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Unexpected Allosteric Network Contributes to LRH-1 Co-regulator Selectivity*

  • Paul M. Musille
    Footnotes
    Affiliations
    From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322 and
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  • Bradley R. Kossmann
    Footnotes
    Affiliations
    the Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30302
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  • Jeffrey A. Kohn
    Footnotes
    Affiliations
    From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322 and
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  • Ivaylo Ivanov
    Affiliations
    the Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30302
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  • Eric A. Ortlund
    Correspondence
    To whom correspondence should be addressed. Tel.: 404-727-5014; Fax: 404-727-2738;
    Affiliations
    From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322 and
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant RO1DK095750 from NIDDK (to E. A. O.), Grant R01GM110387 from NIGMS (to I. I.), and Emory-NIEHS Graduate and Postdoctoral Training in Toxicology Grant T32ES012870 (to P. M. M.), American Heart Association Predoctoral Grant 12PRE12060583, National Science Foundation CAREER Award MCB-1149521, and a Molecular Basis Diseases fellowship (to B. R. K.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    1 These authors contributed equally to this work.
Open AccessPublished:November 09, 2015DOI:https://doi.org/10.1074/jbc.M115.662874
      Phospholipids (PLs) are unusual signaling hormones sensed by the nuclear receptor liver receptor homolog-1 (LRH-1), which has evolved a novel allosteric pathway to support appropriate interaction with co-regulators depending on ligand status. LRH-1 plays an important role in controlling lipid and cholesterol homeostasis and is a potential target for the treatment of metabolic and neoplastic diseases. Although the prospect of modulating LRH-1 via small molecules is exciting, the molecular mechanism linking PL structure to transcriptional co-regulator preference is unknown. Previous studies showed that binding to an activating PL ligand, such as dilauroylphosphatidylcholine, favors LRH-1's interaction with transcriptional co-activators to up-regulate gene expression. Both crystallographic and solution-based structural studies showed that dilauroylphosphatidylcholine binding drives unanticipated structural fluctuations outside of the canonical activation surface in an alternate activation function (AF) region, encompassing the β-sheet-H6 region of the protein. However, the mechanism by which dynamics in the alternate AF influences co-regulator selectivity remains elusive. Here, we pair x-ray crystallography with molecular modeling to identify an unexpected allosteric network that traverses the protein ligand binding pocket and links these two elements to dictate selectivity. We show that communication between the alternate AF region and classical AF2 is correlated with the strength of the co-regulator interaction. This work offers the first glimpse into the conformational dynamics that drive this unusual PL-mediated nuclear hormone receptor activation.

      Introduction

      Phospholipids (PLs)
      The abbreviations used are: PL, phospholipid; LRH-1, liver receptor homolog-1; DLPC, dilauroylphosphatidylcholine; AF, activation function; PPAR, peroxisome proliferator-activated receptor; PDB, Protein Data Bank; DLPE, dilauroylphosphatidylethanolamine; DCIA, 7-diethylamino-3-((4′-(iodoacetyl)amino)phenyl)-4-methylcoumarin; PC, phosphatidylcholine; NR, nuclear receptor; PS, phosphatidylserine; SHP, the small heterodimer partner; PG, phosphatidylglycerol; MD, molecular dynamics; r.m.s.d., root mean square deviation; HDX, hydrogen-deuterium exchange.
      are best known for their structural role in membranes and as synthesis material for potent signaling molecules, such as diacylglycerol, leukotrienes, and inositol phosphates. Recent evidence, however, suggests intact PLs are able to directly modulate the activity of transcription factors involved in lipid homeostasis, such as sterol regulatory element-binding protein 1 (SREBP-1), and some members of the nuclear receptor (NR) family of ligand-regulated transcription factors, including peroxisome proliferator-activated receptor α (PPARα; NR1C1), steroidogenic factor 1 (NR5A1), and human liver receptor homolog-1 (LRH-1; NR5A2) (
      • Musille P.M.
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      Phospholipid–driven gene regulation.
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      ,
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      • Walker A.K.
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      • Shioda T.
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      • Yang F.
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      • Tzoneva M.
      • Hart A.C.
      • Näär A.M.
