Please select what you would like included for printing: Copy the text below and then paste that into your favorite email application This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply Service map data © OpenStreetMap contributors Metrics details Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) in an inverse agonist/corepressor-bound transcriptionally repressive conformation Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume Paramagnetic relaxation enhancement (PRE) NMR and chemical crosslinking mass spectrometry confirm the repressive helix 12 conformation PRE NMR also defines the mechanism of action of the corepressor-selective inverse agonist T0070907 and reveals that apo-helix 12 exchanges between transcriptionally active and repressive conformations—supporting a fundamental hypothesis in the NR field that helix 12 exchanges between transcriptionally active and repressive conformations these structures—some of which are stabilized by crystal contacts between symmetry-related molecules within the crystal lattice or by mutations or coregulator peptides that reduce helix 12 dynamics to facilitate crystal formation for structure determination—are not fully representative of the dynamic NR LBD conformational ensemble Here we report two crystal structures of PPARγ bound to T0070907 and corepressor peptides that reveal a unique a transcriptionally repressive conformation compared with all other reported structures of corepressor-bound NRs A comparative structural analysis to the transcriptionally active conformation and the apo-PPARγ conformational ensemble using solution paramagnetic relaxation enhancement (PRE) NMR validates the unique repressive conformation and provides evidence that helix 12 in an apo-NR exchanges between transcriptionally active and repressive conformations a Fluorescence polarization coregulator interaction assay shows a robust interaction between PPARγ LBD with corepressor ID2 peptides from NCoR and SMRT as well as a TRAP220 coactivator peptide (n = 6) b Mammalian two-hybrid assay in HEK293T cells measuring the effect of T0070907 (10 μM) on the interaction between the Gal4-PPARγ LBD and the VP16-NCoR RID (n = 6; mean ± s.d.) c Luciferase transcriptional reporter assay measuring the ligand-dependent change in PPARγ transcription (n = 4; mean ± s.d.) d TR-FRET biochemical assay measuring the ligand-dependent change in the interaction of PPARγ LBD with SMRT ID2 and TRAP220 ID2 peptides (n = 3; mean ± s.e.m.) Source data are provided as a Source Data file We therefore posited that T0070907 could be used to facilitate crystallization of the PPARγ LBD bound to peptides derived from NCoR and SMRT ID2 motifs a, b Helical structural elements that show notable conformational changes in the crystal structures of a PPARγ LBD bound to rosiglitazone and TRAP220 ID2 peptide (PDB 6ONJ) in the active conformation and b PPARγ LBD bound to T0070907 and NCoR ID2 peptide (PDB 6ONI) in the repressive conformation c Structural overlay of the active and repressive conformation PPARγ LBD crystal structures with arrows depicting the movement of the helical structural elements Ligand-binding pocket volumes (red surfaces) were calculated and displayed using the program CASTp. Ligands were removed from the structures for all calculations. Helix 12 was removed from the repressive conformation structure (PDB 6ONI) to assess the relative pocket volume to the active conformation structures. Calculations for the active conformation structure (PDB 6ONJ) which displays density for the Ω-loop region were performed with and without the Ω-loop region for comparison to the repressive conformation structure d Cell-based luciferase reporter assay in HEK293T cells treated with DMSO control or 5 μM T0070907 measuring the ligand-dependent change in wild-type or mutant PPARγ transcription; data normalized to each WT or mutant construct vehicle control (DMSO) condition (n = 4; mean ± s.e.m.) These corepressor interaction interface structure-guided mutants support the PPARγ crystal structures whereby many interactions contribute to binding corepressor peptide whereas the coactivator interaction is predominately mediated by the “charge clamp” residues h Mammalian two-hybrid assay measuring the effect of T0070907 (10 μM) on the interaction between the Gal4-PPARγ LBD and the VP16-NCoR RID (n = 6; mean ± s.e.m.) i Luciferase transcriptional reporter assay in HEK293T cells treated with DMSO control or 5 μM T0070907 measuring the ligand-dependent change in wild-type or mutant PPARγ transcription; data normalized to each WT or mutant construct vehicle control (DMSO) condition (n = 4–12; mean ± s.e.m.) and +GAP extension all show no increase in NCoR ID2 affinity in the presence of T0070907 and many of the others show diminished corepressor-selective responsiveness to T0070907 mutant significantly weakens NCoR ID2 peptide affinity in both the apo- and T0070907-bound forms The M364 thiol interacts with both the T0070907 nitro group (3.3 and 4.3 Å) and the C-terminal carboxylate of Y477 (3.5 Å) the latter of which may be important for stabilizing the repressive helix 12 conformation in the orthosteric pocket There is generally good agreement between the biochemical and cellular NCoR interaction profiles with the ability of T0070907 to repress the transcription of the mutant variants the mutants that show relatively no T0070907-dependent change in NCoR ID2 peptide binding affinity either do not show T0070907-dependent transcriptional repression or in many cases show activation in the presence of T0070907 mutants that show a T0070907-dependent increase in NCoR ID2 peptide binding affinity generally show T0070907-dependent transcriptional repression This suggests that the repressive helix 12 conformation within the orthosteric pocket may be the primary contribution to the T0070907-dependent increase in corepressor binding affinity and repression of PPARγ transcription is not shown as this conformation is not consistent with the PRE NMR data) conformation PPARγ LBD crystal structures but also indicate the apo-helix 12 exchanges between conformations similar to transcriptionally repressive (T0070907 and NCoR ID2 peptide bound) and active (rosiglitazone and TRAP220 ID2 peptide bound) states a Distances between K474 on helix 12 and several lysine residues structurally proximal to helix 12 (K265 and K275) or further away (K301 and K457) in the crystal structure of PPARγ LBD bound to T0070907 and NCoR ID2 peptide (PDB 6ONI) b Relative peak area of four K474-crosslinked peptides normalized to a control uncrosslinked peptide and to the highest mean peak area within each condition (mean ± s.e.m.; n = 3) which likely inhibits the K301-K474 crosslink; K301 is part of the charge clamp involved in binding coactivator and corepressor-peptide motifs active conformation K474-K457 (helix 12-helix 11) crosslinks were dimensioned in the NCoR ID2 peptide-bound form and further decreased in the presence of T0070907 These XL-MS data support the repressive helix 12 conformation within the orthosteric pocket that we captured in the crystal structures of T0070907-bound PPARγ LBD bound to corepressor peptides and validated with PRE NMR a Structural overlay highlighting the T0070907 binding pose in the crystal structure of PPARγ LBD bound to T0070907 and NCoR ID2 peptide (orange; PDB 6ONI) compared with the crystal structure of T0070907-bound PPARγ LBD without a coregulator peptide (blue; PDB 6C1I) c 2D [1H,15N]-TROSY-HSQC spectra from the PRE analysis of MTSL-labeled T0070907-bound [K474C]-PPARγ LBD without coregulator peptide Purple boxes in (b) highlight residues shown the zoomed-in snapshots (c) showing that only one of two slowly exchanging populations corresponding to the active (labeled “a”) and repressive (labeled “r”) is affected by the MTSL spin label on helix 12 These PRE NMR findings are consistent with helix 12 occupying both the repressive conformation (e.g. and G346 in the β-sheet are affected) and the active conformation (e.g. one of the two peaks for G399 in the AF-2 surface is affected) when of PPARγ is bound to T0070907 solvent exposed) and repressive helix 12 conformation (i.e. within the orthosteric pocket) may influence corepressor selectivity the rate of exchange between active and repressive helix 12 conformations is relatively fast (intermediate NMR time scale) Binding of T0070907 slows the rate of exchange considerably allowing population of a long-lived helix 12 conformation within the orthosteric ligand-binding pocket that affords high corepressor binding affinity and transcriptional repression GW9662-bound PPARγ likely populates a shorter-lived repressive helix 12 population within the orthosteric pocket that exchanges with an active-like conformation more quickly relative to T0070907-bound PPARγ but slower than apo-PPARγ resulting in an intermediate NCoR ID2 affinity between T0070907-bound PPARγ and apo-PPARγ In our corepressor-peptide-bound structures of PPARγ the corepressor-selective inverse agonist T0070907 adopts a binding pose near the AF-2 surface that leaves a large portion of the orthosteric pocket open—double the pocket volume of the coactivator-bound active conformation—which along with a kink in helix 3 relative to the active conformation enables helix 12 to bind deep within the pocket near the β-sheet in a repressive conformation This helix 12 conformation is unique compared with all other published NR crystal structures and enables PPARγ to interact with the ID2 motif of NCoR and SMRT corepressor proteins with high affinity likely by sequestering helix 12 away from the AF-2 surface where it would physically clash with the bound corepressor ID2 peptide the SR10171- and SR11023-bound crystal structures do not report on the conformation of helix 12 in an activity-dependent Structural studies in the presence of corepressor peptides may provide additional insight into the mechanism of action of these ligands compared with covalent inverse agonists and antagonists (T0070907 and GW9662) that enable helix 12 to exchange between a solvent-exposed conformation and a buried conformation within the orthosteric ligand-binding pocket It is therefore possible that other NRs could share a similar helix 12-dependent mechanism for transcriptional repression though future studies would need to explore this in detail but do not stabilize an active helix 12 conformation Noncovalent orthosteric antagonists may maintain a conformation resulting in a transcriptionally neutral coregulator affinity balance that mimics the coregulator affinity of apo-PPARγ or PPARγ bound to endogenous ligands noncovalent orthosteric inverse agonists may function by two related mechanisms: competing with endogenous PPARγ ligands that activate PPARγ transcription and inducing a conformation that weakens coactivator binding without significantly increasing corepressor binding affinity which in total maintains a relative preference for corepressor binding the corepressor-selective inverse agonist mechanism of T0070907 we describe here is different Our work suggests an corepressor-selective ligand design model in which ligands that are able to completely displace helix 12 from the AF-2 surface—e.g. by stabilizing helix 12 within the orthosteric pocket—will robustly increase corepressor binding affinity resulting in transcriptional repression of PPARγ Wild type or mutant human PPARγ ligand-binding domain (LBD) protein was expressed from a pET46 Ek/LIC plasmid (Novagen) as a TEV-clevable N-terminal hexahistag fusion protein in Escherichia coli BL21(DE3) cells using autoinduction ZY media (unlabeled protein) or using M9 minimal media (for NMR studies) supplemented with 15N ammonium chloride with or without 13C-glucose and D2O at 37 °C cells were induced with 1.0 mM isopropyl β-D-thiogalactoside (M9) at an OD600nm of 0.6 grown for an additional 24–48 h at 18 °C then harvested cells were grown for 5 h at 37 °C and additional 12–18 h at 22 °C then harvested Cells were lysed and 6xHis-PPARγ LBD was purified using Ni-NTA affinity chromatography and gel filtration chromatography TEV protease was used to cleave the histag for most experiments except protein used for TR-FRET and fluorescence polarization The purified proteins were concentrated to 10 mg mL−1 in a buffer consisting of 20 mM potassium phosphate (pH 7.4) Purified protein was verified by SDS-PAGE as >95% pure Covalent binding of T0070907 to PPARγ LBD (wild type or mutant variants) was analyzed by ESI-MS using a LTQ XL linear Ion trap mass spectrometer (Thermo Scientific); samples were incubated with or without 2 molar equivalents of T0070907 (unless otherwise indicated below) at 4 °C overnight and diluted to 2–3 μM in 0.1% formic acid for ESI-MS analysis HEK293T (ATCC CRL03216) were cultured in Dulbecco’s minimal essential medium (DMEM Gibco) supplemented with 10% fetal bovine serum (FBS) and 50 units ml−1 of penicillin Cells were plated 20,000 cells/well in a 96-well flat bottom cell culture plate and co-transfected with 100 ng pG5-UAS and 25 ng pCMV-Gal4-PPARγ (human residues 185–477 containing the hinge and LBD) along with pACT empty vector (Promega) expressing the VP16 transactivation domain only pACT with VP16 fused to the mouse NCoR receptor interaction domain (RID or pAct with VP16 fused to the NCoR RID with each critical residue of the ID2 motif (LEDIIRKAL) mutated to threonine (TEDTTRKAT) Transfection solutions were prepared in Opti-MEM with Mirus Bio TransIT-LT1 transfection reagent After 24 h incubation at 37 °C in a 5% CO2 incubator DMSO or T0070907 was added at a final concentration of 0.01% or 10 μM britelite plus (PerkinElmer) was added to each well then transferred to a white-bottom 384-well plate and read on a BioTek Synergy Neo multimode plate reader Data were plotted and analyzed using GraphPad Prism software Cells were grown to 90% confluency in T-75 flasks; from this 2 million cells were seeded in a 10-cm cell culture dish for transfection using X-tremegene 9 (Roche) and Opti-MEM (Gibco) with full-length human PPARγ (isoform 2) expression plasmid (4 μg) and a luciferase reporter plasmid containing the three copies of the PPAR-binding DNA response element (PPRE) sequence (3xPPRE-luciferase) (4 μg) cells were transferred to white 384-well cell culture plates (Thermo Fisher Scientific) at 10,000 cells/well in 20 μL total volume/well cells were treated in quadruplicate with 20 μL of either vehicle control (1.5% DMSO in DMEM media) twofold serial dilution of TZDs for dose response experiments cells were harvested with 20 μL Britelite Plus (PerkinElmer) and luminescence was measured on a BioTek Synergy Neo multimode plate reader Data were plotted in GraphPad Prism as luminescence vs ligand concentration and fit to a sigmoidal dose response curve Time-resolved fluorescence resonance energy transfer (TR-FRET) assays were performed in low-volume black 384-well plates (Greiner) using 23 μL final well volume For the TR-FRET coregulator peptide interaction assay each well contained 4 nM protein (WT or mutant 6xHis-PPARγ LBD) 1 nM LanthaScreen Elite Tb-anti-His Antibody (ThermoFisher #PV5895) NCoR and SMRT peptide in TR-FRET buffer (20 mM potassium phosphoate each well contained 1 nM protein (wild type or mutant 6xHis-PPARγ LBD) 1 nM LanthaScreen Elite Tb-anti-HIS Antibody (ThermoFisher #PV5895) and 5 nM Fluormone Pan-PPAR Green (Invitrogen; #PV4894) in TR-FRET buffer at pH 8 compounds stocks were prepared via serial dilution in DMSO and plates were read using BioTek Synergy Neo multimode plate reader after incubation at 25 °C for several time points between 1 and 24 h and the acceptor FITC emission was measured at 520 nm Data were plotted using GraphPad Prism as TR-FRET ratio (520 nm/495 nm) vs The coregulator interaction data were fit to sigmoidal dose response curve equation and ligand displacement data were fit to the one site–Fit Ki binding equation using the binding affinity of Fluormone Pan-PPAR Green (2.8 nM; Invitrogen #PV4894 product insert) Wild type or mutant 6xHis-PPARγ LBD (with or without T0070907) was serially diluted in assay buffer (20 mM potassium phosphate pH 8) and plated with 180 nM FITC-labeled TRAP220 ID2 or NCoR ID2 peptides in low-volume black 384-well plates (Greiner) in triplicate For single PPARγ LBD concentration experiments the final concentration of FITC-labeled SMRT (ID1 and ID2) and TRAP220 ID2 peptides was 180 nM and wild-type 6xHis-PPARγ LBD concentration was 25 μM and fluorescence polarization was measured on a BioTek Synergy Neo multimode plate reader at 485 nm emission and 528 nm excitation wavelengths Data were plotted using GraphPad Prism as fluorescence polarization signal in millipolarization units vs protein concentration and fit to a one site—total binding equation To visualize the location of MTSL for the PRE NMR studies residue K474 was first modeled into cysteine (C474) then the MTSL ligand was imported into Coot as a SMILES string: CC1(C)C(CSS(C)(=O)=O)=CC(C)(C)N1[O] The MTSL sulfinic acid leaving group was removed and placed adjacent to C474 after which it was regularized and covalently attached to C474 The parameters for pLink2 search were as follows: three missed cleavage sites for trypsin/chymotrypsin for each chain; peptide length 4–100 amino acid; pLink2 search results were filtered by requiring precursor tolerance (±10 p.p.m.) and fragment tolerance (±15 p.p.m.) and FDR below 5% was required for all identified XL-MS peaks The ion intensities and peak areas of the crosslinked peptides from different experimental conditions were manually calculated and compared The highest peak intensity across all indicated experimental conditions within each bar plot panel was normalized as 100%—first to raw peak area of a control peptide lacking a lysine residue (S289-VEAVQEITE-Y299) to normalize for sample injection then to the highest mean peak area within each condition Further information on research design is available in the Nature Research Reporting Summary linked to this article All other datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request Structural overview of the nuclear receptor superfamily: insights into physiology and therapeutics Activation of nuclear receptors: a perspective from structural genomics The antiparasitic drug ivermectin is a novel FXR ligand that regulates metabolism Discovery of second generation rorγ inhibitors composed of an azole scaffold A unique secondary-structure switch controls constitutive gene repression by retinoic acid receptor A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator Ternary crystal structure of human RORγ ligand-binding-domain an inhibitor and corepressor peptide provides a new insight into corepressor interaction Ternary complex of human RORγ ligand-binding domain inverse agonist and SMRT peptide shows a unique mechanism of corepressor recruitment Molecular determinants of the recognition of ulipristal acetate by oxo-steroid receptors Structure of Rev-erbα bound to N-CoR reveals a unique mechanism of nuclear receptor-co-repressor interaction Molecular switch in the glucocorticoid receptor: active and passive antagonist conformations X-ray crystal structures of the estrogen-related receptor-gamma ligand binding domain in three functional states reveal the molecular basis of small molecule regulation Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARα Structural basis for retinoic X receptor repression on the tetramer A revisited version of the apo structure of the ligand-binding domain of the human nuclear receptor retinoic X receptor alpha Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma