of structural elements. In contrast, label-free quantitative crosslinking mass spectrometry revealed that binding of SR11023 to PPARγ enhances interaction with co-repressor motifs by pushing H12 away from the agonist active conformation toward the H2-H3 loop region (i.e., the omega loop), revealing the molecular mechanism for active antagonism of PPARγ.

The AAA + (ATPases associated with a variety of cellular activities) superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. As part of a large oligomeric complex, ClpC controls an array of cellular processes by recognizing, unfolding, and providing misfolded and aggregated proteins as substrates for the ClpP peptidase. ClpC is unique compared to other HSP100/Clp proteins, as it requires an adaptor protein for all fundamental activities. The NMR solution structure of the N-terminal repeat domain of ClpC (N-ClpCR) comprises two structural repeats of a four-helix motif. NMR experiments used to map the MecA adaptor protein interaction surface of N-ClpCR reveal that regions involved in the interaction possess conformational flexibility and conformational exchange on the microsecond-to-millisecond timescale. The electrostatic surface of N-ClpCR differs substantially from the N-domain of Escherichia coli ClpA and ClpB, suggesting that the electrostatic surface characteristics of HSP100/Clp N-domains may play a role in adaptor protein and substrate interaction specificity, and perhaps contribute to the unique adaptor protein requirement of ClpC.

Many genomes contain families of paralogs--proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD's ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors' response element-binding specificity were far from the proteins' DNA-binding interface and interacted epistatically to change the DBD's function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.

A subset of nuclear receptors (NRs) function as obligate heterodimers with retinoid X receptor (RXR), allowing integration of ligand-dependent signals across the dimer interface via an unknown structural mechanism. Using nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry, here we show an allosteric mechanism through which RXR co-operates with a permissive dimer partner, peroxisome proliferator-activated receptor (PPAR)-γ, while rendered generally unresponsive by a non-permissive dimer partner, thyroid hormone (TR) receptor. Amino acid residues that mediate this allosteric mechanism comprise an evolutionarily conserved network discovered by statistical coupling analysis (SCA). This SCA network acts as a signalling rheostat to integrate signals between dimer partners, ligands and coregulator-binding sites, thereby affecting signal transmission in RXR heterodimers. These findings define rules guiding how NRs integrate two ligand-dependent signalling pathways into RXR heterodimer-specific responses., Some nuclear receptors dimerize with retinoid X receptor to allow ligand-dependent signalling. Here, Kojetin et al. use structural and biophysical techniques to identify structural changes that guide these complex signalling networks.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPARγ by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently we reported the development of PPARγ antagonist SR1664, designed to block the obesity induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPARγ, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow derived mesenchymal stem cells (MSCs) with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand mediated PPARγ repression, and suggest a therapeutic approach to promote bone formation.

SUMMARY RORγt is well recognized as the lineage-defining transcription factor for T helper 17 (TH17) cell development. However, the cell-intrinsic mechanisms that negatively regulate TH17 cell development and autoimmunity remain poorly understood. Here, we demonstrate that the transcriptional repressor REV-ERBα is exclusively expressed in TH17 cells, competes with RORγt for their shared DNA consensus sequence, and negatively regulates TH17 cell development via repression of genes traditionally characterized as RORγt dependent, including Il17a. Deletion of REV-ERBα enhanced TH17-mediated proinflammatory cytokine expression, exacerbating experimental autoimmune encephalomyelitis (EAE) and colitis. Treatment with REV-ERB-specific synthetic ligands, which have similar phenotypic properties as RORγ modulators, suppressed TH17 cell development, was effective in colitis intervention studies, and significantly decreased the onset, severity, and relapse rate in several models of EAE without affecting thymic cellularity. Our results establish that REV-ERBα negatively regulates proinflammatory TH17 responses in vivo and identifies the REV-ERBs as potential targets for the treatment of TH17-mediated autoimmune diseases., Graphical Abstract, In Brief Roles for the circadian protein REV-ERBα have not been extensively explored in the immune system. Amir et al. demonstrate that REV-ERBα acts as a negative regulator of proinflammatory TH17 cell development and function, and REV-ERBα ligands are efficacious in mouse models of autoimmunity.