      A conserved SREBP-1/phosphatidylcholine feedback circuit regulates lipogenesis in metazoans.
      ). LRH-1 regulates the expression of genes central to embryonic development, cell cycle progression, steroid synthesis, lipid and glucose homeostasis, and local immune function (
      • Fernandez-Marcos P.J.
      • Auwerx J.
      • Schoonjans K.
      Emerging actions of the nuclear receptor LRH-1 in the gut.
      • Moore D.D.
      • JaeMan L.
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      Targeting nuclear receptors to treat type 2 diabetes.
      ,
      • Oosterveer M.H.
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      Hepatic glucose sensing and integrative pathways in the liver.
      ,
      • Zhang C.
      • Large M.J.
      • Duggavathi R.
      • DeMayo F.J.
      • Lydon J.P.
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      • Kovanci E.
      • Murphy B.D.
      Liver receptor homolog-1 is essential for pregnancy.
      ,
      • Gerrits H.
      • Paradé M.C.
      • Koonen-Reemst A.M.
      • Bakker N.E.
      • Timmer-Hellings L.
      • Sollewijn Gelpke M.D.
      • Gossen J.A.
      Reversible infertility in a liver receptor homolog-1 (LRH-1)-knockdown mouse model.
      ,
      • Kelly V.R.
      • Xu B.
      • Kuick R.
      • Koenig R.J.
      • Hammer G.D.
      Dax1 up-regulates Oct4 expression in mouse embryonic stem cells via LRH-1 and SRA.
      ,
      • Venteclef N.
      • Jakobsson T.
      • Steffensen K.R.
      • Treuter E.
      Metabolic nuclear receptor signaling and the inflammatory acute phase response.
      • Stein S.
      • Schoonjans K.
      Molecular basis for the regulation of the nuclear receptor LRH-1.
      ). Thus, LRH-1 is an enticing pharmaceutical target for the treatment of metabolic and neoplastic diseases (
      • Moore D.D.
      • JaeMan L.
      • Binging D.
      Targeting nuclear receptors to treat type 2 diabetes.
      ).
      Although the endogenous ligand for hLRH-1 is currently unknown, oral treatment with the exogenous PL agonist dilauroylphosphatidylcholine (PC 12:0–12:0; DLPC) lowers serum lipid levels, reduces liver fat accumulation, and improves glucose tolerance in an LRH-1-dependent manner in a diabetic mouse model (
      • Lee J.M.
      • Lee Y.K.
      • Mamrosh J.L.
      • Busby S.A.
      • Griffin P.R.
      • Pathak M.C.
      • Ortlund E.A.
      • Moore D.D.
      A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects.
      ). Activation of LRH-1 by DLPC drives increased glucose uptake by muscle and increases the rate of both glycolysis and glycogen synthesis with a concomitant reduction in fatty acid metabolism (
      • Bolado-Carrancio A.
      • Riancho J.A.
      • Sainz J.
      • Rodríguez-Rey J.C.
      Activation of nuclear receptor NR5A2 increases Glut4 expression and glucose metabolism in muscle cells.
      ). These observations suggest LRH-1 agonists may resolve glucose homeostasis-related diseases. New evidence suggests that LRH-1 may also be targeted to relieve chronic endoplasmic reticulum stress. Activation of LRH-1 by synthetic DLPC or the small molecule RJW100 induces Plk3, which is required for the activation of ATF2 and the induction of its target genes, which play a key role in resolving ER stress (
      • Mamrosh J.L.
      • Lee J.M.
      • Wagner M.
      • Stambrook P.J.
      • Whitby R.J.
      • Sifers R.N.
      • Wu S.P.
      • Tsai M.J.
      • Demayo F.J.
      • Moore D.D.
      Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution.
      ). Given its potential therapeutic value, LRH-1 has been the subject of multiple attempts to identify small molecule modulators (
      • Whitby R.J.
      • Dixon S.
      • Maloney P.R.
      • Delerive P.
      • Goodwin B.J.
      • Parks D.J.
      • Willson T.M.
      Identification of small molecule agonists of the orphan nuclear receptors liver receptor homolog-1 and steroidogenic factor-1.
      • Whitby R.J.
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      • Leesnitzer L.M.