NFκB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity Structural basis for the deactivation of the estrogen-related receptor gamma by diethylstilbestrol or 4-hydroxytamoxifen and determinants of selectivity Small molecule modulation of nuclear receptor conformational dynamics: implications for function and drug discovery Review of the structural and dynamic mechanisms of PPARγ partial agonism Partial agonists activate PPARγ using a helix 12 independent mechanism Structural basis for the activation of PPARγ by oxidized fatty acids Ligand and receptor dynamics contribute to the mechanism of graded PPARγ agonism Ligand-induced stabilization of PPARγ monitored by NMR spectroscopy: implications for nuclear receptor activation The nuclear receptor corepressors NCoR and SMRT decrease peroxisome proliferator-activated receptor gamma transcriptional activity and repress 3T3-L1 adipogenesis a selective ligand for peroxisome proliferator-activated receptor gamma functions as an antagonist of biochemical and cellular activities A structural mechanism for directing corepressor-selective inverse agonism of PPARγ Defining a conformational ensemble that directs activation of PPARγ PPARγ in complex with an antagonist and inverse agonist: a tumble and trap mechanism of the activation helix Structural basis of the transactivation deficiency of the human PPARγ F360L mutant associated with familial partial lipodystrophy Nitroxide labeling of proteins and the determination of paramagnetic relaxation derived distance restraints for NMR studies Chemical crosslinking mass spectrometry reveals the conformational landscape of the activation helix of PPARγ; a model for ligand-dependent antagonism Distance restraints from crosslinking mass spectrometry: mining a molecular dynamics simulation database to evaluate lysine-lysine distances Pharmacological repression of PPARγ promotes osteogenesis Cooperative cobinding of synthetic and natural ligands to the nuclear receptor PPARγ Crystal structure of the ligand binding domain of the human nuclear receptor PPARγ Structural mechanism for signal transduction in RXR nuclear receptor heterodimers Analysis of ligand binding and protein dynamics of human retinoid X receptor alpha ligand-binding domain by nuclear magnetic resonance The effect of antagonists on the conformational exchange of the retinoid X receptor alpha ligand-binding domain Prediction of the tissue-specificity of selective estrogen receptor modulators by using a single biochemical method Unique ligand binding patterns between estrogen receptor alpha and beta revealed by hydrogen-deuterium exchange Hydrogen/deuterium exchange reveals distinct agonist/partial agonist receptor dynamics within vitamin D receptor/retinoid X receptor heterodimer Helix 11 dynamics is critical for constitutive androstane receptor activity Defining the communication between agonist and coactivator binding in the retinoid X receptor alpha ligand binding domain Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM Overview of the CCP4 suite and current developments The Phenix software for automated determination of macromolecular structures Coot: model-building tools for molecular graphics CASTp 3.0: computed atlas of surface topography of proteins UCSF Chimera—a visualization system for exploratory research and analysis Using chemical shift perturbation to characterise ligand binding NMRFx Processor: a cross-platform NMR data processing program Using NMRView to visualize and analyze the NMR spectra of macromolecules Identification of cross-linked peptides from complex samples Download references We thank Paola Munoz-Tello and Ted Kamenecka for helpful discussions and input This work was supported by National Institutes of Health (NIH) grants F32DK108442 (R.B.) and R01DK101871 (D.J.K.); Richard and Helen DeVos graduate fellowship award (S.M.); and National Science Foundation (NSF) award 1359369 that funds the SURF program at Scripps Research Florida (S.M.) Use of the Stanford Synchrotron Radiation Lightsource Office of Basic Energy Sciences under Contract No The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research National Institute of General Medical Sciences (including P41GM103393) The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIDDK Department of Integrative Structural and Computational Biology Skaggs Graduate School of Chemical and Biological Sciences Summer Undergraduate Research Fellows (SURF) program solved the crystal structures and performed NMR and biochemical assays performed the mammalian two-hybrid assay; A.N performed chemical crosslinking mass spectrometry experiments supervised the research and wrote the manuscript along with J.S and input from all authors who approved the final version The authors declare no competing interests Peer review information Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-020-14750-x Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science Amazon’s first robotics centre in the Czech Republic has officially launched its operations and is up and running with a total area just shy of 190,000 square metres and is outfitted with cutting-edge robotic technology it stands as one of the Czech Republic's most state-of-the-art logistics hubs Designated as "BRQ2," the centre officially commenced operations in June of this year It heralds the introduction of the latest technologies aimed at simplifying daily tasks for Amazon employees while 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2,000 good new permanent roles when fully operational,” said Michal Šmíd adding: “Amazon provides some of the most advanced workplaces of their kind in the world and systems to ensure the wellbeing and safety of all employees and I am delighted to launch this new building.” Amazon has invested over 26 billion Czech crowns in the Czech Republic A portion of this investment is dedicated to implementing advanced technologies that support safer work methods and improve wellbeing by reducing the need for heavy lifting Amazon’s European Advanced Technology team has played a pivotal role in creating more than 550 new pieces of technology with over €400 million invested across Amazon sites in Europe in three years Bethel’s Core Value Mark of Distinction award highlights employees who consistently live out the core values of our university these four Bethel community members were named as the 2019 award recipients Doug Kojetin is honored as a Christ-follower and character-builder for his 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colleagues and students for the joy and hospitality she radiates in the Education Department Johnson is a source of light and God’s love to her department and students Associate Professor of Education Geri Von Grey works alongside Johnson “professing Jesus is Savior and Lord and modeling His character to others through such manners as being kind but rather rejoicing in the right while staying rooted in Scripture.” Johnson is a light in the Education Department affecting everyone she comes in contact with as she works hard to ensure their success Students consistently applaud her consistent ability to lift others up She works hard to prepare the teachers that will come out of Bethel as people who will be ready to make a difference in the lives of their students.   Director of Conference & Event Services Avis Soderstrom is the face of Bethel to many people outside our community through her role in Conference & Event Services Soderstrom has built a team that not only advances Bethel’s 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the basis of race Within the intellectual powerhouse that is Vanderbilt University and Vanderbilt University Medical Center thrives a group of doctors computer scientists and others who also are visionaries Several of these change-makers also donate their time and expertise through the Vanderbilt Innovation Ambassadors Program to help colleagues in their departments recognize and pursue the world-altering potential of their own ideas.  “We’re like the kindling that gets the fire going,” said Ambassador Bryan Hartley, MD, associate professor of Clinical Radiology & Radiological Sciences at VUMC whose company, Pulmera is developing novel imaging technology to improve the diagnosis of early-stage lung cancer An extension of long-standing, campuswide efforts to encourage innovation and commercialization activities, the Innovation Ambassador Program was launched in 2022 by the Center for Technology Transfer and Commercialization (CTTC) in collaboration with VUMC, Vanderbilt University School of Medicine and The Wond’ry “Having an ambassador program shows that innovation and entrepreneurship are valued and encouraged,” noted Philip Swaney who co-directs the program with CTTC director and Vanderbilt University assistant vice chancellor Alan Bentley When faculty members see their peers “having an impact in this space,” Swaney said “they feel empowered and supported to act.” Innovation Ambassadors receive about six hours of initial training in commercialization as well as relevant university policies and procedures They’re also kept abreast of new programs and processes that are being rolled out across campus The goal is to enable them share with others in their departments the information and resources that can help them protect and commercialize their ideas and inventions Ambassadors commit to serve two-year terms In addition to communicating what they’ve learned they meet quarterly to brainstorm ways to boost innovation and entrepreneurship An example of their outreach is the Sullivan Family Biomedical Ideator program, launched in January by Ambassador Daniel Fabbri director of Informatics Innovation at VUMC Program participants learn ways to evaluate the commercial value and tech-transfer potential of their research Other examples from the Department of Otolaryngology-Head and Neck Surgery are the national Haynes Award, honoring resident physicians who have made notable innovations, and the Haynes Innovation Symposium, which, according to the department’s innovation webpage “provides practical insights to aspiring innovators on the strategies and pathways toward commercialization and dissemination.” Launched with the help of Alexander Langerman, MD, SM, the Otolaryngology Department’s Ambassador, both the award and symposium are named for David Haynes holder of the Directorship in Otolaryngology for Relationship Development and Chief Patient Experience Officer at VUMC The brainchild of department chair Eben Rosenthal the award and symposium celebrate the “best and the brightest of the next generation of leaders,” and highlight VUMC’s innovative work an accomplished innovator in his own right Langerman and Fabbri are among the first recipients of Vanderbilt University’s Innovation Catalyst Fund for innovations they have brought to their respective fields the fund helps streamline faculty research into real-world applications “The Innovation Ambassador Program provides a great resource for faculty to learn more about inventions and technology commercialization directly from their peers,” said Kenneth Holroyd, MD, MBA, CTTC’s medical director, and lead of the Brock Family Center for Applied Innovation at VUMC.  “It is an important and growing part of building VUMC’s entrepreneurial culture,” said Holroyd who also is VUMC’s Vice President for Tech Transfer “We are looking for additional faculty volunteers to serve from departments that are not currently participating in this program.”  Of the 20 current Innovation Ambassadors, 13 are primarily affiliated with VUMC, including Shabnam Eghbali a first-year resident physician in Internal Medicine who plans a dual career in medicine and biopharma investment said she hopes as an Innovation Ambassador to help other physicians in specialty training learn how they can apply their knowledge to affect large-scale changes in health care represent the departments of Biochemistry and Pharmacology in the School of Medicine Basic Sciences TS Harvey, PhD, co-recipient of a $1 million grant from the Robert Wood Johnson Foundation to study cultural factors in health inequities represents the Department of Anthropology in the College of Arts and Science Ambassador Christopher Vanags directs the Peabody Research Office in the Vanderbilt Peabody College of Education and Human Development The Ambassadors program “is a grassroots approach to building out our innovations Ambassador program leadership is helping a half dozen other universities launch similar ambassador-like programs of their own Vanderbilt has long been a hotbed of invention thanks in large part to the creativity of its faculty and the support of the CTTC and 593 patents and 923 licenses were issued generating more than $286 million in revenue.  Vanderbilt and VUMC ranked sixth in the country in adjusted gross income from technology licensing according to the latest survey conducted by AUTM a nationwide community of university technology transfer offices. In FY2023 licensing revenue topped a record $96 million.  technology transfer is more than a revenue stream — it can open the door to inventors who want to help change the world for the better.  Physicians want to have a positive impact on their patients’ lives. The Innovation Ambassadors show that “they can also have a great impact through innovation and entrepreneurship,” said Ambassador Ryan Buckley, MD, who also directs the Medical Innovators Development Program for Vanderbilt medical students Yet “people really need examples, advice and informal conversations with fellow entrepreneurs to be successful,” said Webster, the Richard A. Schroeder Professor of Mechanical Engineering who has co-founded companies that introduced innovations to the robotic surgery and flexible endoscopy fields Last year the National Institutes of Health awarded a four-year, $4 million grant to Webster and Charleson Bell, PhD, director of entrepreneurship and biomedical innovation at the Wond’ry, to establish the Mid-South Research Evaluation and Commercialization Hub (REACH) Leveraging an additional $8 million in state and institutional matching funds mentorship and financial support for aspiring entrepreneurs to hundreds of community colleges and minority-serving institutions in Kentucky accustomed to receiving government support for their research and publishing results in professional journals are reluctant to leap into entrepreneurship But unless they take steps to protect their intellectual property “Basically, you’re killing it,” warned Ambassador Robert Carnahan, PhD, associate director of the Vanderbilt Vaccine Center, who helped develop business modules for scientists offered through the School of Medicine’s BRET Office of Career Development “Most things require a commercial platform,” Carnahan explained “If (companies) are going to put a million dollars into it they need to know they’re going to get a return on their investment you’ve got to put some protections around it.” Entrepreneurship is not for the faint of heart “It is not easy to take a proof-of-principle result from the bench to the market,” cautioned Kostoulas professor of the Practice of Engineering Management who has extensive experience as a marketing professional in the semiconductor industry “Most of the work and expense happens during that transition.” Faculty inventors don’t have to shoulder this burden alone By pointing the way to the tech transfer office and investment community Innovation Ambassadors encourage crucial collaborations that can help turn vision into reality “The Innovation Ambassadors program provides a dynamic network for all of those interactions to happen.” Webster said “By building this into the fabric of what we do at Vanderbilt we will ultimately better achieve Vanderbilt’s core missions of discovery and translating those discoveries to impact the real world.” Colin Barker Barker is an international leader in interventional cardiology and percutaneous heart valve therapies His research has led to development of devices to prevent strokes and improve treatment of congestive heart failure Ryan Buckley For Buckley, who also directs the Medical Innovators Development Program for Vanderbilt medical students innovation is about bringing people together to find new ways to alleviate the suffering of others Robert Carnahan Professor of Pediatrics and of Radiology and Radiological Sciences A leader in developing monoclonal antibody treatments for a host of viral diseases Carnahan also is known internationally for his contributions to rational vaccine design Edward Chaum A self-described “serial entrepreneur,” Chaum specializes in the medical and surgical management of retinal diseases drug development and biomedical engineering Shabnam Eghbali Eghbali, who worked for two investment funds while in medical school, is co-founder of Theia Healthcare a nonprofit dedicated to inspiring and empowering women entrepreneurs and investors in healthcare Her goal is a dual career as physician and biopharma investor Daniel Fabbri Holder of the DBMI Directorship in Informatics Innovation at VUMC Biomedical Informatics and Computer Science Fabbri has developed and marketed tools to improve the security and efficient use of medical records and envisioned an “Ideator” program to evaluate the marketability of faculty research Bryan Hartley Clinical Radiology & Radiological Sciences A Vanderbilt-trained radiologist, Hartley was inspired by his experience as a Stanford Biodesign Innovation Fellow to “take the next step” to entrepreneurship. He inspires others as a contributor to the BackTable Innovation podcast series TS Harvey Vanderbilt University’s first Ford Foundation Senior Fellow Harvey’s research in Guatemala and elsewhere focuses on expanding scientific partnerships and building local capacities to collaboratively tackle large-scale public health and environmental challenges experienced by vulnerable populations.  