Nuclear receptor (NR) transcription factors bind various coreceptors, small-molecule ligands, DNA response element sequences, and transcriptional coregulator proteins to affect gene transcription. Small-molecule ligands and DNA are known to influence receptor structure, coregulator protein interaction, and function; however, little is known on the mechanism of synergy between ligand and DNA. Using quantitative biochemical, biophysical, and solution structural methods, including 13C-detected nuclear magnetic resonance and hydrogen/deuterium exchange (HDX) mass spectrometry, we show that ligand and DNA cooperatively recruit the intrinsically disordered steroid receptor coactivator-2 (SRC-2/TIF2/GRIP1/NCoA-2) receptor interaction domain to peroxisome proliferator-activated receptor gamma-retinoid X receptor alpha (PPARγ-RXRα) heterodimer and reveal the binding determinants of the complex. Our data reveal a thermodynamic mechanism by which DNA binding propagates a conformational change in PPARγ-RXRα, stabilizes the receptor ligand binding domain dimer interface, and impacts ligand potency and cooperativity in NR coactivator recruitment.

As one of the most important eukaryotic DNA-binding motifs, the homeodomain has been identified in over one thousand proteins. Transcription factors containing homeodomains play critical roles in diverse biological processes and are often implicated in human genetic disorders. The human Pitx2 protein is a central downstream transcription factor in both the TGF-β and Wnt/β-catenin signaling pathways, and involved in the regulation of cell differentiation, cell pattern formation and tissue maintenance. To better understand how Pitx2 regulates its target genes, we are investigating the mechanisms that confer DNA-binding specificity on its homeodomain. Here, we report complete (1)H, (13)C and (15)N protein chemical shift assignments of the human Pitx2 homeodomain in complex with a non-consensus DNA sequence.

The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5 lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.

Resveratrol has beneficial effects on aging, inflammation and metabolism, which are thought to result from activation of the lysine deacetylase, sirtuin 1 (SIRT1), the cAMP pathway, or AMP-activated protein kinase. In this study, we report that resveratrol acts as a pathway-selective estrogen receptor-α (ERα) ligand to modulate the inflammatory response but not cell proliferation. A crystal structure of the ERα ligand-binding domain (LBD) as a complex with resveratrol revealed a unique perturbation of the coactivator-binding surface, consistent with an altered coregulator recruitment profile. Gene expression analyses revealed significant overlap of TNFα genes modulated by resveratrol and estradiol. Furthermore, the ability of resveratrol to suppress interleukin-6 transcription was shown to require ERα and several ERα coregulators, suggesting that ERα functions as a primary conduit for resveratrol activity. DOI: http://dx.doi.org/10.7554/eLife.02057.001, eLife digest Resveratrol is a compound found in significant quantities in red wine, grapes, and peanuts. Many health benefits have been linked to it, including protecting against certain types of cancer and reducing the risk of cardiovascular disease. How resveratrol could produce these very different effects is unknown, but evidence is emerging that it is involved in a wide range of biological processes. However, the ability of resveratrol to bind with, and activate, proteins called estrogen receptors has largely been overlooked. These receptors have a range of roles. For example, estrogen receptors fight against inflammation by preventing the transcription of the gene that encodes a signaling protein called interleukin-6. However, estrogen receptors do not work alone: other molecules called coregulators interact with them and alter how effectively they can prevent gene expression. Resveratrol has also been associated with anti-inflammatory effects, particularly in tissues that contain large numbers of an estrogen receptor called ERα, though this connection has been little studied. Nwachukwu et al. now reveal that the two are linked—the anti-inflammatory response of resveratrol relies on it being bound to ERα. This binding changes the shape of the receptor in a way that controls which coregulator molecules help it to regulate transcription. Additionally, this binding complex does not produce the cancer-causing side effects often associated with activated ERα. Nwachukwu et al. also found that the effect of resveratrol on the inflammatory response depends on specific other coregulators being present. The role of ERα in enhancing and activating resveratrol's effects is important because resveratrol has poor bioavailability in humans, and so it is not easily absorbed into the bloodstream. This makes it difficult for someone to get a dose high enough to produce beneficial effects. Further research targeting ERα may produce similar beneficial compounds to resveratrol, but with improved bioavailability. DOI: http://dx.doi.org/10.7554/eLife.02057.002