      • Orband-Miller L.A.
      • Williams S.P.
      • Willson T.M.
      • Xu R.
      • Zuercher W.J.
      • Cai F.
      • Ingraham H.A.
      Small molecule agonists of the orphan nuclear receptors steroidogenic factor-1 (SF-1, NR5A1) and liver receptor homolog-1 (LRH-1, NR5A2).
      ,
      • Corzo C.A.
      • Mari Y.
      • Chang M.R.
      • Khan T.
      • Kuruvilla D.
      • Nuhant P.
      • Kumar N.
      • West G.M.
      • Duckett D.R.
      • Roush W.R.
      • Griffin P.R.
      Antiproliferation activity of a small molecule repressor of liver receptor homolog 1.
      • Benod C.
      • Carlsson J.
      • Uthayaruban R.
      • Hwang P.
      • Irwin J.J.
      • Doak A.K.
      • Shoichet B.K.
      • Sablin E.P.
      • Fletterick R.J.
      Structure-based discovery of antagonists of nuclear receptor LRH-1.
      ). These attempts have been met with mixed success due in part to our limited understanding of LRH-1's mechanism of activation.
      We have shown that DLPC is able to bind directly to the LRH-1 ligand binding domain (LBD) and activate the receptor by affecting receptor dynamics in an alternate activation function (AF) region, encompassing the β-sheet-H6 region of the protein, to alter co-regulator binding preference (
      • Musille P.M.
      • Pathak M.C.
      • Lauer J.L.
      • Hudson W.H.
      • Griffin P.R.
      • Ortlund E.A.
      Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation.
      ). Importantly, it seems that DLPC may promote activation by relieving LRH-1 from repression by the non-canonical co-repressor NR SHP, which mimics a co-activator using the canonical Leu-Xaa-Xaa-Leu-Leu (where Xaa is any amino acid) nuclear co-activator interaction motif (
      • Goodwin B.
      • Jones S.A.
      • Price R.R.
      • Watson M.A.
      • McKee D.D.
      • Moore L.B.
      • Galardi C.
      • Wilson J.G.
      • Lewis M.C.
      • Roth M.E.
      • Maloney P.R.
      • Willson T.M.
      • Kliewer S.A.
      A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.
      ,
      • Zhi X.
      • Zhou X.E.
      • He Y.
      • Zechner C.
      • Suino-Powell K.M.
      • Kliewer S.A.
      • Melcher K.
      • Mangelsdorf D.J.
      • Xu H.E.
      Structural insights into gene repression by the orphan nuclear receptor SHP.
      ). In the absence of ligand, the alternate AF is highly dynamic and mutations that restrict motion in this region ablate transactivation (
      • Musille P.M.
      • Pathak M.C.
      • Lauer J.L.
      • Hudson W.H.
      • Griffin P.R.
      • Ortlund E.A.
      Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation.
      ). SHP is a robust co-repressor of LRH-1-mediated transactivation in the liver and can recognize both apo-LRH-1 and LRH-1 when bound to a non-ideal ligand such as bacterial PLs in vitro (
      • Goodwin B.
      • Jones S.A.
      • Price R.R.
      • Watson M.A.
      • McKee D.D.
      • Moore L.B.
      • Galardi C.
      • Wilson J.G.
      • Lewis M.C.
      • Roth M.E.
      • Maloney P.R.
      • Willson T.M.
      • Kliewer S.A.
      A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.
      ,
      • Goodwin B.
      • Watson M.A.
      • Kim H.
      • Miao J.
      • Kemper J.K.
      • Kliewer S.A.
      Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-α.
      ,
      • Lu T.T.
      • Makishima M.
      • Repa J.J.
      • Schoonjans K.
      • Kerr T.A.
      • Auwerx J.
      • Mangelsdorf D.J.
      Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors.
      ). It is unclear how LRH-1 discriminates between SHP and co-activators such as TIF2 that bind using a similar LXXLL motif to recognize the active NR orientation. Furthermore, how does human LRH-1 recognize co-activators in the absence of ligand? How do PLs varying only in their acyl tail composition show differing abilities to drive transactivation? Which ligand/co-regulator states are appropriate for in silico ligand design?