Douglas Kojetin Kojetin, who joined the Vanderbilt faculty in 2023, uses “structural pharmacology” and computational biology methods to define the molecular mechanisms of drug action and to probe the regulation of nuclear receptors, which play a role in a broad spectrum of human disease.Yiorgos Kostoulas Professor of the Practice of Engineering Management Division of Engineering Science and Management Kostoulas is a researcher (optical spectroscopy) who also has extensive professional experience in marketing in the semiconductor industry Alexander Langerman Associate Professor of Otolaryngology-Head & Neck Surgery Langerman is a head-and-neck surgeon and was co-founder of Surgical Explorer now part of GHX (Global Healthcare Exchange) which operates a platform that enables surgical teams to access remote medical device support directly from suppliers George Nicholson Trained in interventional cardiology procedures Nicholson and his colleagues work with biomedical engineers in the design and development of tools techniques and devices tailored to improve outcomes for children with congenital heart disease Chris Vanags Research Assistant Professor of Earth and Environmental Sciences  Vanags helped develop and implement widely adapted STEM enrichment programs Jonathan Wanderer Professor of Anesthesiology and Biomedical Informatics Associate Chief Medical Information Officer, VUMC Wanderer innovates clinical informatics-driven, perioperative interventions, around the time of surgery. He directs the eStar Physician Builders Program which supports the devising of  new content and tools for VUMC’s health and information technology system Alex Waterson Research Professor of Pharmacology and Chemistry Associate Director for Medicinal Chemistry Vanderbilt Center for Cancer Drug Discovery Waterson’s drug discovery collaborations span the cancer and include service on the scientific advisory board of Nashville-based Cumberland Emerging Technologies Robert Webster Schroeder Professor of Mechanical Engineering Webster is investigating the use of surgical robotics in ear James Weimer Assistant Professor of Computer Science and Biomedical Engineering A focus of Weimer’s research is cyber-physical systems in health care. He has cofounded companies that developed a non-invasive continuous stroke monitoring system (Neuralert), and novel technology to monitor the risk of postpartum hemorrhage during labor and delivery (Vasowatch) Lauren Williamson Research Assistant Professor of Pediatrics Williamson’s method of stabilizing cell populations that play essential roles in autoimmune disorders won second place in the 2022 Innovation Cup sponsored by the German science and technology company Merck KGaA She also consults for the biotech industry Jesse Wrenn Wrenn’s research is focused on applying computer science and informatics methods to improve clinical care. He is part of a team investigating whether a strategy to individualize diuretic therapy will improve outcomes for patients hospitalized with acute heart failure Adam Yock Holder of the Directorship in Technology and Innovation for Radiation Oncology Yock is a medical physicist whose group is developing quality assurance devices and treatment planning software to improve the accuracy and outcomes of radiation therapy assistant professor of Chemical and Biomolecular Engineering at Vanderbilt University received a $50,000 research grant at the 2019 Vanderbilt-Ingram Cancer Center Ambassador Breakfast Many clinical trials are stopped prematurely because they fail to recruit enough study participants Vanderbilt University Medical Center has received a five-year $14 million grant from the National Center for Advancing Translational Sciences at the National Institutes of Health to address this Vanderbilt-Ingram Cancer Center Ambassadors surpassed the $1 million mark with the awarding of the group’s latest research grants His Mass of Christian Burial will be 11:00 am Mary in Willmar with interment in the church cemetery.  Visitation will be Tuesday with a Knights of Columbus Rosary at 5:00 pm at Harvey Anderson Funeral Home in Willmar.  www.hafh.org Visitation will continue one hour prior to mass at the church on Wednesday.  Memorials may be given to the Willmar Area Food Shelf or the Andrew Hatlestad Memorial Nursing Scholarship and Angela (Kojetin) Frank in Renville County.  He was the eldest of five children Michael’s Catholic Church in Morgan.  They made their home on a farm in Henryville Township in Renville County.  Myron and Elaine worked hard and enjoyed life on the farm along with their 10 children.  Myron’s specialty was raising pullets and sugar beets.  Myron was a member of the Knights of Columbus Catherine’s Church Council in Redwood Falls and served as a board member for the Danube Farmers Elevator and Oil Company.  Myron was also one of the founding members of Southern Minnesota Beet Sugar Cooperative Myron served as treasurer of the board for the Willmar Senior Citizens and playing cards.  He shared his love and knowledge of the land hard work and faith with his children and grandchildren.  He also taught them many card games and was notorious for winning most of the time.  Myron loved to drive around his farm and the countryside to check how the crops were growing.  Such drives made his day complete even up until his last days children; Kathleen (Steve) Chesney of Brooklyn Park and Brenda Frank (Doug Knight) of Denver CO; Also surviving are his siblings: Donald (Veronica) Frank of Redwood Falls Betty Miller of Redwood Falls and Susie Drexler of Willmar.  Also surviving are his beloved 13 grandchildren and 19 great-grandchildren.  Credit: Photo courtesy of The Scripps Research Institute 2014 – Scientists from the Florida campus of The Scripps Research Institute (TSRI) have been awarded $2.1 million from the National Institutes of Health to study the therapeutic potential of safer and more effective alternatives to the current crop of anti-diabetic drugs which have been limited in their use due to side effects including bone loss and congestive heart failure is the principal investigator for the new five-year study The study will take a cue from the two mainstays of type 2 diabetes treatment—pioglitazone (Actos) and rosiglitazone (Avandia) Both drugs raise the body's sensitivity to insulin increasing the amount of glucose or sugar absorbed by the cells that while these and other recently developed drugs are designed to bind to a specific site on the PPAR gamma (PPARG) nuclear receptor "This unexpected finding opens a lot of potential opportunities," Kojetin said "We're looking to design a molecule that blocks both sites and can be used to probe what this alternative binding does on a molecular level—with the hope that this information will help us come up with a better drug model." are not responsible for the accuracy of news releases posted to EurekAlert by contributing institutions or for the use of any information through the EurekAlert system Copyright © 2025 by the American Association for the Advancement of Science (AAAS) Photo Contest by | Oct 2024 we asked local residents to scroll down their digital files or flip through their film—whatever it took to find their favorite shots of the Lake Minnetonka community From scenic landscapes to candid portraits artistic abstractions to vibrant close-ups Lake Minnetonka Magazine readers delivered their favorite scenes from the past year We would like to extend our thanks to returning contestants and newcomers alike we’re excited to share the winning photos with you Please note that judges reserve the right to recategorize submissions if they feel the photograph is better suited to a different category Title: Sunset Cruise – Lake Minnetonka Title: Minnetonka Yacht Club In The Clouds Sign up for our newsletter and receive email updates with our top stories , , , , © Copyright 2026 Local. All Rights Reserved | Privacy Policy Read the May 2025 Lake Minnetonka Magazine Digital Edition            Metrics details 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 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 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 it is poorly understood how ligand binding to one LBD controls co-regulator recruitment to its dimer partner within a NR heterodimer complex The structural mechanisms that generate this spectrum of signalling outcomes are unknown where helix 5 functions more generally as a signalling rheostat that integrates signals with the dimer interface (a) CV-1 cells co-transfected with PPARγ expression plasmid RXRα expression plasmid and a PPAR-responsive luciferase reporter 10 μM 9cRA and/or 10 μM rosiglitazone for 24 h Magenta and black bars are coloured to match NMR data in d Luciferase activity is shown normalized to vehicle-treated cells and was performed in quadruplicate plotted with the average (±s.e.m) and representative of at least three experiments (b) CV-1 cells co-transfected with TRβ expression plasmid RXRα expression plasmid and a TR-responsive luciferase reporter 100 nM LG100268 (LG268) and/or 1 μM T3 for 24 h plotted with the average (±s.e.m) and representative of at least two experiments Magenta and black bars are coloured to match NMR data in e (c) Structural location of RXR residues mentioned in the NMR analysis (d) Overlay of 2D [1H,15N]-TROSY-HSQC NMR data for [2H,13C,15N]-RXRα LBD in the apo form and coloured magenta—with the same bound to 9cRA and coloured black (e) Overlay of 2D [1H,15N]-TROSY-HSQC NMR data for [2H,13C,15N]-RXRα LBD bound to 9cRA and heterodimerized to apo-PPARγ and coloured magenta—with the same bound to rosiglitazone (Rosi) and coloured black (f) Overlay of 2D [1H,15N]-TROSY-HSQC NMR data for [2H,13C,15N]-RXRα LBD bound to 9cRA and heterodimerized to apo-TRβ and coloured magenta—with the same bound to T3 and coloured black indicating this may be a general feature for ligand activation of NRs To determine the mechanism through which PPARγ acts as a permissive dimer partner, we performed differential NMR analysis by adding unlabelled apo-PPARγ to 9cRA-bound isotopically labelled RXRα, with and without addition of the full PPARγ agonist, rosiglitazone (Fig. 1b,d) NMR chemical shift changes in RXRα are observed on addition of apo-PPARγ to the 9cRA-bound RXRα consistent with complete formation of a heterodimer complex Addition of rosiglitazone causes subtle but significant NMR chemical shift changes in RXRα (for example S355 and G429 in the dimer interface) and only minor changes in NMR resonance linewidths for select residues although heterodimerization with and ligand binding to PPARγ perturbs the conformation of RXRα neither of these events dramatically affects the μs–ms dynamics of RXRα This is in contrast to what occurs with the non-permissive RXR partner To determine the mechanism through which TRβ acts as a non-permissive RXR heterodimer partner, we performed differential NMR analysis by adding unlabelled apo-TRβ to 9cRA-bound isotopically labelled RXRα, with and without addition of the TRβ agonist, T3 (Fig. 1b,e) addition of apo-TRβ exerts a profound effect on the μs–ms conformational dynamics of 9cRA-bound RXRα where a large number of agonist-bound RXRα NMR resonances revert to an apo-like NMR profile NMR resonances that are destabilized—missing or have broad linewidths indicating increased μs–ms motion—correspond to RXRα residues in the ligand-binding pocket (for example G413 and G429) and other nearby regions such as helix 8 (for example Even more striking is that the addition of the TRβ agonist re-stabilizes these agonist-bound RXRα residues by decreasing motion on the μs–ms timescale resulting in a reappearance of NMR resonances for these regions many of the missing NMR resonances in the apo-TRβ/agonist-RXRα heterodimer correspond to residues in the apo-RXRα homodimer that are stabilized on binding 9cRA Our NMR studies indicate that these residues are not affected by PPARγ heterodimerization or ligand binding to PPARγ but they are significantly affected by TRβ heterodimerization and ligand binding to TRβ these data indicate that the mechanism through which RXRα is allosterically silenced by TRβ but not PPARγ involves conformational dynamics on the μs–ms timescale (a) Structure of the TR/RXR LBD heterodimer is shown as ribbon with T3 as space filled and the bound SRC-2 peptide is coloured blue and only binds to T3·TR with the dimer interface coloured coral and helix 12 coloured purple and RXR helix 12 adopts an inactive conformation positioned into the AF-2 co-activator-binding surface (b) TR is superimposed on the RXR LBD homodimer (PDB 1MVC) except dimers are superimposed via the RXR protomer illustrating the shift in the TR dimer interface relative to the other RXR promoter in the RXR homodimer The magenta circle highlights the only region that superimposes similarly between TR and RXR The amino-terminal end of TRβ helix 11 is oriented similarly towards RXRα 10 and the carboxyl-terminal part of helix 11 are substantially shifted this altered dimer interface induces conformational changes in RXRα (a) Helix 11 of the dimer interface shown as Cα traces for TRβ·T3·SRC-2/apo-RXRα RXR/PPARγ (PDB 1FM6) and RXR/LXR (PDB 1UHL) heterodimers were superimposed on the TRβ·T3·SRC-2/apo-RXRα structure using the RXR promoter molecule and are coloured grey (b–d) The active conformation RXR homodimer (PDB 1MVC) superimposed on the TRβ·T3·SRC-2/apo-RXRα structure via RXR and coloured as in a (b) TR helix 11 (green) induces a shift in the RXR helix 11 (coral) relative to the RXR homodimer (grey) (c) The unique position of TR T426 in helix 11 induces a shift in RXR P423 in helix 11 and a rotation of the RXR helix 11 backbone (d) The location of TR A433 in helix 11 away from the dimer interface compared with the equivalent residue in RXR allows RXR L430 and the RXR helical backbone to rotate in context of the TRβ·T3·SRC-2/apo-RXRα heterodimer (e) RXR in the active conformation (PDB 1MVC) with RXR ligand (MBS649) shown as space filled and SRC-2 peptide bound to the AF-2 co-activator-binding surface coloured red RXR W305 in helix 5 mediates contacts with the ligand M454 in helix 12 and the co-activator-binding site via L276 in helix 3 Colour is used to help differentiate secondary structural elements and provide depth for overlapping elements; helix 3 and 4 in cyan (f) TRβ·T3·SRC-2/apo-RXRα was superimposed with the active conformation RXR homodimer (PDB 1MVC) and shown as Cα trace The rotation of helix 5 in TRβ·T3·SRC-2/apo-RXRα repositions W305 such that it clashes with the active conformation of RXR L276 but showing the active conformation of helix 12 and the clash with the rotated position of W305 in the TRβ·T3·SRC-2/apo-RXRα heterodimer Thus in the TRβ·T3/apo-RXRα structure the TRβ-induced rotation of RXRα helix 11 and helix 5 disables the active conformation of RXRα although the structural mechanism that drives this effect at the atomic level remained unknown (a) The TRβ·T3·SRC-2/apo-RXRα structure (green and coral) superimposed with the active conformation RXR homodimer (PDB 1MVC) via RXR and shown in grey E307) are part of a network of co-evolved amino acids identified using a statistical coupling analysis (SCA) (b) The SCA network amino acids shown as space filled on the TRβ·T3·SRC-2/apo-RXRα heterodimer link the dimer interface ligand-binding pocket and AF-2 co-activator-binding site (c) Active conformation RXR homodimer (PDB 1MVC) superimposed with RXR from the TRβ·T3·SRC-2/apo-RXRα heterodimer Shown are helix 3 and helix 4 of the AF-2 surface The rotation of helix 5 induces an altered conformation of the AF-2 surface via the SCA network amino acids (d) Regions in RXR that are protected from HDX on heterodimerizaton with TR The changes in HDX support our model where these regions direct allosteric signalling within the heterodimer resulting in the silencing of RXR by TR (a,b) CV-1 cells co-transfected with TRβ expression plasmid (a) RXRα or (b) RXRα L276V mutant expression plasmid or the indicated dose of T3±1 μM 9cRA for 24 h (c) Drosophila SL2 cells co-transfected with TRβ expression plasmid RXRα or RXRα E434-mutant expression plasmid the RXR agonist LG100268 (LG268; 100 nM) and/or 1 μM TR agonist (T3) for 24 h (d) CV-1 cells co-transfected with TRβ expression plasmid RXRα or RXRα E434N mutant expression plasmid Cells were treated with vehicle or the indicated dose of T3 overnight (e) Drosophila SL2 cells co-transfected with VDR expression plasmid the RXR agonist LG100268 (LG268; 100 nM) and/or 1 μM VDR agonist (vitamin D3) for 24 h Luciferase activity is shown normalized to vehicle-treated cells and was performed in quadruplicate; plotted with the average±s.e.m and representative of at least three experiments the C terminus of RXRα helix 11 plays a critical role in regulating the response of the TRβ/RXRα heterodimer to T3 Although RXR ligand has no activity on its own within the context of the VDR/RXRα heterodimer it is conditionally permissive because it enhances vitamin D3-induced transactivation The helix 11 mutations selectively modulate the conditional activation by the combination of vitamin D3 and RXR agonists and lead to both gain- and loss-of-function these data suggest that similar helix 11-mediated mechanisms control allosteric signalling across the dimer interfaces of TR/RXR and VDR/RXR heterodimers and that there are several mechanisms for heterodimer signal integration NMR data are coloured grey for 9cRA-bound RXRα; black for 9cRA-bound RXRα heterodimerized to apo-PPARγ or the same bound to the following PPARγ ligands: rosiglitazone (magenta) MRL24 (orange) or SR1664 (green); plotted on PPARγ/RXRα (PDB 1FM9) (a) NMR data (left) focusing on residues in RXRα helix 7 and helix 10/11 dimer interface that are perturbed by ligand binding to PPARγ which are plotted onto the PPARγ/RXRα crystal structure and coloured according to structural location (yellow for helix 7; blue for helix 10/11); coloured dark if shown in the NMR data to the left or light if not (b) NMR data (left) focusing on residues in core of RXRα that are perturbed by ligand binding to PPARγ which are plotted onto the PPARγ/RXRα crystal structure and coloured red; and coloured dark if shown in the NMR data to the left or light if not (c) NMR data (left) focusing on residues in RXRα helix 12 the AF-2 surface and the ligand-binding pocket that are perturbed by ligand binding to PPARγ which are plotted onto the PPARγ/RXRα crystal structure and coloured according to structural location (green for AF-2/helix 12; orange for the ligand-binding pocket); coloured dark if shown in the NMR data to the left or light if not RXRα K431 in helix 11 forms a hydrogen bond with PPARγ Y477 RXRα residues affected differently by the graded PPARγ ligands are structurally close to this region the effect of PPARγ full agonists on RXRα residues in the dimer interface are likely mediated through stabilization of PPARγ helix 12 and its interaction with RXRα but notable changes in RXRα helix 3 residue L276 as well as AF-2 surface residues K284 and S290 which are part of a region that forms electrostatic interactions with the bound co-activator peptide in the crystal structures RXRα helix 5 is a key part of the LBD core that transmits PPARγ ligand-induced allosteric signals from the dimer interface to the RXRα ligand-binding pocket and the AF-2 surface also mediates PPARγ ligand-induced allosteric signalling across the dimer interface in a similar direction Helix 9 forms part of the core that contacts the dimer interface and stabilizes the AF-2 surface via interaction with helix 3 and helix 4 residues PPARγ ligand-induced NMR chemical shift changes in RXRα helix 11 (for example suggest that helix 9 may also transmit allosteric information from the dimer interface to the AF-2 surface Additional NMR resonances showing specific changes in response to PPARγ ligands include A457 at the C terminus of RXRα helix 12 and A327 in the RXRα ligand-binding pocket These effects are not only present at the dimer interface but also extend through the core of the RXRα LBD these structural regions involve a network of co-evolved amino acids in NRs which are energetically coupled and mediate allosteric signalling in RXR heterodimers (a) NMR chemical shift perturbations in PPARγ on heterodimerization with RXRα mapped onto the structure of the PPARγ LBD (PDB 2PRG) (b) LXR homodimer (PDB 3IPU) coloured grey and superimposed with the LXR promoter from the LXR/RXR heterodimer (PDB 1UHL) coloured green and coral shows that RXR induces rotation of LXR helix 11 (c) The RXR-induced shift in LXR helix 11 (PDB 1UHL) induces a rotation of LXR helix 5 relative to the LXR homodimer (PDB 3IPU) allowing W443 in helix 12 to adopt an alternative conformation with greater van der Waals contacts and increased buried surface area supporting a general role for helix 5 rotation in allosteric control of RXR heterodimers helix 5 rotation and the evolutionarily conserved SCA network of amino acid residues provide a structural conduit for signalling from the dimer partner to the ligand-binding pocket and co-regulator-binding surface most of the structural features for allosteric signal integration have remained a mystery limited in part by our insufficient structural understanding of