The nuclear receptors REV-ERB (consisting of REV-ERBα and REV-ERBβ) and retinoic acid receptor-related orphan receptors (RORs; consisting of RORα, RORβ and RORγ) are involved in many physiological processes, including regulation of metabolism, development and immunity as well as the circadian rhythm. The recent characterization of endogenous ligands for these former orphan nuclear receptors has stimulated the development of synthetic ligands and opened up the possibility of targeting these receptors to treat several diseases, including diabetes, atherosclerosis, autoimmunity and cancer. This Review focuses on the latest developments in ROR and REV-ERB pharmacology indicating that these nuclear receptors are druggable targets and that ligands targeting these receptors may be useful in the treatment of several disorders.

The HSP100/AAA+ superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. We present here the backbone and side-chain assignments of the N-terminal repeat domain (residues 1-145) of ClpC from Bacillus subtilis.

Nuclear receptors (NR) plieotropically regulate a number of physiological processes including metabolism, immune function, reproduction, and development. Therefore NRs have significant roles in the pathology of human diseases including diabetes, cancer, autoimmune disease, and aging. As a result, targeted modulation of NR activity has tremendous pharmacological utility. Receptor ligands can be classified into two prototypical categories: 1) agonists, which promote receptor transcriptional activity above basal levels; and 2) antagonists, which essentially block receptor–ligand interactions. Recent emphasis in the nuclear receptor field has focused on producing functionally selective nuclear receptor modulators which deviate from those of the classical agonist or antagonist. These selective modulators direct receptor activity toward desired/therapeutic pathways or “biased signaling.” Understanding the molecular mechanism of action of these selective modulators has and will continue to provide significant insight toward the development of the next generation of NR modulators. Although most nuclear receptor structural studies have traditionally focused on obtaining ligand–receptor co-crystal structures, recent studies implicate an important role for protein dynamics in the mechanism of action of nuclear receptor ligands. Here we will discuss biased nuclear receptor signaling and the techniques used to study conformational dynamics of the nuclear receptor ligand-binding domain (LBD).

The homeodomain-containing transcription factor Pitx2 (pituitary homeobox protein 2) is present in many developing embryonic tissues, including the heart. Its homeodomain is responsible for the recognition and binding to target DNA sequences and thus constitutes a major functional unit in the Pitx2 protein. Nuclear magnetic resonance techniques were employed to determine the solution structure of the native Pitx2 homeodomain and a R24H mutant that causes autosomal dominantly inherited ring dermoid of the cornea syndrome. The structures reveal that both isoforms possess the canonical homeodomain fold. However, the R24H mutation results in a 2-fold increase in DNA binding affinity and a 5 °C decrease in thermal stability, while changing the dynamic environment of the homeodomain only locally. When introduced into full-length Pitx2c, the mutation results in an only 25% loss of transactivation activity. Our data correlate well with clinical observations suggesting a milder deficiency for the R24H mutation compared to those of other Pitx2 homeodomain mutations.

Activation of p53 function leading to cell-cycle arrest and/or apoptosis is a promising strategy for development of anti-cancer therapeutic agents. Here, we describe a novel mechanism for stabilization of p53 protein expression via activation of the orphan nuclear receptor, RORα. We demonstrate that treatment of cancer cells with a newly described synthetic ROR agonist, SR1078, leads to p53 stabilization and induction of apoptosis. These data suggest that synthetic ROR agonists may hold utility in the treatment of cancer.

Research efforts spanning the past two decades have established a clear link between nuclear receptor function, regulation of the circadian clock and lipid homeostasis. As such, this family of receptors represents an important area of research. Recent advances in the field have identified two nuclear receptor subfamilies, the REV-ERBs and the ‘retinoic acid receptor-related orphan receptors’ (RORs), as critical regulators of the circadian clock with significant roles in lipid homeostasis. In this review, the latest information garnered from cutting-edge research on these two nuclear receptor subfamilies will be discussed. Through direct targeting of the REV-ERBs and RORs with synthetic ligands, generation of novel tools aimed at characterizing their function in vivo have been developed, which may lead to novel therapeutics for the treatment of metabolic disorders.