      This incomplete understanding of what dictates LRH-1's PL and co-regulator selectivity limits our ability to guide the design of robust small molecule modulators for this intriguing pharmacological target. To address these questions, we have generated a novel crystal structure of the LRH-1·TIF2 complex in an apo-state, as well as a higher resolution structure of LRH-1 bound to Escherichia coli PLs. These crystal structures, in combination with novel lipid binding assays, molecular dynamics simulations, and principal component analysis (PCA) have allowed us to identify an unexpected allosteric network that may contribute to PL-mediated NR signaling and co-regulator selectivity.

      Discussion

      Robust signaling pathways must not only respond to activating ligands but must discriminate against the wrong ones to reduce noise (
      • Atkins W.M.
      Biological messiness vs. biological genius: mechanistic aspects and roles of protein promiscuity.
      ). For LRH-1, this challenge is amplified because its ligands include highly abundant intact PLs that include a large fraction of cell membranes. It is possible that LRH-1 displays an intrinsic set of selection criteria for PL isoforms, that PL delivery to the receptor is facilitated by soluble lipid transport proteins, or a combination of the two. Our results show that LRH-1 is able to bind a wide range of PLs in vitro but can extract only PCs, PGs, and phosphatidylinositols from a membrane/vesicle without assistance from a molecular chaperone. Inclusion of a nonspecific lipid chaperone, β-cyclodextrin, permits the binding of all glycerophospholipids tested. This is in line with structural studies because the majority of recognition occurs via contacts with the lipid tails and phosphoglycerol backbone. Thus, LRH-1 lipid preference is driven more so by the composition of the PL tails than by the headgroup, which protrudes from the receptor surface. Remarkably, although LRH-1 can readily accommodate a range of medium chain saturated PLs, affinity is highest for the 11- and 12-carbon PCs shown to selectively drive receptor activation in cells (
      • Lee J.M.
      • Lee Y.K.
      • Mamrosh J.L.
      • Busby S.A.
      • Griffin P.R.
      • Pathak M.C.
      • Ortlund E.A.
      • Moore D.D.
      A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects.
      ).

      Lipid-mediated Allosteric Control of a Protein-Protein Binding Interface

      Intact PLs are unusual ligands, and LRH-1 has evolved to respond to them via a novel allosteric pathway to support appropriate interaction with co-regulators depending on the ligand status. The idea that ligand binding can drive the selective recruitment of different co-regulators has been hypothesized before; previous MD studies have indicated that the SHP/LRH-1 interaction is weakened upon the binding of PS to the apo-receptor, although binding of DAX-1 and PROX1 is strengthened (
      • Burendahl S.
      • Treuter E.
      • Nilsson L.
      Molecular dynamics simulations of human LRH-1: the impact of ligand binding in a constitutively active nuclear receptor.
      ), suggesting that an avenue exists for communication between the ligand binding pocket and the AF-2 cleft. Although no studies have demonstrated a role for PS in the regulation of LRH-1's target genes, recent HDX studies that compared LRH-1 bound to E. coli PLs and DLPC demonstrated increased flexibility in both the mouth of the ligand binding pocket and the AF-2 region in DLPC-bound LRH-1 (
      • Musille P.M.
      • Pathak M.C.
      • Lauer J.L.
      • Hudson W.H.
      • Griffin P.R.
      • Ortlund E.A.
      Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation.
      ). Furthermore, stabilizing the mouth of the LBP in apo-hLRH-1 by replacing residues 419–424 with the corresponding mouse LRH-1 sequence enhances binding of the co-activators TIF-2 and PGC1α (
      • Musille P.M.
      • Pathak M.
      • Lauer J.L.
      • Griffin P.R.
      • Ortlund E.A.
      Divergent sequence tunes ligand sensitivity in phospholipid-regulated hormone receptors.
      ). In the absence of PLs, the receptor accesses a greater amount of conformational space and readily interacts with co-repressors. Medium chain PLs appear to promote productive motions that favor co-activator interaction and disfavor SHP interaction, perhaps by suppressing non-activating (non-productive) motions to drive selective interaction with co-regulators. LRH-1's allosteric network connecting the β-sheet-H6 region may be an evolutionary adaptation that allowed LRH-1 to sense these unusually large ligands and discriminate against fatty acids and cholesterol-derived ligands, which would also fit in the receptor's large hydrophobic pocket.