signalling within the individual domains (a) The SCA network residues plotted on RXR using the PPARγ/RXRα heterodimer structure as a model (PDB 1FM6) (b) Schematic diagram summarizing our TRβ·T3·SRC-2/apo-RXRα crystal structure showing how TR structurally silences RXR The signal that emanates from TR (i) induces a shift in RXR helix 11 (ii) leading to a rotation of helix 5 (iii) resulting in structural arrangements that cause RXR helix 12 to adopt an inactive conformation (iv) (c) Summary of residues affected in the NMR analysis of ligand-selective signalling in PPARγ/RXRα Helix numbers are indicated for elements of interest Arrows indicate the flow of the allosteric signal These regions in general employ the network of co-evolved residues predicted by SCA PPARγ full agonists appear to cause a more prominent effect but the specific role of this structural conduit is not clear It could mean that PPARγ full agonists may provide additional stabilization to the RXRα AF-2 surface or alter the shape of the AF-2 to give preferences for certain co-activators Our NMR data further suggests that this interlocking relay system is also modulated by the ligand as one of the two tryptophan residues in the RXR LBD was differentially sensitive to PPAR ligands We thus envision that structural elements in helix 5 of RXR and the dimer partner can move in a coordinated way with the C-terminal region of the helix 11 dimer interface to coordinate both receptor- and ligand-specific signals into an integrated transcriptional response with the co-evolved amino acids playing a primary role Our data suggest an extension of this model where the position of helix 11 is also controlled by the heterodimer partner helix 11 Our mutagenesis data further suggests that the C terminus of helix 11 is also positioned by the type of dimer partner contributing to permissive versus non-permissive heterodimer signal integration 15N and 13C′ (carbonyl) NMR chemical shift differences between monomer and heterodimer and mapped onto the PPARγ LBD crystal structure (PDB 2PRG) Each HDX experiment was carried out in triplicate and the intensity-weighted average m/z value (centroid) of each peptide isotopic envelope was calculated Data-dependent tandem mass spectroscopy was performed in the absence of exposure to deuterium for peptide identification in a separate experiment using a 60-min gradient Peptides with a Mascot score of ≥20 were included in the peptide sets used for HDX PPREx3-luc (PPAR-luc) or ADH-mSppx3-luc (VDR-luc) RXR mutant expression plasmids were generated using the Stratagene QuikChange Site-Directed Mutagenesis kit and verified by DNA sequencing Statistical analyses were performed with Graphpad Prism Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 4ZO1 Retinoid x receptor heterodimers in the metabolic syndrome Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response Molecular determinants for the tissue specificity of SERMs Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARgamma by Cdk5 The therapeutic potential of nuclear receptor modulators for treatment of metabolic disorders: PPARgamma DNA binding site sequence directs glucocorticoid receptor structure and activity DNA binding alters coactivator interaction surfaces of the intact VDR-RXR complex The glucocorticoid receptor dimer interface allosterically transmits sequence-specific DNA signals A novel role for helix 12 of retinoid X receptor in regulating repression Structural determinants of 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recognition of agonist ligands by RXRs Crystal structure of the heterodimeric complex of LXRalpha and RXRbeta ligand-binding domains in a fully agonistic conformation Asymmetry in the PPARgamma/RXRalpha crystal structure reveals the molecular basis of heterodimerization among nuclear receptors Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry Structural basis for autorepression of retinoid X receptor by tetramer formation and the AF-2 helix Effect of heterodimer partner RXRalpha on PPARgamma activation function-2 helix in solution Heterodimeric interaction between retinoid X receptor alpha and orphan nuclear receptor OR1 reveals dimerization-induced activation as a novel mechanism of nuclear receptor activation Novel roles of retinoid X receptor (RXR) and RXR ligand in dynamically modulating the activity of the thyroid hormone receptor/RXR heterodimer Modulation of RXR function through ligand design Molecular mechanism of allosteric communication in the human PPARalpha-RXRalpha heterodimer An alternate binding site for PPARγ ligands Benzoyl 2-methyl indoles as selective PPARgamma modulators NMRPipe: a multidimensional spectral processing system based on UNIX pipes PHENIX: building new software for automated crystallographic structure determination Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis X-ray structure determination at low resolution Considerations for the refinement of low-resolution crystal structures Optimal description of a protein structure in terms of multiple groups undergoing TLS motion Presenting your structures: the CCP4mg molecular-graphics software Methods for the analysis of high precision differential hydrogen deuterium exchange data HDX workbench: software for the analysis of H/D exchange MS data HD desktop: an integrated platform for the analysis and visualization of H/D exchange data Download references Mangelsdorf (the University of Texas Southwestern Medical Center) for discussions related to SCA Support is acknowledged from an endowed fellowship from the Frenchman’s Creek Women for Cancer Research (S.S.); a fellowship from the Landenberger Foundation (D.P.M.); a NIH Pharmacological Sciences Training Grant (A.I.S); a NIH Medical Scientist Training Program Grant (A.I.S.); a New Investigator Award 1KN-09 from the James and Esther King Biomedical Research Program Florida Department of Health (D.J.K.); the National Institutes of Health (NIH) National Research Service Award (NRSA) DK097890 (T.S.H.) and Pathway to Independence Award DK103116 (T.S.H.); NIH grants DK101871 (D.J.K.) RR027270 (P.R.G.) and CA132022 (K.W.N.); and the State of Florida for institutional Scripps Florida start-up funds Present address: Present address: Division of Pediatric Rheumatology The Scripps Research Institute-Scripps Florida University of Texas Southwestern Medical Center expressed/purified protein and/or performed assays wrote the manuscript with input from all the authors Kendall Nettles is a consultant for Genentech Supplementary Figures 1-8 and Supplementary Tables 1-2 (PDF 593 kb) Download citation went to be with our heavenly Father on April 10 A memorial service will be held at the Harvey Anderson & Johnson Funeral Home in Willmar Visitation will be held one hour prior to the service at the funeral home in Redwood Falls the daughter of Charles and Dorothy (Sanger) Malecek received her first Communion and was confirmed at St Mary's Catholic Church in Bechyn She grew up in Bechyn and attended country primary school and attended Danube High School graduating in the class of 1961 being a very independent individual with a sense of adventure and wanting to get away from the cold Minnesota winters purchased a used vehicle and headed west by herself to California.  Judy had several stops along the way: first in Colorado Springs At both stops she took waitressing jobs until she had enough money to continue her ultimate destination – California.  William (Bill) Zaccheo.  Judy and Bill enjoyed 51 years of married life until Bill passed away in 2016.  While in California Judy attended Pima Community College where she earned an Associate of Science Respiratory Therapist Degree She worked as a respiratory therapist at hospitals in California Judy was an excellent seamstress making most of her own clothes.  Judy also very much enjoyed making dolls and embroidering pillows and towels as gifts for family and friends.  Both Judy and Bill enjoyed traveling and took a cruise to Alaska and sightseeing train trips.  However Judy's biggest joys were being around family and friends.  Judy would always have a big smile to greet someone and then she would follow up her smile with her sense of humor Judy is survived by her siblings:  Diane Wohnoutka Charlene Malecek (special friend John Gilchrist) and Rick Malecek; brother-in-law: Richard Zaccheo; sisters-in-law: Kathleen Zaccheo and Virginia Zaccheo; plus nieces and nephews Charles and Dorothy (Sanger) Malecek; siblings:  baby Gordy Malecek; brothers-in-law: John Kojetin Dominick Zaccheo; and sisters-in-law:  Sandy Malecek and Connie Malecek                                                       Blessed be her memory.  Metrics details Nuclear receptors (NRs) are thought to dynamically alternate between transcriptionally active and repressive conformations Most NR ligand series exhibit limited bias primarily consisting of transcriptionally active agonists or neutral antagonists but not repressive inverse agonists—a limitation that restricts understanding of the functional NR conformational ensemble we report a NR ligand series for peroxisome proliferator-activated receptor gamma (PPARγ) that spans a pharmacological spectrum from repression (inverse agonism) to activation (agonism) where subtle structural modifications switch compound activity While crystal structures provide snapshots of the fully repressive state NMR spectroscopy and conformation-activity relationship analysis reveals that compounds within the series shift the PPARγ conformational ensemble between transcriptionally active and repressive conformations that are natively populated in the apo/ligand-free ensemble Our findings reveal a molecular framework for minimal chemical modifications that enhance PPARγ inverse agonism and elucidate their influence on the dynamic PPARγ conformational ensemble studies into the mechanisms of graded NR transcriptional repression have been hampered because most NR ligand series consist of compounds of a single pharmacological type (e.g. Less common are reports of transcriptionally repressive NR inverse agonists and compounds that span the full pharmacological spectrum which display distinct coregulator recruitment profiles or ligand bias these static structures do not inform on the structural mechanism behind the differential graded activity observed within a ligand series during structure-activity relationship development as they do not elucidate how compounds with graded activity influence the dynamic PPARγ LBD conformational ensemble we show that the 2-chloro-5-nitrobenzamide scaffold offers an opportunity to understand how ligands with graded activity across the entire pharmacological spectrum influence the PPARγ LBD conformational ensemble We found that relatively minimal chemical modifications to this scaffold relative to the parent compound T0070907 yielded a ligand series spanning graded inverse agonism to agonism Crystal structures of PPARγ LBD bound with ligands and corepressor peptide along with density functional theory (DFT) calculations provide structural insight into the structural basis of improved inverse agonist efficacy NMR studies reveal that compounds within the ligand series influence the function of PPARγ by shifting the LBD conformational ensemble between transcriptionally active- and repressive-like states that are natively populated in the apo-LBD ensemble Correlation analysis shows that biochemical activities and NMR structural analysis using purified PPARγ LBD protein can explain ligand-dependent functions of full-length PPARγ-mediated transcription and gene expression in cells a AF-2 surface differences between PPARγ LBD in the transcriptionally active conformation bound to agonist rosiglitazone and TRAP220/MED1 coactivator peptide (PDB 6ONJ) and repressive conformation bound to inverse agonist T0070907 and NCoR1 corepressor peptide (PDB 6ONI) peptides Coactivator and corepressor peptides are shown in green and pink b Pi stacking between the polar T0070907 pyridyl group and three residues (His323 and Tyr473) and a bridging water molecule form in the transcriptionally repressive conformation (PDB 6ONI) c Compound scaffold of parent molecules containing polar pyridyl (T0070907) and hydrophobic phenyl (GW9662) groups in relation to the polar benzamide group in ZINC5672437 d TR-FRET NCoR1 corepressor biochemical interaction assay (n = 3 biological replicates; mean ± s.e.m.) e Cellular luciferase transcriptional reporter assay in HEK293T cells treated with 10 µM compound (n = 4 biological replicates; mean ± s.e.m.) f Crystal structures showing the compound flipped binding modes in transcriptionally active vs repressive conformations when bound to GW9662 (PDB 3B0R and 8FHE) g ZINC5672437 pi-stacking interaction in the repressive state (PDB 8FHE) where the benzamide replaces the bridging water in the T0070907-bound structure (PDB 6ONI) h 2D [1H,15N]-TROSY-HSQC NMR focused on Gly399 of 15N-labeled PPARγ LBD bound to GW9662 or ZINC5672437 in the absence (solid lines) or presence (dashed lines) of NCoR1 corepressor peptide i Data-informed design hypothesis to improve PPARγ inverse agonism the compound analogs have a wide range of graded activities spanning transcriptional repression with increased corepressor recruitment and binding affinity with increased coactivator recruitment and binding affinity Approximately half of the compounds display similar or improved inverse agonism compared to T0070907; of these compounds 3 (SR33068) and 7 (SR36708) are the most efficacious in repressing aP2 expression in 3T3-L1 cells to levels similar to undifferentiated cells Several analogs show properties of PPARγ agonism via increased coactivator peptide interaction and increased transcription and aP2 expression compared to the orthosteric full agonist rosiglitazone the activity of compounds 17-20 classify these analogs as partial agonists Some general features of chemical modifications that confer inverse agonism or partial agonism are apparent all the improved inverse agonists contain a polar pyridyl aromatic ring most of which are appended with a polar substitution (cyano analogs with activities ranked worse than GW9662—the least efficacious inverse agonists and partial agonists— all contain a hydrophobic non-polar phenyl ring Compounds 16 (SR36705) and 17 (SR33487) have extensions off a similar position of a polar pyridyl ring indicating extensions at these positions may be unfavorable for inverse agonism SAR on a larger ligand series would be needed to confirm these observations Compounds are numbered via the rank order in efficacy in the TR-FRET NCoR1 corepressor peptide interaction assay data shown in Fig. 3. All of the crystallized compounds contain a pyridyl nitrogen, which in T0070907 is at the fourth position, with additional R1 groups that interact with the aromatic triad residues (His323, His449, Tyr473) via pi-stacking interactions (Fig. 5b) Compounds 3 and 11 contain a cyano group at the fourth position with a pyridyl nitrogen at the third and second positions which substitutes for the water that bridges the interaction of T0070907 with the side chain of His323 Compound 2 contains a pyridyl nitrogen at the third position pointing towards the NCoR1 corepressor peptide bound at the AF-2 surface and a cyano group at the fifth position that points towards helix 11 with no water-bridged interaction with the side chain of His323 Compound 5 contains a pyridyl nitrogen at the third position that points towards the NCoR1 corepressor peptide but contains a fluorine at the fifth position instead of a cyano group that points towards helix 11 compound 4 contains a pyridyl nitrogen at the fourth position similar to T0070907 which retains the water-bridged interaction with His232 and a methyl substitution at the third position that points towards the NCoR1 corepressor peptide the ligands could shift the ensemble towards and stabilize only the repressive-like conformation while agonists would shift the ensemble towards an active-like conformation we compared 2D [1H,15N]-TROSY-HSQC NMR data of 15N-labeled PPARγ LBD bound to the ligand series Gly338 (β-sheet in the ligand-binding pocket) This indicates the entire LBD is sensitive to the ligand-induced graded shift in the conformational ensemble between repressive- and active-like states our findings demonstrate a platform that can assess and potentially predict the cellular transcriptional activity of PPARγ compounds ranging from agonism to inverse agonism via biochemical and NMR-detected structural biology studies focused on the PPARγ LBD T0070907 (CAS 313516-66-4) and GW9662 (CAS 22978-25-2) Details for the compounds synthesized in this study can be found in Supplementary Methods Peptides derived from human NCoR1 ID2 (2256-2278; DPASNLGLEDIIRKALMGSFDDK) and human TRAP220/MED1 ID2 (residues 638–656; NTKNHPMLM NLLKDNPAQD) were synthesized by LifeTein with an amidated C-terminus for stability with or without a N-terminal FITC label and a six-carbon linker (Ahx) Ligand concentrations for final functional profiling assays described below were chosen based on the observed potency values in biochemical TR-FRET coregulator and cellular transcriptional reporter assays after ~2 h ligand treatment (see Source Data 1) Because 2-chloro-5-nitrobenzamide compound analogs bind via a covalent mechanism their potency values will progressively left shift (become more potent) with time HEK293T (ATCC #CRL-11268) and 3T3-1L (ATCC #CL-173) cells were cultured according to ATCC guidelines HEK293T cells were grown at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium Gibco) supplemented with 10% fetal bovine serum (FBS 100 μg/mL of streptomycin (Gibco) until 90–95% confluence in T-75 flasks prior to subculture or use 3T3-L1 cells were grown at 37 °C and 5% CO2 in DMEM pyruvate (Gibco) supplemented with 10% bovine calf serum (BCS 100 μg/mL of streptomycin (Gibco) until 70% confluence in T-75 flasks prior to subculture or use was expressed as a TEV-cleavable N-terminal hexa-his-tag fusion protein (6xHis-PPARγ LBD) Expression was performed in BL21(DE3) Escherichia coli cells in either autoinduction ZY media (unlabeled protein) or using M9 minimal media supplemented with 15N ammonium chloride (for NMR studies) and 18 °C for 16 h before harvesting by centrifugation (4000 g cells were grown until the OD600 was 0.6 before adding 0.5 mM (final concentration) isopropyl β-D-thiogalactoside (IPTG) and incubating at 18 °C for 16 h before harvesting by centrifugation (4000 g Cells were resuspended in lysis buffer (50 mM potassium phosphate (pH 7.4) 10 mM imidazole) and lysed by sonication on ice Cell lysate was clarified by centrifugation (20,000 g the protein was purified using Ni-NTA affinity chromatography followed by size exclusion chromatography (Superdex 75) on an AKTA pure in assay buffer (20 mM potassium phosphate the His-tag was cleaved with TEV protease in dialysis buffer (20 mM potassium phosphate pH 7.4 The cleaved protein was reloaded on the Ni-NTA column and further purified by size exclusion chromatography (Superdex 75) in assay buffer (20 mM potassium phosphate Protein was confirmed to be >95% pure by SDS-PAGE Assays were performed in black 384-well plates (Greiner) with 23 μL final well volume 1 nM LanthaScreen Elite Tb-anti-His Antibody (Thermo Fisher; dilution/amount used per product guidelines) and 400 nM FITC-labeled NCoR1 ID2 or MED1 ID2 peptide in a buffer containing 20 mM potassium phosphate (pH 7.4) Ligands were added as a single concentration (10 μM) or in dose-response format to determine EC50/IC50 values and plates were read using BioTek Synergy Neo