REV-ERBα is a member of the nuclear receptor superfamily that functions as a receptor for the porphoryin heme. REV-ERBα suppresses transcription of its target genes in a heme-dependent manner. Recently, the first nonporphyrin synthetic ligand for REV-ERBα, GSK4112, was designed, and it mimics the action of heme acting as agonist. Here, we report the identification of the first REV-ERB antagonist, SR8278. SR8278 is structurally similar to the agonist but blocks the ability of the GSK4112 to enhance REV-ERBα-dependent repression in a cotransfection assay. Additionally, whereas GSK4112 suppresses the expression of REV-ERBα target genes involved in gluconeogenesis, SR8278 stimulates the expression of these genes. Thus, SR8278 represents a unique chemical tool for probing REV-ERB function and may serve as a point for initiation of further optimization to develop REV-ERB antagonists with the ability to explore circadian and metabolic functions.

The mammalian clock is regulated at the cellular level by a transcriptional/translational feedback loop. BMAL1/CLOCK (or NPAS2) heterodimers activate the expression of the PERIOD (PER) and CRYPTOCHROME (CRY) genes acting as transcription factors directed to the PER and CRY promoters via E-box elements. PER and CRY proteins form heterodimers and suppress the activity of the BMAL1/CLOCK (or NPAS2) completing the feedback loop. The circadian expression of BMAL1 is influenced by retinoic acid receptor-related orphan receptor α (RORα) and REV-ERBα, two nuclear receptors that target a ROR-response element in the promoter of the BMAL1 gene. Given that BMAL1 functions as an obligate heterodimer with either CLOCK or NPAS2, it is unclear how the expression of the partner is coordinated with BMAL1 expression. Here, we demonstrate that NPAS2 is also a RORα and REV-ERBα target gene. Using a ChIP/microarray screen, we identified both RORα and REV-ERBα occupancy of the NPAS2 promoter. We identified two functional ROREs within the NPAS2 promoter and also demonstrate that both RORα and REV-ERBα regulate the expression of NPAS2 mRNA. These data suggest a mechanism by which RORα and REV-ERBα coordinately regulate the expression of the positive arm of the circadian rhythm feedback loop.

The homeodomain is one of the most important eukaryotic DNA-binding motifs and has been identified in over one thousand proteins. Homeodomain proteins play critical roles in diverse biological processes, including cell differentiation and cell pattern formation. The human Pitx2 homeodomain binds several different DNA sequences and is a pivotal component of both the TGF-β and Wnt/ β-catenin signaling pathways. As the recognition of specific DNA sequences represents an essential biochemical function of all DNA-binding proteins, we have chosen the Pitx2 homeodomain model to investigate the mechanisms that convey biological specificity in these protein-DNA interactions. Here, we report complete chemical shift assignments of the human Pitx2 homeodomain and the R24H mutation that induces ring dermoid of the cornea syndrome.

The two-component signal transduction system is a ubiquitous signaling module present in most prokaryotic and some eukaryotic systems. Two conserved components, a histidine protein kinase (HPK) protein and a response regulator (RR) protein, function as a biological switch, sensing and responding to changes in the environment, thereby eliciting a specific response. Extensive studies have classified the HPK and RR proteins using primary sequence characteristics, domain identity, domain organization, and biological function. We propose that structural analysis of the surface properties of the highly conserved receiver domain of RRs can be used to build on previous classification methods. Our studies of the OmpR subfamily RRs in Bacillus subtilis and Escherichia coli reveal a notable correlation between the RR receiver domain surface classification and previous classification of cognate HPK proteins. We have extended these studies to analyze the receiver domains of all predicted RR proteins in the marine-dwelling bacterium Vibrio vulnificus.