      Ideally, structure-function work should be performed and interpreted in the context of the full-length protein. Obtaining a structure of the intact receptor has been challenging, likely due to the large amount of disorder in the linker region connecting the DNA and ligand binding domains. Thus, we modeled systems for which there was empirical structural and biochemical data. In addition, LRH-1 transactivation has been shown to be affected by post-translational modifications located on the hinge (i.e. phosphorylation, acetylation, and SUMOylation) (
      • Stein S.
      • Schoonjans K.
      Molecular basis for the regulation of the nuclear receptor LRH-1.
      ). Phosphorylation of the serine residues Ser-238 and Ser-243 in the hinge region of the human LRH-1 by the mitogen-activated protein kinase ERK1/2 enhances its activity (
      • Lee Y.K.
      • Choi Y.H.
      • Chua S.
      • Park Y.J.
      • Moore D.D.
      Phosphorylation of the hinge domain of the nuclear hormone receptor LRH-1 stimulates transactivation.
      ). LRH-1 also been shown to be acetylated in the basal state and is bound by SHP·sirtuin 1 (SIRT1) transrepressive complex. Surprisingly SIRT1 does not modulate LRH-1 directly, thus what is driving the acetylation and deacetylation of LRH-1 is not established (
      • Chanda D.
      • Xie Y.B.
      • Choi H.S.
      Transcriptional co-repressor SHP recruits SIRT1 histone deacetylase to inhibit LRH-1 transactivation.
      ). LRH-1 transactivation is also controlled by small ubiquitin-like modifier conjugation to lysine 289 (
      • Chalkiadaki A.
      • Talianidis I.
      SUMO-dependent compartmentalization in promyelocytic leukemia protein nuclear bodies prevents the access of LRH-1 to chromatin.
      ). SUMOylation was shown to drive LRH-1 localization in nuclear bodies, whereby small ubiquitin-like modifier-conjugated LRH-1 is preferentially sequestered in these bodies preventing it from binding to DNA (
      • Chalkiadaki A.
      • Talianidis I.
      SUMO-dependent compartmentalization in promyelocytic leukemia protein nuclear bodies prevents the access of LRH-1 to chromatin.
      ). Recently, the K. Schoonjans lab showed that SUMOylated LRH-1 interacts with PROX-1, a co-repressor, to control 25% of LRH-1 gene targets in the liver. Mutation of lysine 289 to an arginine specifically ablates PROX-1 interaction, without affecting other canonical co-regulator interactions.
      Emerging evidence suggests that NR activation does not occur via the classically described “mouse trap” model, whereby the AF-H swings from an inactive to active state upon agonist binding. Both experimental and modeling studies are inconsistent with radical repositioning of H12 away from the AF-2 in apo-NRs (
      • Hughes T.S.
      • Chalmers M.J.
      • Novick S.
      • Kuruvilla D.S.
      • Chang M.R.
      • Kamenecka T.M.
      • Rance M.
      • Johnson B.A.
      • Burris T.P.
      • Griffin P.R.
      • Kojetin D.J.
      Ligand and receptor dynamics contribute to the mechanism of graded PPARγ agonism.
      • Martínez L.
      • Polikarpov I.
      • Skaf M.S.
      Only subtle protein conformational adaptations are required for ligand binding to thyroid hormone receptors: simulations using a novel multipoint steered molecular dynamics approach.
      ,
      • Batista M.R.
      • Martínez L.
      Dynamics of nuclear receptor Helix-12 switch of transcription activation by modeling time-resolved fluorescence anisotropy decays.
      • Mackinnon J.A.
      • Gallastegui N.
      • Osguthorpe D.J.
      • Hagler A.T.
      • Estébanez-Perpiñá E.
      Allosteric mechanisms of nuclear receptors: insights from computational simulations.
      ). Rather, subtle local conformational adaptations are observed in H12 as well as other regions within the ligand binding pocket such as the H11-H12 loop, H3, and H5 (
      • Mackinnon J.A.