multimode plate reader after incubation at 25 °C for 1 h The Tb donor was excited at 340 nm; the Tb donor emission was measured at 495 nm ligand concentration; fit to a three-parameter sigmoidal dose-response equation to obtain potency values; and are representative of two or more independent experiments Assays were performed using 6xHis-PPARγ LBD preincubated with or without 2 molar equivalents of compound at 4  °C overnight and buffer exchanged via centrifugal concentration using Amicon Ultra centrifugal filters to remove excess ligand Protein samples were serially diluted into a buffer containing 20 mM potassium phosphate (pH 8) and 0.01% Tween 20 and plated with 180 nM FITC-labeled NCoR1 ID2 or TRAP220/MED1 ID2 peptide in black 384-well plates (Greiner) and FP was measured on a BioTek Synergy Neo multimode plate reader at 485 nm emission and 528 nm excitation wavelengths Data were plotted using GraphPad Prism as FP signal in millipolarization units vs protein concentration (n = 3 biological replicates); fit to a one site—total binding equation using a consistent fixed Bmax value determined from a fit of the high-affinity interactions as binding for some conditions did not saturate at the highest protein concentration used (45 µM); and are representative of two or more independent experiments HEK293T cells were cultured in Dulbecco’s minimal essential medium (DMEM Gibco) supplemented with 10% fetal bovine serum (FBS) and 50 units mL−1 of penicillin 2 million cells were seeded in a 10-cm cell culture dish for transfection using X-tremegene 9 (Roche) and Opti-MEM (Gibco) with full-length human PPARγ isoform 2 expression plasmid (4 μg) and a luciferase reporter plasmid containing the three copies of the PPAR-binding DNA response element (PPRE) sequence (3xPPRE-luciferase; 4 μg) cells were treated in quadruplicate with 20 μL of either vehicle control (1.5% DMSO in DMEM media) or 5 μM ligand or in dose-response format to determine EC50/IC50 values where each ligand treated condition had separate control wells to account for plate location-based artifacts Data were plotted in GraphPad Prism as luciferase activity vs ligand concentration (n = 4 biological replicates); fit to a three-parameter sigmoidal dose-response equation to obtain potency values; and are representative of two or more independent experiments 3T3-L1 cells were cultured in DMEM medium supplemented with 10% FBS and 50 units mL−1 of penicillin Cells were grown to 70% confluency and then seeded in 12-well dishes at 50,000 cells per well and incubated overnight at 37 °C cells were treated with media supplemented with 0.5 mM 3-iso-butyl-1-methylxanthine cells were treated with 10 μM compound in media supplemented with 877 nM insulin for 24 h RNA was extracted using quick-RNA MiniPrep Kit (Zymo) and used to generate complementary DNA using qScript cDNA synthesis kit (Quantabio) Expression levels of the PPARγ target gene aP2/FABP4 (forward primer: 5′-AAGGTGAAGAGCATCATAACCCT-3′) and the housekeeping gene TBP (forward primer: 5′-ACCCTTCACCAATGACTCCTATG-3′) used for normalization was measured using Applied Biosystems 7500 Real-Time PCR system Relative gene expression was calculated via the ddCt method using Applied Biosystems Relative Quantitation Analysis Module Software which reported values as mean with upper and lower limits Data were plotted in GraphPad Prism and are representative of two or more independent experiments Three of the compounds in our ligand series—GW9662 and 11 (SR33486)—and the control agonist rosiglitazone were not available or at the time these experiments were performed and left out of this analysis Population-weighted 1H NMR chemical shift analysis was performed focusing on the NMR peaks observed for Gly399 in each ligand-bound NMR spectrum Peak volumes for well-resolved peaks (e.g. two peaks = two states/conformations in slow exchange) were calculated using an elliptical peak fitting algorithm Population weighted average 1H NMR chemical shift values were calculated with Python using Jupyter Notebook using NumPy and Pandas packages using an equation (“lambda x: np.average(x.H1_P weights=x.weighted_vol)”) with two inputs: peak volumes estimated the relative population sizes (weighted_vol) of each state and the1H NMR chemical shift values of each state (H1_P) the binding free energy was calculated by subtracting the free energy of the complex from the free energy sum of the ligand and binding site Correlation data plotting and analysis of Spearman (s) correlation coefficients and two-sided alternative hypothesis p value testing (without adjustments for multiple comparisons) of the biochemical and cellular ligand profiling data were performed in Python using Jupyter Notebook using several libraries including Seaborn Reported two-sided p values (p) represent the probability that the absolute value of the Spearman or Pearson coefficient of random (x y) value drawn from the population with zero correlation would be greater than or equal to abs(r or s) according to the Scipy stats documentation Data in figure legends are reported as (n = X biological replicates Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article A comprehensive map of molecular drug targets The coregulator exchange in transcriptional functions of nuclear receptors and allostery in nuclear receptors as transcription factors A structural mechanism of nuclear receptor biased agonism Chapter 4—Biased signaling and conformational dynamics in nuclear hormone receptors Interactions governing transcriptional activity of nuclear receptors The nuclear receptor superfamily: a structural perspective The role of dynamic conformational ensembles in biomolecular recognition Ligand-induced stabilization of PPARgamma monitored by NMR spectroscopy: implications for nuclear receptor activation A dynamic mechanism of nuclear receptor activation and its perturbation in a human disease Quantitative structural assessment of graded receptor agonism Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662 Probing the complex binding modes of the PPARγ partial agonist 2-chloro-N-(3-chloro-4-((5-chlorobenzo[d]thiazol-2-yl)thio)phenyl)-4-(trifluoromethyl)benzenesulfonamide (T2384) to orthosteric and allosteric sites with NMR spectroscopy Modification of the orthosteric PPARγ covalent antagonist scaffold yields an improved dual-site allosteric inhibitor Endogenous vitamin E metabolites mediate allosteric PPARγ activation with unprecedented co-regulatory interactions Identification of a new type of covalent PPARγ agonist using a ligand-linking strategy A molecular switch regulating transcriptional repression and activation of PPARγ Biochemical and structural basis for the pharmacological inhibition of nuclear hormone receptor PPARγ by inverse agonists Discovery and structure-based design of potent covalent PPARγ inverse-agonists BAY-4931 and BAY-0069 Discovery and characterization of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists Bladder-cancer-associated mutations in RXRA activate peroxisome proliferator-activated receptors to drive urothelial proliferation Pparg signaling controls bladder cancer subtype and immune exclusion Genomic activation of PPARG reveals a candidate therapeutic axis in bladder cancer Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer Definition of functionally and structurally distinct repressive states in the nuclear receptor PPARγ The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors The coactivator LXXLL nuclear receptor recognition motif PPARγ corepression involves alternate ligand conformation and inflation of H12 ensembles Structural mechanism underlying ligand binding and activation of PPARγ a first-in-class inverse agonist of the peroxisome proliferator-activated receptor gamma (PPARG) lineage transcription factor to potentially treat patients with the luminal subtype of advanced urothelial cancer (UC) Abstract 2802: development of a surrogate tissue pharmacodynamic (PD) assay for clinical use with FX-909 a novel inhibitor of the urothelial luminal lineage transcription factor peroxisome proliferator-activated receptor gamma (PPARG) GoodVibes: automated thermochemistry for heterogeneous computational chemistry data Download references This work was supported in part by the National Institutes of Health (NIH) grants R01DK124870 (D.J.K.) from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and R35GM146982 (Z.J.Y.) from the National Institute of General Medical Sciences (NIGMS) The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH NIGMS grant P30GM133894 These authors contributed equally: Brian S Scripps Research and The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology Department of Chemical and Biomolecular Engineering expressed and purified protein and solved crystal structures supervised the research and wrote the manuscript along with B.S Nature Communications thanks Daniel Merk and the other anonymous reviewer(s) for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-025-57325-4 Metrics details The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity Crystal structures have defined multiple low-energy LBD structural conformations of the activation function-2 (AF-2) co-regulator-binding surface yet it remains unclear how ligand binding influences the number and population of conformations within the AF-2 structural ensemble we present a nuclear receptor co-regulator-binding surface structural ensemble in solution viewed through the lens of fluorine-19 (19F) nuclear magnetic resonance (NMR) and molecular simulations and the response of this ensemble to ligands co-regulator peptides and heterodimerization We correlate the composition of this ensemble with function in peroxisome proliferator-activated receptor-γ (PPARγ) utilizing ligands of diverse efficacy in co-regulator recruitment While the co-regulator surface of apo PPARγ and partial-agonist-bound PPARγ is characterized by multiple thermodynamically accessible conformations the full and inverse-agonist-bound PPARγ co-regulator surface is restricted to a few conformations which favor coactivator or corepressor binding experimental evidence describing this ensemble is lacking and it remains poorly understood how binding of pharmacologically distinct ligands affects the ensemble of co-regulator-binding surface and helix 12 conformations a direct observation of the ligand-dependent ensemble implied by these data is lacking This raises the question: are there multiple long-lived conformations that correlate with functional efficacy (e.g. co-regulator affinity) in nuclear receptors using 19F NMR combined with biochemical co-regulator interaction analysis and molecular simulations we define the ligand-dependent conformational ensemble of the co-regulator interaction surface which controls the transcriptional activity of PPARγ The data presented here indicate that helix 12 and the co-regulator-binding surface of apo PPARγ and partial-agonist-bound PPARγ is found in a broad energy well with multiple local minima of similar potential energy separated by relatively small kinetic barriers allowing exchange on the μs to ms time scale when PPARγ is bound to a full agonist or inverse agonist helix 12 and the co-regulator-binding surface occupies narrow energy wells with fewer thermodynamically accessible conformations simulations define some of the probable structures that compose these ensembles These data better elucidate how ligands induce functional effects via nuclear receptors the DMSO concentration is constant across the titration both in this figure and all other TR-FRET data presented Q322C and K502C yielded well-functioning protein with pronounced ligand-inducible changes; we used these mutants to probe the conformational ensemble of the PPARγ co-regulator-binding surface (i.e. any unlabeled portion of PPARγK502C-BTFA should not affect FP or TR-FRET results these data indicate a diverse ligand-dependent helix 12 conformational ensemble Helix 12 solvent exposure is distinct for the active and inactive helix 12 conformations a 19F NMR spectra of delipidated apo PPARγC313A,K502C-BTFA bound to 0.2 molar equivalent of GW1929 (left) and T0070907-bound PPARγK502C-BTFA with excess free BTFA Due to the high binding affinity of GW1929 (4 nM) all three expected peaks (two apoprotein peaks and one GW1929 peak) are present in slow exchange on the NMR time scale allowing analysis of the three conformations simultaneously change 19F NMR chemical shift values for various peaks; a dotted gray line is shown to highlight no D2O-induced change in 19F NMR chemical shift these data indicate that some inverse agonists do more than simply displace activating lipids but instead induce a distinct PPARγ LBD state with higher affinity for corepressors than delipidated apoprotein Agonists reduce the conformational complexity of the co-regulator-binding surface a Fluorine NMR spectra of PPARγC313A,Q322C-BTFA bound to the indicated ligands The small sharp left-shifted peak in all the spectra is free BTFA b Trajectory frame from a simulation of PPARγC313A,Q322C-BTFA bound to a coactivator peptide (MED1; green) with 322C-BTFA shown in orange as spheres (fluorine atoms are turquoise) A single well-populated 19F NMR peak is observed for BTFA probes placed in two areas of the co-regulator-binding surface (helices 3 and 12) when PPARγ-BTFA is bound to a strong agonist such as GW1929 or rosiglitazone multiple-well-populated 19F NMR peaks are observed in slow exchange on the NMR time scale indicating that the peaks represent distinct co-regulator-binding surface conformations with lifetimes on the order of milliseconds or longer Chemical exchange saturation transfer indicates slow exchange between multiple helix 12 conformations a A selective Gaussian pulse was used to saturate the 19F spectra bound to the indicated ligands at locations indicated by pink circles The height of the pink circles indicates the height of the peak indicated by the black arrow when the selective pulse was carried out at the chemical shift of the pink circle If exchange is occurring between the two peaks b A selective Gaussian pulse was used to saturate the spectrum at an on resonance (a; orange box arrow) and off resonance (a; blue box arrow) location the peak height of the most abundant resonance (a; black arrow) was monitored as a function of the duration of the saturation pulse and the resulting peak intensities were fit to extract the exchange rate Ninety-five percent confidence intervals for the fit of the calculated exchange rates are shown within parentheses except for ciglitazone which used PPARγC313A,K502C-BTFA for increased signal to noise b Plot of mean 19F NMR chemical shift values (PPARγK502C-BTFA) vs and SMRT peptide dissociation constant (Kd) for PPARγK502C-BTFA (top panels) and wt PPARγ LBD (bottom panels) as measured by fluorescence polarization for a subset of the ligands in a Linear regression fit is shown as a solid line and the correlation coefficient (R2) for the fitted line is indicated The ligands with the highest efficacy for MED1 and NCoR recruitment for PPARγK502C-BTFA are highlighted Error bars represent standard deviation of two (K502C-BTFA Kd and wt SMRT Kd) or three (wt MED1 and NCoR Kd) independent experiments Agonist-bound PPARγ is changed little by MED1 binding whereas NCoR binding changes the spectrum drastically and vice versa for inverse-agonist (T0070907)-bound PPARγ *SMRT-induced mean chemical shift in GW1929-bound PPARγK502C-BTFA is the same as NCoR Heterodimerization of PPARγ LBD with RXRα LBD favors coactivator binding a 19F NMR of PPARγK502C-BTFA bound to T0070907 and PPARγC313A,502C-BTFA bound to MRL24 or GW1929 or with no ligand bound was performed in the presence (orange) or absence (black) of RXRα LBD d The 19F NMR signal from the MRL24 ligand Broadening and consequent reduction in signal intensity is expected as a consequence of the increased rotational correlation time of the heterodimer complex c TR-FRET was used to measure interaction between wt PPARγ LBD and MED1 or NCoR in the presence or absence of equimolar concentrations of RXRα Error bars represent standard deviation of two technical replicates within a single experiment The experiment was repeated twice and gave similar results each time e Heterodimerization favors MED1 binding (p = 0.0017) and disfavors NCoR binding (p = 0.0076) to apo PPARγ visual and statistical comparison of NCoR and MED1 recruitment to PPARγ LBD saturated with ligand (four highest concentrations of ligands) indicates that RXRα affects co-regulator recruitment to T0070907-bound PPARγ (NCoR All p values are derived from a two-tailed t test e A model for helix 12 conformational diversity based on simulation and experiment Apo PPARγ or partial-agonist-bound PPARγ helix 12 is found in many similar conformations of varying helical structure producing broad NMR peaks and rapid hydrogen deuterium exchange while full agonist is found in a tighter cluster of conformations Co-binding of the inverse agonist T0070907 and the corepressor NCoR produces one main conformation similar to an active conformation Fuzziness implies intermediate exchange (μs to ms) between the conformations these data reveal a correlation between ligand efficacy and the prevalence of at least three distinct structural ensembles using a diverse set of 16 pharmacologically distinct PPARγ ligands it is encouraging that these non-converged simulations qualitatively agree with 19F NMR and provide a glimpse of possible conformations that comprise a portion of the observed 19F NMR spectra; however quantitative comparison between the 19F NMR spectra and converged simulations remains a future challenge Addition of NCoR or SMRT shifts the population from cluster 2 to cluster 3 while the mutations and labeling may shift the equilibrium population in the opposite direction toward the antagonist/partial agonist cluster (cluster 2) it may be that helix 12 of wt PPARγ LBD bound to T0070907 is found in a conformation represented by the inverse agonist cluster (cluster 3; left-shifted narrower peak) to a larger degree than detected by PPARγK502C-BTFA This work adds detail to how ligands in general control the activity of nuclear receptors Our data not only confirm that helix 12 and the co-regulator-binding surface exist as a ligand-specific dynamic structural ensemble but also reveal the relative populations of sub-ensembles that comprise the overall structural ensemble and correlate function with this ensemble Further definition of the conformational ensemble of the entire protein and the kinetics and thermodynamics of exchange between the members of the ensemble will build an accurate model of how ligands produce functional outputs via nuclear receptors and allow greater control of their function via ligands A pET-46 plasmid carrying the genes for ampicillin resistance and N terminally 6xHis-tagged PPARγ containing a tobacco etch virus (TEV) nuclear inclusion protease recognition site between the His tag and protein of interest was transformed into chemically competent E Cells were grown in either ZYP-5052 autoinduction media or terrific broth (TB) Cells grown in TB at 37 °C were induced at an OD600 of approximately 0.8 by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and the temperature lowered to 22 °C Induction proceeded for 16 h prior to harvesting Harvested cells were homogenized into 50 mM phosphate (pH 8.0) and lysed using a C-5 Emulsiflex high-pressure homogenizer (Avestin) Lysates were then clarified and passed through two Histrap FF 5 ml columns in series (GE Healthcare) Protein was eluted using a