The two‐component signal transduction system is a ubiquitous signaling module present in most prokaryotic and some eukaryotic systems. Two conserved components, a histidine protein kinase (HPK) protein and a response regulator (RR) protein, function as a biological switch, sensing and responding to changes in the environment, thereby eliciting a specific response. Extensive studies have classified the HPK and RR proteins using primary sequence characteristics, domain identity, domain organization, and biological function. We propose that structural analysis of the surface properties of the highly conserved receiver domain of RRs can be used to build on previous classification methods. Our studies of the OmpR subfamily RRs in Bacillus subtilis and Escherichia coli reveal a notable correlation between the RR receiver domain surface classification and previous classification of cognate HPK proteins. We have extended these studies to analyze the receiver domains of all predicted RR proteins in the marine‐dwelling bacterium Vibrio vulnificus .

Calbindin-D(28K) is a Ca2+-binding protein, performing roles as both a calcium buffer and calcium sensor. The NMR solution structure of Ca2+-loaded calbindin-D(28K) reveals a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca2+ in loops on EF1, EF3, EF4 and EF5. Target peptides from Ran-binding protein M and myo-inositol monophosphatase, along with a new target from procaspase-3, are shown to interact with the protein on a surface comprised of alpha5 (EF3), alpha8 (EF4) and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D(28K) adopts discrete hydrophobic states as it binds Ca2+. The structure, binding interface and hydrophobic characteristics of Ca2+-loaded calbindin-D(28K) provide the first detailed insights into how this essential protein may function. This structure is one of the largest high-resolution NMR structures and the largest monomeric EF-hand protein to be solved to date.

The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and microESI-MS. NMR studies show that the divalent metals Ca(2+), Mg(2+) and Mn(2+) primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu(2+) show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the beta 4-alpha 4 loop and the beta 5/alpha 5 interface, particularly around and including H101. microESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu(2+), multiple metal-bound species are observed. Subsequent experiments reveal that Mg(2+) supports phosphotransfer between KinA and Spo0F, while Cu(2+) fails to support KinA phosphotransfer. Additionally, the presence of Cu(2+) at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.

The marine bacterium Vibrio harveyi controls its bioluminescence by a process known as quorum sensing. In this process, autoinducer molecules are detected by membrane-bound sensor kinase/response regulator proteins (LuxN and LuxQ) that relay a signal via a series of protein phosphorylation reactions to another response regulator protein, LuxO. Phosphorylated LuxO indirectly represses the expression of the proteins responsible for bioluminescence. Integral to this quorum sensing process is the function of the phosphotransferase protein, LuxU. LuxU acts to shuttle the phosphate from the membrane-bound proteins, LuxN and LuxQ, to LuxO. LuxU is a 114 amino acid residue monomeric protein. Solution NMR was used to determine the three-dimensional structure of LuxU. LuxU contains a four-helix bundle topology with the active-site histidine residue (His58) located on alpha-helix C and exposed to solution. The active site represents a cluster of positively charged residues located on an otherwise hydrophobic protein face. NMR spin-relaxation experiments identify a collection of flexible residues localized on the same region of LuxU as His58. The studies described here represent the first structural characterization of an isolated, monomeric bacterial phosphotransferase protein.

Ca2+ channel beta subunits modify alpha1 subunit gating properties through direct interactions with intracellular linker domains. In a previous report (Helton and Horne, 2002), we showed that alternative splicing of the beta4 subunit had alpha1 subunit subtype-specific effects on Ca2+ channel activation and fast inactivation. We extend these findings in the present report to include effects on slow inactivation and block by the peptide toxin omega-conotoxin (CTx)-MVIIC. N-terminal deletion and site-directed mutagenesis experiments revealed that the effects of alternative splicing on toxin block and all aspects of gating could be attributed to a proline-rich motif found within N-terminal beta4b amino acids 10-20. Interestingly, this motif is conserved within the third postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1 domain of the distantly related membrane-associated guanylate kinase homolog, PSD-95. Sequence identity of approximately 30% made possible the building of beta4a and beta4b three-dimensional structural models using PSD-95 as the target sequence. The models (1) reveal that alternative splicing of the beta4 N terminus results in dramatic differences in surface charge distribution and (2) localize the proline-rich motif of beta4b to an extended arm structure that flanks what would be the equivalent of a highly modified PSD-95 carboxylate binding loop. Northern blot analysis revealed a markedly different pattern of distribution for beta4a versus beta4b in the human CNS. Whereas beta4a is distributed throughout evolutionarily older regions of the CNS, beta4b is concentrated heavily in the forebrain. These results raise interesting questions about the functional role that alternative splicing of the beta4 subunit has played in the evolution of complex neural networks.