gradient from 15 to 500 µM imidazole Fast protein liquid chromatography was performed on either an NGC Scout system (Bio-Rad) or an ÄKTA Start (GE Healthcare) Eight milligrams of recombinant 6xHis-tagged TEV was added to eluted protein followed by dialysis into 50 mM Tris (pH 8.0) The protein was again passed through HisTrap FF columns in order to separate cleaved protein from TEV as well as the cleaved 6xHis tag The cleavage step was only performed on protein which would be used for NMR or FP but the protein used for TR-FRET did not have the 6xHis tag removed The protein was then further purified by gel filtration using a HiLoad 16/600 Superdex 200 PG (GE Healthcare) Size exclusion was performed in 25 mM MOPS (pH 8.0) Protein was then dialyzed into 25 mM 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.4) Protein purity in excess of 95% was determined by gradient 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (NuSep) Protein concentration was determined using ε280 = 12,045 M−1 cm−1 15N-labeled protein was grown in M9 minimal media containing 99% 15NH4Cl (Cambridge Isotope Laboratories) as the sole nitrogen source cells were grown at 37 °C and 180 rpm until an OD600 of approximately 1.0 was reached the temperature was dropped to 22 °C for 1 h protein expression was induced by the addition of 500 µM IPTG during which induction cells remained at 22 °C Protein expression and purification was then accomplished utilizing the same protocol as outlined above purified protein was diluted to 0.8 mg ml−1 and batched with Lipidex 1000 (Perkin-Elmer) at an equal volume This mixture was batched for 1 h at 37 °C and 100 rpm protein was pulled through a gravity column by syringe it was found that the speed of elution was important; protein could not remain on the resin at room temperature in excess of 3 min Two more column volumes of pre-warmed 25 mM MOPS and 1 mM EDTA were also pulled through in the same manner Quality of delipidation was then estimated by 19F NMR and loss of lipid can be most easily detected by a reduction in the peak at −84.1 ppm When non-delipidated protein is used in this report it is labeled as bound to E Mutations in PPARγ LBD were generated using the Quikchange Lightning site-directed mutagenesis kit (Agilent) using primers listed in Supplementary Table 6 The presence of expected mutations and absence of spurious mutations was confirmed by Sanger sequencing (Eurofins) protein was incubated for 30–60 min and then buffer exchanged at least 100x using 10 kDa Amicon Ultra-15 concentrators (Merck Millipore) to remove excess unbound BTFA and then mixed with appropriate amounts of H2O buffer that had been adjusted to read pH 7.4 ligands were added to the appropriate concentration as usual The samples which contained only 10% D2O were prepared as usual with the standard addition of 10% buffered D2O to the final sample Samples of PPARγK502C-BTFA were already loaded with ligand and labeled appropriately with BTFA prior to exchange into deuterated buffers FP peptide binding assays were performed by plating a mixture of 50 nM peptide with an N-terminal FITC tag 12-point serial dilutions of PPARγ-LBD (wt PPARγ-LBD and PPARγ ligands were added at a 1:1 ratio This mixture was added to wells of low-volume 384-well black plates (Grenier Bio-one catalog number 784076) to a final volume of 16 μL Peptides were synthesized by Lifetein LLC (Somerset sequence: NTKNHPMLMNLLKDNPAQD; and the NCoR peptide sequence: GHSFADPASNLGLEDIIRKALMG (2251–2273) Other peptides were purchased from ThermoFisher (Waltham sequence: NTKNHPMLMNLLKDNPAQD (catalog number PV4549); CBP peptide sequence: AASKHKQLSELLRGGSGSS (catalog number PV4596); and SMRT sequence: HASTNMGLEAIIRKALMGKYDQW (catalog number PV4424) All dilutions were made in 25 mM MOPS (pH 7.4) 0.01% fatty-acid-free bovine serum albumin (BSA) (EMD Millipore Assay titrations were performed in duplicate Plates were incubated in the dark at room temperature for 2 h before being read on a Synergy H1 microplate reader (BioTek) FP was measured by excitation at 485 nm/20 nm and emission at 528 nm/20 nm for FITC Data were fit using nonlinear regression (agonist vs response – variable slope 4 parameters) in Prism 7.0b we did two technical replicates and repeated these experiments independently in the lab for once (Fluormone) or 2 or more times FP and TR-FRET We chose these number of technical replicates and independent experiment replicates based on our experience with the limited variability inherent in these biochemical assays TR-FRET peptide recruitment assays were performed by plating a mixture of 8 nM 6xHis-PPARγ-LBD (wt 0.9 nM LanthaScreen Elite Tb-anti-His antibody (LifeTechnologies catalog number PV5863) 200 nM peptide (N terminally biotinylated and C terminally amidated) and 12-point serial dilutions of PPARγ ligands from 50 μM to 1 pM catalog number 784076) to a final volume of 20 μL USA) for MED1 peptide; sequence: VSSMAGNTKNHPMLMNLLKDNPAQ; and NCoR peptide TR-FRET was measured by excitation at 330 nm/80 nm and emission at 620 nm/10 nm for terbium and 665 nm/8 nm for d2 Change in TR-FRET was calculated by 665 nm/620 nm ratio so as to exclude most effects of the second transition the 50 μM SR2088 point was not run because we did not have sufficient ligand PPARγ ligand inhibition constants (Ki) were measured using a protocol adapted from LanthaScreen TR-FRET PPARγ competitive binding assay (Invitrogen Assay was performed by plating a mixture of 8 nM 6xHis-PPARγ-LBD 2.5 nM LanthaScreen Elite Tb-anti-His antibody 5 nM LanthaScreen Fluormone Pan-PPAR Green (Invitrogen and 12-point serial dilutions of PPARγ ligands from 50 μM to 140 fM This mixture was added to wells of low-volume 384-well black plates (Grenier Bio-one) to a final volume of 16 μL Plates were incubated in the dark for 2 h at room temperature before being read on a Synergy H1 microplate reader (BioTek) TR-FRET was measured by excitation at 330 nm/80 nm and emission at 495 nm/10 nm for terbium and 520 nm/25 nm for Fluormone Change in TR-FRET was calculated by 520 nm/495 nm ratio Nonlinear curve fitting was performed using Prism 7.0b (Graphpad Software Inc.) as described above for the TR-FRET data including manual exclusion of highest two concentrations for nTZDpa Thirty of the 1224 total data points for all three proteins (wt and PPARγC313A,K502C-BTFA) were automatically excluded by Prism in the fits where IC50 is the concentration of the ligand that produces 50% displacement of the Fluormone tracer Lo is the concentration of Fluormone in the assay (5 nM) and Kd is the binding constant of Fluormone to wt or the two BTFA-labeled mutants and Lb is the concentration of bound Fluormone in the assay with no addition of test ligand The affinity of Fluormone for the two BTFA-labeled mutant proteins was determined via TR-FRET by titration of Fluormone into each mutant bound to Elite Tb-anti-His antibody Dissociation constants of Fluormone for wt was measured as 7.9 ± 0.2 for PPARγ LBD and the variants were measured as 26 ± 3 nM for PPARγK502C-BTFA and 44 ± 4 nM for PPARγC313A,K502C-BTFA and 12 ± 1 nM PPARγC313A Intermediate exchange effects and field inhomogeneity are likely present in some of these spectra which will result in inaccuracies in the fitted models; however the deconvolution method provides an objective view of the possible underlying spectral structure and populations Two-dimensional [1H,15N]TROSY-HSQC NMR data were obtained using the trosyf3gpphsi19.2 pulse program Select NMR spectra were replicated in two different ways: (1) Some NMR samples were measured via NMR initially and then days to weeks later to determine if certain parts of the spectrum changed Any changes would indicate that non-reversible processes contribute to that part of the signal such as unfolding or degradation of the protein (2) Some spectra were run twice utilizing protein from the same batch as utilized for the first spectra or from an entirely different protein preparation All production simulations were carried out using pmemd.cuda or pmemd.cuda.MPI The best scoring docked binding mode overlaid well with GI262570 In this docked model in the helix 12-interacting region RESP charges for GW1929 were derived using the RED server and force modification files were generated using GAFF parameters 1FM9 was modified (or not) to incorporate cysteine-BTFA (parameterized as described above) in place of K502 and docked with GW1929 in a similar way to that described above to build PPARγK502C-BTFA and PPARγ bound to GW1929 1PRG chain B was used to create the build for apo and 3BOR chain B was used to build T0070907-bound PPARγ LBD 3BOR contains GW9662 which differs from T0070907 by one atom T0070907 has a nitrogen in place of a carbon atom in one of the ligand rings This change was made in chimera and the ligand parameterized and incorporated into the structure as described above for BTFA NCoR (same sequence as used in NMR and TR-FRET including N-terminal acetylation and C-terminal amidation) was added to these builds utilizing chimera using the following procedure The core helix structure from NCoR (from 2OVM) was aligned to SMRT on a PPARα SMRT structure (1KKQ) and then apo PPARγ chain B (1PRG) was aligned to PPARα and the PPARα/SMRT structure deleted leaving the aligned NCoR on apo PPARγ chain B A similar procedure was used with the 3BOR structure to create T0070907 co-bound with NCoR on PPARγ These PDBs were then used to create the final solvated and equilibrated structure in a manner similar to that described above and FP are publically available at https://osf.io/rqdpz/ Any other datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request Binding of ligands and activation of transcription by nuclear receptors The structural basis of estrogen receptor/co-activator recognition and the antagonism of this interaction by tamoxifen Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-alpha Nuclear receptor structure: implications for function Distinct properties and advantages of a novel peroxisome proliferator-activated protein [gamma] selective modulator (19)F-modified proteins and (19)F-containing ligands as tools in solution NMR studies of protein interactions The role of ligands on the equilibria between functional states of a G protein-coupled receptor The dynamic process of β2-adrenergic receptor activation Structural insights into the dynamic process of β2-adrenergic receptor signaling Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR Current applications of 19F NMR to studies of protein structure and dynamics An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma) A novel N-aryl tyrosine activator of peroxisome proliferator-activated receptor-γ reverses the diabetic phenotype of the Zucker diabetic fatty rat a selective ligand for peroxisome proliferator-activated receptor-γ functions as an antagonist of biochemical and cellular activities Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) gamma activators and Pan-PPAR partial agonists Deconvolution of complex 1D NMR spectra using objective model selection Tryptophan solvent exposure in folded and unfolded states of an SH3 domain by 19F and 1H NMR Partial agonists activate PPARγ using a Helix 12 independent mechanism 19F NMR studies of solvent exposure and peptide binding to an SH3 domain Mechanistic elucidation guided by covalent inhibitors for the development of anti-diabetic PPARγ ligands Genome-wide profiling of PPARγ:RXR and RNA polymerase II occupancy reveals temporal activation of distinct metabolic pathways and changes in RXR dimer composition during adipogenesis The shear viscosities of common water models by non-equilibrium molecular dynamics simulations Conformational diversity of the helix 12 of the ligand binding domain of PPARγ and functional implications Molecular determinants of nuclear receptor–corepressor interaction Activation of the A2A adenosine G-protein-coupled receptor by conformational selection Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation Use of glass electrodes to measure acidities in deuterium oxide Detecting outliers when fitting data with nonlinear regression—a new method based on robust nonlinear regression and the false discovery rate Colloidal drug formulations can explain “bell-shaped” concentration–response curves Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer Pharmacologic Analysis of Drug/Receptor Interaction 2nd edn (Raven Simulation of NMR pulse sequences during equilibrium and non-equilibrium chemical exchange in Introduction to Protein Structure Predictiction: Methods and Algorithms (eds Rangwala Comparative protein modelling by satisfaction of spatial restraints H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations Server: A web service for deriving RESP and ESP charges and building force field libraries for new molecules and molecular fragments Application of RESP charges To calculate conformational energies Development and testing of a general amber force field ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB Comparison of simple potential functions for simulating liquid water Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data AutoDock Vina: improving the speed and accuracy of docking with a new scoring function Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes Download references We thank James Aramini at the City University of New York Advanced Science Research Center (CUNY ASRC) for assistance in setting up the saturation transfer difference Roe (NIH Laboratory of Computational Biology NHLBI) for providing the minimization and equilibration script and simulation advice NMR data presented herein were collected at the CUNY ASRC Biomolecular NMR Facility This work was supported in part by National Institutes of Health (NIH) grants K99DK103116 (to T.S.H.) and DK105825 (to P.R.G.); and National Science Foundation (NSF) award 1359369 (PI Karbstein) that funds the SURF program at Scripps Florida These authors contributed equally: Michelle D Biochemistry and Biophysics Graduate Program Center for Biomolecular Structure and Dynamics Department of Biomedical and Pharmaceutical Sciences Summer Undergraduate Research Fellows (SURF) Program Department of Pharmacology & Physiology conceived the study and designed experiments prepared samples and performed experiments prepared samples and performed preliminary experiments and characterization of ligand pharmacology with molecular dynamics simulations and ligand parameterization analyzed data and wrote the manuscript with input from all authors Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-018-04176-x Metrics details Small chemical modifications can have significant effects on ligand efficacy and receptor activity but the underlying structural mechanisms can be difficult to predict from static crystal structures alone Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor (PPAR) gamma converts a transcriptionally neutral antagonist (GW9662) into a repressive inverse agonist (T0070907) relative to basal cellular activity and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for corepressor-selective inverse agonism NMR spectroscopy reveals that PPARγ exchanges between two long-lived conformations when bound to T0070907 but not GW9662 including a conformation that prepopulates a corepressor-bound state priming PPARγ for high affinity corepressor binding Our findings demonstrate that ligand engagement of Arg288 may provide routes for developing corepressor-selective repressive PPARγ ligands and it remains poorly understood how to design inverse agonists Crystal structures of PPARγ bound to T0070907 or GW9662 reveal no major overall structural differences that explain the difference in efficacy a water-mediated hydrogen bond network that uniquely links R288 to the T0070907 pyridyl group—an interaction that cannot occur with GW9662 which lacks a hydrogen bond acceptor—is essential for corepressor-selective cellular repression of PPARγ NMR analysis shows that T0070907-bound PPARγ populates two long-lived structural conformations one of which resembles the state populated by GW9662 and a unique state that is similar to the corepressor-bound state thus revealing a structural mechanism for directing corepressor-selective repression of PPARγ a Chemical structures of GW9662 and T0070907 b Cell-based full-length PPARγ luciferase transcriptional assay showing the effect of activating and repressive PPARγ ligands (5 µM) on full-length PPARγ transcription in HEK293T cells Individual points (n = 12) normalized to DMSO control (mean) are plotted as white circles on top of a box-and-whiskers plot; the box represents 25th and the whiskers plot the entire range of values c Relative quantitation of fold change in PPARγ target gene expression determined by qPCR normalized to TBP expression of 3T3-L1 cells treated with transcriptionally activating plotted as relative log2 of data calculated using the 2-ΔΔCt method (n = 3) with error bars representative of the upper and lower limits Data are representative of at least 3 independent experiments Coregulator binding profiles of the activating a Fluorescence polarization coregulator interaction assay showing the effect of ligands on the interaction between the PPARγ LBD and peptides derived from the TRAP220 coactivator or NCoR1 corepressor plotted as the mean (n = 2) with error bars representative of s.d b Kd values derived from the coregulator interaction assay Dotted orange and purple lines note the DMSO/apo-PPARγ values Orange and purple shaded areas note the affinity regions for an ideal inverse agonist (i.e. orange circles in the orange square; for higher NCoR1 affinity c Superposition of crystal structures of the PPARγ LBD bound to GW9662 (PDB code 3B0R; ligand in green cartoon in blue) and SR1664 (PDB code 4R2U; ligand in yellow) The location of the simple substitution between GW9662 (methine) vs T0070907 (nitrogen) is marked with a black circle and the SR2595 and SR10221 tert-butyl extension within the helix 12 subpocket towards F282 (blue) from the SR1664 parent compound is marked with a yellow circle suggesting the corepressor-selective inverse agonist transcriptionally repressive profile of T0070907 may originate from a previously uncharacterized structural mechanism A water-mediated hydrogen bond network connects Arg288 to T0070907 a Overall structure of T0070907-bound PPARγ LD (PDB code 6C1I; gray) and overlay with GW9662-bound crystal structure (PDB code 3B0R; blue) b A water-mediated hydrogen bond network in the T0070907-bound crystal structure (chain B is shown) links the pyridyl group in T0070907 to the R288 side chain which forms a bipartite hydrogen bond with the E295 side chain c The GW9662-bound crystal structure (chain B is shown) lacks the R288-ligand hydrogen bond network but contains the R288-E295 hydrogen bond d 2D strip corresponding to the R288 Nε-Hε group from a 3D 15N-NOESY-HSQC NMR experiment (τmix = 100 ms) of T0070907-bound 15N-labeled PPARγ LBD reveals a NOE signal at 4.77 p.p.m e Pyridyl-water network hydrogen bonds populated during molecular dynamics simulations for T0070907- and GW9662-bound structures starting from crystallized (xtal) and modeled (model) conformations from 3–4 replicate simulations as well as a model generated of T0070907-bound PPARγ from the GW9662-bound PPARγ crystal structure that was independently solvated without the crystallized waters (model) the pyridyl group of T0070907 was hydrogen bonded to a water molecule for a significant fraction of the simulation (65–95%) as was the water-bridged R288-T0070907 pyridyl (5–46%) a direct interaction between R288 and the pyridyl group of T0070907 not mediated by water was lowly populated (<2%) A direct (4–64%) and water-bridged (3–61%) R288-E295 interaction was also confirmed The extensive pyridyl-based water-mediated hydrogen bond network is not possible to the hydrophobic phenyl group of GW9662 revealing a unique chemical feature in T0070907 that could confer corepressor-selective inverse agonism Mutagenesis validates the R288-pyridyl interaction for conferring corepressor-selective inverse agonism a HEK293T cells transfected with full-length PPARγ expression plasmid along with a 3 × -PPRE-luciferase reporter plasmid and treated with the indicated ligands (5 µM) Individual points (n = 6) normalized to DMSO control (mean) are plotted on top of a box-and-whiskers plot; the box represents 25th c Affinities determined from a fluorescence polarization assay of wild-type and mutant PPARγ LBDs preincubated with a covalent ligand (GW9662 or T0070907) or vehicle (DMSO) binding to FITC-labeled b TRAP220 or c NCoR1 Data plotted as the Kd value and error from fitting data of two experimental replicates using a one site binding equation d Legend to the web of efficacy radar chart diagrams Corepressor-selective inverse agonism is associated with data points populating the periphery of the plots e Radar web of efficacy plots displaying assay data for wild-type PPARγ and mutant variants Data normalized within the range of values for each assay (a–c) Consistent with the cell-based transcription assay the R288K and E295A mutants maintained the coregulator binding profile of T0070907 and rank ordering the R288A and R288L mutants showed similar affinity for TRAP220 and NCoR1 when covalently bound to T0070907 or GW9662 This indicates the pyridyl-water network directs the corepressor-selective inverse agonism profile of T0070907 the lack of which results in an antagonist GW9662-like profile This dramatic result reveals that R288-mediated pyridyl-water network directs the corepressor-selective inverse agonism profile conferred by T0070907 We therefore used NMR spectroscopy to assess the impact of T0070907 and GW9662 on the dynamics of the PPARγ LBD NMR detected exchange between two long-lived T0070907-bound conformations a Overlay of 2D [1H,15N]-TROSY-HSQC NMR spectra of 15N-labeled PPARγ LBD bound to GW9662 or T0070907 b Binding of T0070907 but not GW9662 stabilizes intermediate exchange (µs-ms time scale) dynamics (residues labeled in a shown in green spheres) and causes peak doubling (tan and pink spheres; G399 is colored pink for emphasis) Data plotted on the T0070907-bound PPARγ crystal structure and important structural regions are highlighted as follows: AF-2 surface (black oval); an extended pyridyl-water hydrogen bond network (blue spheres beyond the key pyridyl-water interaction (red sphere) c Snapshot overlays of 2D [1H,15N]-TROSY-HSQC spectra of 15N-labeled PPARγ LBD bound to T0070907 or GW9662 shows co-linear shifting of peaks at the different temperatures The spectral region displayed shows peaks conserved when PPARγ is bound to GW9662 or T0070907 (green arrows); a unique set of doubled peaks when bound to T0070907 (purple arrows); and absent peaks due to intermediate exchange on the NMR time scale when bound to GW9662 (dotted rectangles) d Snapshots of ZZ-exchange [1H,15N]-HSQC NMR spectra (delay = 1 s) of T0070907-bound 15N-labeled PPARγ LBD focused on G399 at the indicated temperatures Two G399 conformational states are denoted as A and B with the ZZ-exchange transfer crosspeaks as A–B and B–A e ZZ-exchange NMR analysis build-up curve from for G399 at 310 K generated by plotting peak intensities of the state A and B peaks and exchange crosspeaks (A–B and B-A) as a function of delay time f Schematic of the T0070907-bound PPARγ conformational ensemble defined by the NMR studies including the β-sheet (G338) and helix 6 (R350 L356) within the ligand-binding pocket; a surface comprising helix 2a (R234) and the C-terminal region of helix 7 and the loop connecting helix 7 and 8 (K373 D380); helix 3 near the AF-2 surface (I303); and the loop connecting helix 8 and 9 near the AF-2 surface (S394) the peak for G321 in the GW9662-bound conformation and one of the T0070907-bound conformations shows significant line broadening at the lower temperature indicating these states may share conformational and dynamical features T0070907 populates a shared conformation with GW9662 and a unique conformation which is connected to the AF-2 coregulator interaction surface via water-mediated hydrogen bonds to N312 and D313 but does not directly interact with a coregulator peptide bound to the PPARγ LBD (PDB code 2PRG) b Snapshot overlay of [1H,15N]-TROSY-HSQC NMR spectra of 15N-labeled PPARγ LBD (wild-type or R288 mutants) bound to GW9662 or T0070907 shows that the single GW9662-bound G399 peak has similar chemical shift values to one of the two T0070907-bound G399 peaks (state A) whereas state B is uniquely populated by T0070907 but not T0070907-bound R288A mutant PPARγ LBD significantly populates the unique conformation c Deconvoluted 19F NMR spectra of PPARγ LBD labeled with BTFA on helix 12 and covalently bound to GW9662 or T0070907 the 19F spectral profile of GW9662-bound PPARγ revealed two peaks corresponding to a major state (right peak; 78%) and minor state (left peak; 22%) T0070907-bound PPARγ also showed two peaks with chemical shift values similar to GW9662-bound PPARγ but the population magnitudes of the states are switched and skewed towards the left peak this helix 12/AF-2 surface 19F NMR probe showed similar relative population sizes to that observed in the G399 ZZ-exchange analysis (34% and 66% The right 19F NMR peak abundantly populated by GW9662 and moderately populated by T0070907 likely corresponds to the mutual G399 conformation from the 2D NMR analysis the left 19F NMR peak likely corresponds to the unique G399 conformation; this peak is abundantly populated by T0070907 but lowly populated by GW9662 The low abundance of this peak when bound to GW9662 could explain in part why it was not detected by the 2D NMR analysis which has lower overall sensitivity of signal-to-noise compared to the 19F NMR analysis the BTFA probe attached to helix 12 may also be more sensitive to larger structural changes compared to backbone amide resonances Coregulator binding preferences of the unique and mutual T0070907-bound conformations a Snapshot overlay of 2D [1H,15N]-TROSY-HSQC spectra of T0070907-bound 15N-labeled PPARγ LBD titrated with NCoR1 peptide b Extracted 1D planes of the NCoR1 spectra shown in a show the qualitative binding trends observed by NMR whereas c an overlay of the extracted 1D planes is quantitatively described by d spectra using a 4-state model where the two slowly exchanging receptor populations (R and R*) are capable of binding to the peptide (RP and R*P) and receptor isomerizes in both the free and peptide-bound states (R ↔ R* (e–h) Analysis of performed for T0070907-bound 15N-labeled PPARγ LBD titrated with TRAP220 peptide performed similarly to the NCoR1 analysis described in a–d which is also quantitatively described using the same 4-state model Coregulator binding preferences of the single GW9662-bound conformation detected by NMR a Snapshot overlay of 2D [1H,15N]-TROSY-HSQC spectra of GW9662-bound 15N-labeled PPARγ LBD titrated with NCoR1 peptide b The 15N spectral planes of the 2D NMR titration are quantitatively described using a 3-state model (R + P ↔ RP ↔ R*P) where PPARγ LBD bound to GW9662 and NCoR1 slowly isomerizes between two states (RP and R*P) c Snapshot overlay of 2D [1H,15N]-TROSY-HSQC spectra of GW9662-bound 15N-labeled PPARγ LBD titrated with TRAP220 peptide d The 15N spectral planes of the 2D NMR titration are quantitatively described using a 2-state model (R + P ↔ RP) These results reveal that the two long-lived T0070907-bound conformations have different binding preferences for NCoR1 and TRAP220 The NMR chemical shifts of the unique and mutual T0070907-bound conformations in the absence of coregulator peptide are similar to the NCoR1- and TRAP220-bound forms the unique and mutual T0070907-bound states prepopulate a corepressor-like and coactivator-like bound conformation that afford high-affinity binding to NCoR1 and TRAP220 the chemical shift difference between the unique T0070907 conformation and NCoR1-bound state (i.e. the degree of state B shifting) is much smaller than the mutual conformation and TRAP220-bound state (i.e. indicating that the corepressor-like conformation prepopulated by T0070907 is more similar to the corepressor-bound state NCoR1 binding to GW9662-bound PPARγ introduces a conformational frustration within the AF-2 surface: the AF-2 surface of GW9662-bound PPARγ does not prepopulate the corepressor-bound conformation and upon binding NCoR1is found in two slowly exchanging conformations similar to the corepressor- and coactivator-bound forms of T0070907-bound PPARγ in addition to their utility as nondissociative orthosteric competitive ligands that inhibit binding of other orthosteric PPARγ ligands our data suggest that T00709707 and GW9662 can be classified as a covalent corepressor-selective inverse agonist and a covalent antagonist our work shows that the combination of different but complementary structural methods provides the full picture of ligand mechanism of action Published findings and the data presented here indicate that PPARγ possesses constitutive activity and that binding of ligands differentially influences PPARγ activity if indeed PPARγ lacks constitutive activity our results should only require a minor change in nomenclature as the corepressor-selective activity of T0070907 is well supported by structural and functional evidence provided by us and by others our findings should inspire future work to develop and characterize corepressor-selective inverse agonists to probe the repressive functions of PPARγ Human PPARγ LBD (residues 203–477 in isoform 1 numbering which is commonly used in published structural studies and thus throughout this manuscript; or residues 231–505 in isoform 2 numbering) and full-length retinoid x receptor α (RXRα) were expressed in Escherichia coli BL21(DE3) cells using enriched media (LB or autoinduction) or for NMR studies minimal media (M9 supplemented with 13C-glucose and/or 15NH4Cl) as TEV-cleavable hexahistidine-tagged fusion protein using a pET46 plasmid cells were lysed in lysis buffer (500 mM potassium chloride pH 7.5) supplemented with 5 µg/mL of DnaseI and lysozyme Lysates were cleared by sonication (24,000 × g 1 h) and loaded onto 2 × 5 mL Histrap FF columns (GE Healthcare) Protein was eluted using lysis buffer with 500 mM imidazole protein was incubated at a 1:50 ratio with TEV protease overnight at 4 °C loaded back onto the HisTrap FF column and collecting the flow through Protein was concentrated and loaded onto a Superdex 200 prep grade 26/60 column (GE Healthcare) The final storage buffer for LBD samples following size exclusion chromatography and subsequently frozen at −80 °C was 50 mM potassium chloride (pH 7.4) and 0.5 mM EDTA; for full-length PPARγ was 25 mM MOPS (pH 7.4) and 1 mM EDTA; or for full-length RXRα ws 25 mM MOPS (pH 7.4) In most studies of covalent ligands (except TR-FRET studies) PPARγ protein was pretreated with GW9662 or T0070907 overnight at 4 °C with a 2X molar excess of compound dissolved in d6-DMSO Delipidation was performed using Lipidex 1000 resin (Perkin-Elmer): the protein was diluted to 0.8 mg ml−1 batched with an identical volume of resin at 37 °C and spun at 100 rpm for 45 min and concentrated to a working concentration in 25 mM MOPS (pH 7.4) and was either frozen immediately at −80 °C or used in the same day Mammalian expression plasmids included Gal4-PPARγ-hinge-LBD (residues 185-477 in isoform 1 numbering; 213-505 in isoform 2 numbering) inserted in pBIND plasmid; and full-length PPARγ (residues 1-505; isoform 2) inserted in pCMV6-XL4 plasmid Mutant proteins were generated using site directed mutagenesis of the aforementioned plasmids using primers listed in Supplementary Table 2 Peptides: LXXLL-containing motifs from TRAP220 (residues 638–656; NTKNHPMLMNLLKDNPAQD) and NCoR1 (2256–2278; DPASNLGLEDIIRKALMGSFDDK) without a FITC-label or containing a N-terminal FITC label with a six-carbon linker (Ahx) or NCoR1 (2251-2273; GHSFADPASNLGLEDIIRKALMG) containing an N-terminal biotin label and an amidated C-terminus for stability Ligand (5 µM) or vehicle control was added (20 μL) cells incubated for 18 hr and harvested for luciferase activity quantified using Britelite Plus (Perkin Elmer; 20 μL) or cell viability was tested using Celltiter-glo (Promega: 20 µL) on a Synergy Neo multimode plate reader (BioTek) Data were analyzed using GraphPad Prism (luciferase activity vs ligand concentration) and fit to a sigmoidal dose response curve For western blot analysis of protein levels HEK293T cells were transfected as described above 250,000 cells were transferred to 6-well plates (Corning) Transfected cells were then lysed in TNT buffer (150 mM NaCl Protein concentration was determined by BCA assay (Thermo Fisher) and 20 µg of protein was loaded onto 4–15% gradient gels (Bio-Rad) and wet transferred onto PVDF The membrane was blocked for 1 h at room temperature with Odyssey blocking buffer (Li-COR) the membrane was incubated overnight at 4 °C with primary antibodies diluted in Odyssey blocking buffer: 1:1000 rabbit anti-PPARγ (Cell signaling technology; catalog #2443 S 1:1000 mouse anti-actin (EMD Millipore; catalog #MAB1501 The following day the blot was washed with PBST and treated with secondary antibodies (Li-COR; donkey anti-mouse-IgG IRDye 680LT lot # C71201-15; goat anti-rabbit-IgG IR800CW lot # C80546-08) at 1:2000 dilution in Odyssey blocking buffer for 1 hr at RT The blot was then washed with PBST and visualized via multiplexed detection using the Odyssey 9120 infrared imaging system (Li-COR) The assay was performed in black 384-well plates (Greiner) in assay buffer (20 mM potassium phosphate His-PPARγ LBD was pre-incubated with or without a 2X molar excess of covalent ligand overnight at 4 °C Noncovalent compounds were incubated with a constant concentration of 90 µM equivalent to the maximum protein concentration FITC-labeled NCoR1 and TRAP220 peptides were plated at a final concentration of 100 nM Plates were incubated for 2 h at 4 °C and measured on a Synergy Neo multimode plate reader (BioTek) exciting at 495 nm and reading at 528 nm wavelengths Data were plotted using GraphPad Prism and fit to one-site binding equation We observed no significant changes in coregulator affinity when using native or delipidated protein indicating any bacterial lipids retained during protein purification are bound substoichiometrically FITC-labeled NCoR1 peptide was plated to a final concentration of 50 nM in wells to which was added delipidated full-length PPARγ loaded with a stoichiometric amount of ligand (protein concentration ranged from 50 µM to 24 nM by a 12 point 2-fold dilution) in a buffer containing 25 mM MOPS (pH 7.4) 0.01% fatty acid-free bovine serum albumin (BSA) (Millipore) Plates were incubated in the dark for 2 h at room temperature and measured on a Synergy H1 microplate reader (BioTek) exciting at 495 nm and reading at 528 nm wavelengths LanthaScreen® Elite Tb-anti-HIS Antibody (ThermoFisher; catalog #PV5863 and FITC-labeled NCoR1 peptide was prepared in assay buffer (20 mM potassium phosphate Ligands were prepared as a 2X ligand stock initially prepared via serial dilution in DMSO prior to addition to buffer Equal volumes of the protein mixture and ligand were added to black 384 well plates (Greiner) for final concentrations of 4 nM protein Final concentration of DMSO (vehicle) was constant in all wells at 0.5% Plates were incubated for 2 h at 4 °C and measured on a Synergy Neo multimode plate reader (BioTek) Data were plotted using GraphPad Prism (TR-FRET ratio 520 nm/495 nm vs ligand concentration) and fit to sigmoidal dose response equation Delipidated His-full-length PPARγ/full-length RXRα were plated at 31 nM with 0.9 nM LanthaScreen® Elite Tb-anti-HIS Antibody and 12-point serial dilutions of T0070907 from 50 μM to 1 pM to a final volume of 16 μL Plates were incubated for 4 h at room temperature and measured on Synergy H1 microplate reader (BioTek) at 620 nm for terbium and 665 nm for d2 a 1.25x molar excess of dsDNA of the Sult2A1 PPRE (5′-GTA AAA TAG GTG AAA GGT AA-3′; and its reverse complement) was added to each well and the plate was incubated for 3 h prior to a subsequent reading Data were plotted using GraphPad Prism (TR-FRET ratio 665 nm/620 nm vs ligand concentration) and fit to a sigmoidal dose response equation was used to simulate 1D NMR line shapes using MATLAB software (version R2018a) from the peptide titration experiments for G399 (15N planes vs extracted 15N planes from 2D NMR data) using 2-state (U) All simulations were performed using the 4-state model MATLAB script parts of the 4-state model were turned off as described in the LineShapeKin manual The 4-state simulations were performed considering the exchange rates and molar fractions of the two slowly exchanging PPARγ populations from the ZZ-exchange analysis Scripts or non-commercially available programs used for the analysis in this study are publicly available, including MATLAB scripts used for the ZZ exchange NMR lineshape analyses [https://osf.io/4fmkd/], MATLAB from the LinShapeKin package [http://lineshapekin.net], and decon1d [https://github.com/hughests/decon1d] The crystal structure of T0070907-bound PPARγ LBD is available in the Protein Data Bank (PDB accession code 6C1I). NMR chemical shift assignments used in our analysis were obtained from the Biological Magnetic Resonance Data Bank (BMRB accession codes 17975, 17976, 17977) Thiazolidinediones and the promise of insulin sensitization in type 2 diabetes A novel non-agonist peroxisome proliferator-activated receptor gamma (PPARgamma) ligand UHC1 blocks PPARgamma phosphorylation by cyclin-dependent kinase 5 (CDK5) and improves insulin sensitivity PPARG post-translational modifications regulate bone formation and bone resorption Pharmacological repression of PPARgamma promotes osteogenesis Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma Antagonism of peroxisome proliferator-activated receptor gamma prevents high-fat diet-induced obesity in vivo The PPARgamma antagonist T0070907 suppresses breast cancer cell proliferation and motility via both PPARgamma-dependent and -independent mechanisms Potential of peroxisome proliferator-activated receptor gamma antagonist compounds as therapeutic agents for a wide range of cancer types Inhibition of peroxisome proliferator-activated receptor gamma activity suppresses pancreatic cancer cell motility Defining a conformational ensemble that directs activation of PPARgamma Insights into the mechanism of partial agonism: crystal structures of the peroxisome proliferator-activated receptor gamma ligand-binding domain in the complex with two enantiomeric ligands Crystal structure of the peroxisome proliferator-activated receptor gamma (PPARgamma) ligand binding domain complexed with a novel partial agonist: a new region of the hydrophobic pocket could be exploited for drug design Co-crystal structure guided array synthesis of PPARgamma inverse agonists A functional peroxisome proliferator-activated receptor-gamma ligand-binding domain is not required for adipogenesis Transcription coactivator TRAP220 is required for PPAR gamma 2-stimulated adipogenesis Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) gamma activators and