PPARγ is a target for insulin-sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands.

By recruiting chromatin remodeling machinery to promoter regions of target genes, nuclear receptors (NRs) act as ligand-dependent transcriptional protein scaffolds, exerting powerful influences on all aspects of physiology. The critical mediators that bridge NRs to the transcriptional machinery are called NR coregulators. NR coregulators are broadly classified into coactivators or corepressors depending on their ability to promote or repress gene transcription. Coactivators contain a cluster of "LXXLL" motifs in a flexible/disordered region called the Receptor Interaction Domain (RID) that mediates their interactions with NRs. Because there is very little atomic level structural information on disordered coactivator RIDs, the physical mechanism driving the interaction between coregulator RIDs and NR transcriptional complexes is poorly understood. We are studying the interaction of Steroid Receptor Coactivator-2 (SRC-2) with the PPARγ-RXR transcriptional complex as a model system for understanding NR/coregulator interactions on the atomic level. SRC-2 is a critical coactivator in the PPARγ-driven differentiation of mesenchymal stem cells into fat cells, a pathway that is a target for therapeutic intervention of metabolic syndrome and type 2 diabetes. Using biochemical, biophysical and solution NMR approaches, we have begun to probe the interaction of the SRC-2 RID with the full-length PPARγ-RXR complex bound to DNA (∼150 kDa complex). Our studies reveal that the binding of DNA to PPARγ-RXR imparts a long-range conformational change that impacts the binding and conformation of SRC-2 RID. Importantly, our work extends the recent proposition that DNA can act as an allosteric effector of NR activity, indicating that DNA binding can affect the conformation of a coregulator bound to a NR transcriptional complex.

Disruption of the circadian rhythm leads to increased risk for development of breast cancer in humans. The molecular mechanism underlying the link between circadian rhythm disruption and the increased risk of breast cancer in humans is not clear. The REV-ERBs are nuclear receptors that function as critical regulatory components of the circadian rhythm. We observed that REV-ERBbeta is highly overexpressed in multiple breast cancer cell lines, but not in the non-tumorigenic control breast cell line, MCF-10A. Furthermore, we show that a synthetic REV-ERB agonist that we discovered, SR9011, inhibits the proliferation of multiple breast cancer cell lines including triple negative breast cancers but has no effect on MCF10A cells. We discovered that the REV-ERBs directly regulates the expression of cyclin A2 gene suggesting a possible mechanism for inhibition of proliferation. Treatment of breast cancer cell lines with SR9011 repress the expression of cyclin A2 at both mRNA and protein levels. SR9011 treated SKBR3 cells display a reduced fraction of cells in S phase and in the G2/M phase. Administration SR9011 to synchronized SKBR3 cells results in about 8 hours delay of G2/M phase occurrence. Thus, our data suggests that REV-ERB is a valid target for development of anti-cancer agents and SR9011 holds promise for treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1357. doi:10.1158/1538-7445.AM2011-1357

SummaryLigand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution nuclear magnetic resonance (NMR) studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators.

SummaryUnderstanding the DNA recognition and binding by the AbrB-like family of transcriptional regulators is of significant interest since these proteins enable bacteria to elicit the appropriate response to diverse environmental stimuli. Although these “transition-state regulator” proteins have been well characterized at the genetic level, the general and specific mechanisms of DNA binding remain elusive. We present RDC-refined NMR solution structures and dynamic properties of the DNA-binding domains of three Bacillus subtilis transition-state regulators: AbrB, Abh, and SpoVT. We combined previously investigated DNase I footprinting, DNA methylation, gel-shift assays, and mutagenic and NMR studies to generate a structural model of the complex between AbrBN55 and its cognate promoter, abrB8. These investigations have enabled us to generate a model for the specific nature of the transition-state regulator-DNA interaction, a structure that has remained elusive thus far.