pan-PPAR partial agonists Accurate quantitation of water-amide proton exchange rates using the phase-modulated CLEAN chemical EXchange (CLEANEX-PM) approach with a Fast-HSQC (FHSQC) detection scheme Ligand-directed signalling at beta-adrenoceptors New concepts in pharmacological efficacy at 7TM receptors: IUPHAR review 2 An introduction to NMR-based approaches for measuring protein dynamics NMR line shapes and multi-state binding equilibria The necessary nitrogen atom: a versatile high-impact design element for multiparameter optimization Subtleties in GPCR drug discovery: a medicinal chemistry perspective Tactical approaches to interconverting GPCR agonists and antagonists Modification of the orthosteric PPARgamma covalent antagonist scaffold yields an improved dual-site allosteric inhibitor Probing the Complex Binding Modes of the PPARgamma partial agonist 2-chloro-N-(3-chloro-4-((5-chlorobenzo[d]thiazol-2-yl)thio)phenyl)-4-(trifluorome thyl)benzenesulfonamide (T2384) to orthosteric and allosteric sites with NMR spectroscopy PPARgamma helix 12 exhibits an antagonist conformation Zheng, J. et al. Chemical crosslinking mass spectrometry reveals the conformational landscape of the activation helix of PPARgamma; a model for ligand-dependent antagonism. Structure, in press, https://doi.org/10.1016/j.str.2018.07.007 (2018) PPARgamma in complex with an antagonist and inverse agonist: a tumble and trap mechanism of the activation helix Crystal structure of the ligand binding domain of the human nuclear receptor PPARgamma The differential interactions of peroxisome proliferator-activated receptor gamma ligands with Tyr473 is a physical basis for their unique biological activities Comparative protein structure modeling using Modeller UCSF Chimera--a visualization system for exploratory research and analysis H++: a server for estimating pKas and adding missing hydrogens to macromolecules Server: a web service for deriving RESP and ESP charges and building force field libraries for new molecules and molecular fragments Application of RESP charges to calculate conformational energies Long-time-step molecular dynamics through hydrogen mass repartitioning Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes An improved hydrogen bond potential: impact on medium resolution protein structures Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules A slow conformational switch in the BMAL1 transactivation domain modulates circadian rhythms Download references We thank Paola Munoz-Tello for critical reading of the manuscript This work was supported by National Institutes of Health (NIH) grants R01DK101871 (DJK) and P20GM103546 (computational resources to TH); American Heart Association (AHA) fellowship award 16POST27780018 (RB); and the William R Charitable Trust (TSRI High School Student Summer Internship Program) A portion of this work (ZZ exchange NMR) was performed at the National High Magnetic Field Laboratory (NHMFL/MagLab) which is supported by National Science Foundation (NSF) Cooperative Agreement No 19F NMR data presented herein were collected at the CUNY ASRC Biomolecular NMR Facility High School Student Summer Internship Program and M.D.N performed mutagenesis and/or purified proteins performed cellular assays and mutagenesis assays performed the molecular dynamics simulations conceived the experiments and wrote the manuscript with input from all authors Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-018-07133-w * Assisted living and similar communities (e.g. Residential care communities with missing data were excluded † Participating administrators and directors of residential care communities were asked "What is the total number of residents currently living at this residential care community § Participating administrators and directors of residential care communities were asked Include both occupied and unoccupied beds." there were 22,200 residential care communities serving 713,300 residents across the United States Forty percent of residential care communities were smaller with 4–10 beds but these communities housed only 7% of all residents The largest residential care communities with more than 100 beds were only 9% of all communities but housed 33% of all residents Source: Caffrey C, Harris-Kojetin L, Rome V, Sengupta M. Operating characteristics of residential care communities, by community bed size: United States, 2012. NCHS data brief, no 170. Hyattsville, MD: National Center for Health Statistics; 2014. Available at http://www.cdc.gov/nchs/data/databriefs/db170.htm Reported by: Vincent Rome, MPH, vrome@cdc.gov Alternate Text: The figure above is a bar chart showing that in 2012 Forty percent of residential care communities were smaller with 4-10 beds but these communities housed only 7% of all residents The largest residential care communities with more than 100 beds were only 9% of all communities but housed 33% of all residents How do I view different file formats (PDF, DOC, PPT, MPEG) on this site?  This work, Soldier in Heroic Battle to Receive Silver Star, by SGT Nicole Kojetin, identified by DVIDS, must comply with the restrictions shown on https://www.dvidshub.net/about/copyright This work, Face of Defense: Apache Pilot Receives Distinguished Flying Cross, must comply with the restrictions shown on https://www.dvidshub.net/about/copyright Homeowner blames city for flooding; city says it's climate change Longtime Edina resident Robert Tengdin's legal fight with the city over stormwater flooding his beloved tennis court has spilled into the community newspaper with the two sides arguing their opposing views "I ask you, is this the Edina others envy and of which we have been so proud?" Tengdin, 91, wrote in an open letter in the Edina Sun Current last month The area east of Highlands Lake that abuts Tengdin's tennis court and several neighbors' backyards has become a swamp in recent years and it likely will stay that way if the city wins its appeal next spring was dry for decades and considered part of Highlands Park with the city laying wood chips and maintaining nature walking paths But the city claims it always has been a wetland and this is its natural state "The city can't control the weather," City Manager Scott Neal wrote in a letter responding to Tengdin's full-page ad The back-and-forth is the latest development in a battle now spanning seven years. Tengdin first complained to the city in 2014 when historic flooding in Edina damaged 48 homes. Five years later, neither Tengdin's concerns nor the water had receded, so he gathered nearly 100 signatures on a petition asking the city to fix the flooding. Jeff Doom, a financial planner who moved across the street from Tengdin in 2007, said he was walking his dog Ole in the area until it became impassable two years ago. "Some parts of the trails were muddy, then all of a sudden the trail is 2 feet under water," Doom said. "That's the puzzling thing. How in the heck did this happen? If you say, 'Well, it's a wetland, and this is just part of being a wetland,' ... but we've had a drought. Shouldn't it go back after a drought to at least where it was a few years ago? What's different?" City staff declined to be interviewed for this story, citing the pending litigation. In court documents, Edina argues it is not responsible for rising groundwater levels that have increased by 10 feet over the past decade, and it has no statutory duty "to lower water levels in a wetland that are the clear result of climate change." The 88-year-old said if he were still in his role today, he would drain water into Highlands Lake. "It can be corrected, but it's going to cost a few dollars," Kojetin said. Ken Powell, Wetland Conservation Act supervisor for the Minnesota Board of Water and Soil Resources, said he's seen an increase in similar litigation from homeowners downstream, or where stormwater drains, who are "getting the brunt" from a combination of climate change and more drainage. "I don't know what the solution is. There is not a mechanism to stop the drainage. These landowners downstream have no option but to pursue legal remedies," Powell said. Tengdin's attorney, Tamara O'Neill Moreland, argues the flooding is not only caused by a changing climate, but an increase in impervious surfaces from redevelopment. In addition, she says the basin is part of the city's stormwater management system, so Edina has an obligation to maintain and repair it. "You don't have the ability as a city to sit back and say, 'We're going to store our water on your private property because this is what happens when it rains a lot,' " she said. Now that portion of the park is only accessible when water freezes over. Tengdin refers to it as the Okefenokee Swamp. "All it needs is an alligator," he said. "It's a wonderful place to be able to walk to but not to wade through." Tengdin wants Edina to fix the flooding so residents can enjoy the area again. Such a remedy also would improve the value of his home where he raised three sons. A 2019 assessment from Tengdin's real estate agent listed a recommended selling price of $850,000, but ReMax estimates the house is devalued by $400,000 without the flooding being addressed. In summer 2020, the tennis court he built in 1967 was full of standing water and mallard ducks, instead of the friends and neighbors who played on the court for decades. That year, Tengdin retired after 68 years with Allison-Williams Co. investment banking firm, and he filed his lawsuit. The city sought to throw out the case, arguing it can't be sued for enacting its own policies. Hennepin County District Judge Jacqueline Regis denied the motion, but the city appealed days before an August trial was set to begin. A decision from the appellate court is likely next spring. Regis wrote in her order that the east basin "was dry enough that Edina built and maintained a park and trail system" from the 1960s through at least the 1990s. "In recent years, however, the water level has noticeably risen," she stated, adding that "numerous trees in the east basin died due to flooding and the trails are, at least partly, submerged." A $68,000 Barr Engineering study conducted by the city found flooding wasn't caused by actions or inaction of the city. Four possible solutions from the study were rejected by the city: building a floodwall, draining stormwater into Highlands Lake, more frequent maintenance of the pump at the lake and pumping the east basin. In February, a year after Tengdin sued, the Edina City Council adopted a policy stating that the city only pumps out stormwater "when floodwater threatened a principle (habitable) structure." Before that, City Engineer Chad Millner wrote that "flooded tennis courts are currently not a priority" in a series of 2019 e-mails, after Mayor James Hovland and council members toured Tengdin's property and he raised his concerns with city staff. Attorney Paul Reuvers is defending Edina through the League of Minnesota Cities Insurance Trust. The city made a "judgment call," he said, and drew the line with limited resources. "The city can't pump [at] everyone's request," he said. Tengdin thinks the city is trying to drag out litigation long enough that he dies before the issue is settled in court. But when asked whether he's lived his whole life in Edina, Tengdin replied, "Not yet." Kim Hyatt reports on North Central Minnesota. She previously covered Hennepin County courts. No Section Peek inside homes for sale in the Twin Cities area After falling behind 17-0 at halftime and being dominated most of the game the Bulldogs may have locked up a spot in the College Football Playoff * Denominators used to calculate percentages for adult day services centers and residential care communities were derived from the number of residents/participants on a given day in 2012 Denominators used to calculate percentages for home health agencies and hospices were the number of patients whose episode of care in a home health agency ended at any time in 2011 and the number of patients who received care from Medicare-certified hospices at any time in 2011 † Participating administrators and directors of residential care communities and adult day services centers were asked "Of the residents currently living at this community/participants enrolled at this center about how many have been diagnosed with depression?" the percentage of users of long-term care services with a diagnosis of depression was highest in nursing homes (49%) and home health agencies (35%) and lowest in residential care communities (25%) The percentage of users with a diagnosis of depression in nursing homes (49%) was approximately twice that of those in adult day services centers (24%) or residential care communities (25%) in 2012 Source: Harris-Kojetin L, Sengupta M, Park-Lee E, Valverde R. Long-term care services in the United States: 2013 overview. Hyattsville, MD: US Department of Health and Human Services, CDC; 2013. Available at http://www.cdc.gov/nchs/data/nsltcp/long_term_care_services_2013.pdf For a long time, the sport of bandy was so obscure it barely registered a ripple on the U.S. landscape. After more than three decades of growth in the country, though, the ice sport — consider it a cross between ice hockey, field hockey and soccer — has gained enough traction to create a U.S. Bandy Hall of Fame. ¶ The inaugural class of six, deep with Minnesotans, will be inducted at 6 p.m. Wednesday night at the Aster Cafe in Minneapolis. They are: Bob Kojetin, former head of Edina Park and Recreation, who is credited with helping introduce bandy — an 11-on-11 skating sport with Swedish roots played with a ball and bowed sticks on a rink roughly the size of a soccer field — to the United States; Magnus Skold, longtime General Manager of the U.S. team; Gunnar Fast, who brought the sport to Minnesota; and Chris Halden, Tom Howard, Chris Middlebrook, all longtime standout players. The Hall of Fame will be housed at the John Rose Oval in Roseville, which is also the home rink for the U.S. National Team. "Once we reached the 30-year mark, with hundreds of players, we formed a committee. We're not getting any younger," Halden said. "They decided to do the induction by decade, starting with the guys who really built the sport." Like a lot of U.S. players, Halden is a converted hockey player. He and Middlebrook were college hockey teammates at Gustavus. Both gave bandy a shot in the early 1980s and became hooked. More than 30 years and countless international tournaments later, Halden still is going strong at age 58. Many of those international tournaments were in Russia, where he said the U.S. National Team players were treated like "rock stars" and often play in front of crowds of 25,000. Locally, the 11-on-11 version is played at the Oval, while a modified 4-on-4 version at local hockey rinks is catching on among the next generation of college players looking for a diversion, but still a workout, over the summer. A league this summer attracted several top-tier players, and Halden is hoping to convince some of them to make the leap to the national team someday. "Our formula is to get hockey players to convert to bandy," Halden said. "But we've had a youth program in the past few years with homegrown players. ... It's still a small cult sport. But it's really incredible. It's so fun to play." Wolves Minnesota passing on Curry (twice) or Ant’s list Butler and the Warriors will play at Target Center in a second-round NBA playoff series Owen Marsolek struck out 17 to lead the Hilltoppers to a 3-0 victory Monday at Siebert Field Online retail giant Amazon is now the owner of a newly completed warehouse in central Moravia. Once its operations are in full swing next year, the Kojetín Industrial Park could offer up to 2,000 jobs This will be the online retail giant’s second warehouse in Czechia, following one in Dobrovíz, Central Bohemia that opened in 2015 Built by industrial construction firm Panattoni this is Czechia’s first multi-story distribution center the building boasts a high level of energy self-sufficiency The warehouse will have a special robotic unit to facilitate the work of employees The four-story distribution center has a total area of more than 187,000 square meters “I believe that the state-of-the-art logistics complex will help Amazon in its further expansion in the e-commerce sector which has been experiencing a boom in recent years The project will also be beneficial for the region thanks to a large number of job opportunities,” Sovička added The new distribution hall was built on a brownfield formerly used by a sugar factory for its settling tanks “Investment in the revitalization of aging or dilapidated sites is our specialty I am proud that we have once again succeeded in creating an industrial zone on land with low ecological value which will rank at the very top in the field of environmentally friendly and technological construction,” Accolade Group CEO Milan Kratina said The logistics hall was built to meet strict environmental requirements. Accolade states it is aiming for BREEAM New Construction sustainable approach certification at the "Excellent" level The certification takes place after the building is finished BREEAM (Building Research Establishment Environmental Assessment Method) is the oldest and most widely recognized certification for the sustainability of buildings The certification evaluates not only the energy efficiency of the building and other operational factors but also the environmental impact of the construction process Many of the building panels were made at a factory some 12 kilometers from the construction site and moved by rail The area around the warehouse will feature a lizard habitat and bee hives out of which around 300 will be planted at the warehouse and the rest in Kojetín “We care about the operation being environmentally friendly which is why we installed modern 4 MW photovoltaic panels on the roof this exceeds ordinary home installations by three orders of magnitude,” Michal Šmíd Your morning coffee deserves a great companion. Why not enjoy it with our daily newsletter? News from Czechia, curated insights, and inspiring stories in English. Global internet retail giant Amazon is building a new logistics center in Kojetin industrial park near Prerov in the Olomouc Region which should create over 2,000 jobs when it opens in 2023 and will be equipped with modern technology Photo credit: Freepik (illustrative photo) The ongoing construction of the new Amazon facility in the industrial park in Kojetin is scheduled for completion next year The facility will be equipped with modern technology General Manager of Amazon for the Czech Republic the new distribution centre will allow the company to offer more products and support a growing number of independent small businesses that sell their goods through its e-shop The Kojetin centre will prepare and dispatch smaller items to customers Amazon has already started recruiting the first staff for the centre The full recruitment process is planned for the second quarter of 2022 “The new jobs will complement 4,000 existing permanent jobs that we have already created in the Czech Republic and this is a result we are proud of,” said Šmíd in a press release.  Amazon has been operating in the Czech Republic since 2013 The company’s first distribution centre in the country opened in 2015 and it now employs over 3,000 permanent staff The company employs a further 1,000 specialized staff in corporate offices in the center of Prague Advertise with us Privacy Policy Brno Daily is a Czech media outlet for expats Our partners