      • Gallastegui N.
      • Osguthorpe D.J.
      • Hagler A.T.
      • Estébanez-Perpiñá E.
      Allosteric mechanisms of nuclear receptors: insights from computational simulations.
      ). These subtle conformational differences between structures may be functionally important, representing a shift between conformational ensembles, but are difficult to identify via inspection of superimposed crystal structures. Previous work with both steroid receptors and fatty acid-sensing NRs have also revealed remarkable flexibility in this region comprising bottom half of the ligand binding pocket, including H3, H6-H7, and H11 (
      • Bledsoe R.K.
      • Montana V.G.
      • Stanley T.B.
      • Delves C.J.
      • Apolito C.J.
      • McKee D.D.
      • Consler T.G.
      • Parks D.J.
      • Stewart E.L.
      • Willson T.M.
      • Lambert M.H.
      • Moore J.T.
      • Pearce K.H.
      • Xu H.E.
      Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and co-activator recognition.
      ,
      • Gee A.C.
      • Katzenellenbogen J.A.
      Probing conformational changes in the estrogen receptor: evidence for a partially unfolded intermediate facilitating ligand binding and release.
      ). In the absence of ligand, NRs are partially unfolded. Recent NMR studies focused on PPARγ show that in the apo-state only half of the expected peaks appear on the intermediate exchange time scale (milliseconds-to-microseconds). NMR supports a model whereby NRs sample a range of conformations in the apo-state. Full-agonists drive this equilibrium toward a more classically active conformation by protecting residues comprising the ligand binding pocket and AF-2 from intermediate exchange, although partial agonists only partially stabilize the regions of the receptor (
      • Kojetin D.J.
      • Burris T.P.
      Small molecule modulation of nuclear receptor conformational dynamics: implications for function and drug discovery.
      ). The β-sheet region may also play an important role in mediating PPARγ's response to ligands (
      • Hughes T.S.
      • Chalmers M.J.
      • Novick S.
      • Kuruvilla D.S.
      • Chang M.R.
      • Kamenecka T.M.
      • Rance M.
      • Johnson B.A.
      • Burris T.P.
      • Griffin P.R.
      • Kojetin D.J.
      Ligand and receptor dynamics contribute to the mechanism of graded PPARγ agonism.
      ). Although the dynamics in this region are important for mediating ligand action, activation by partial agonists is mediated by the ability of a solvent-inaccessible serine residue in this region to be phosphorylated (
      • Hughes T.S.
      • Chalmers M.J.
      • Novick S.
      • Kuruvilla D.S.
      • Chang M.R.
      • Kamenecka T.M.
      • Rance M.
      • Johnson B.A.
      • Burris T.P.
      • Griffin P.R.
      • Kojetin D.J.
      Ligand and receptor dynamics contribute to the mechanism of graded PPARγ agonism.
      ).
      Given LRH-1's limited selectivity criteria in vitro, it is possible that access to endogenous ligands is controlled both temporally and spatially by phospholipid transfer proteins. For example, phospholipid transfer proteins such as phosphatidylinositol transfer protein α and phosphatidylcholine transfer protein are both capable of transporting intact PLs into the nucleus (
      • Nile A.H.
      • Bankaitis V.A.
      • Grabon A.
      Mammalian diseases of phosphatidylinositol transfer proteins and their homologs.
      ,
      • Kang H.W.
      • Kanno K.
      • Scapa E.F.
      • Cohen D.E.
      Regulatory role for phosphatidylcholine transfer protein/StarD2 in the metabolic response to peroxisome proliferator activated receptor alpha (PPARα).
      ). The effect of tail unsaturation has also not yet been studied, but it is likely that the bends introduced by cis unsaturation would allow the LRH-1 ligand binding pocket to accommodate longer chain acyl tails promoting potent receptor activation. Given the diverse composition of PL tails in vivo, these studies are best guided by lipidomics-based identification of endogenous PL ligands. Current limitations in the ability to isolate LRH-1 from mammalian tissue have limited the field's ability to identify endogenous ligands, although these studies are underway.

      Author Contributions

      P. M. M., B. R. K., J. A. K., I. I., and E. A. O. participated in research design. P. M.M., B. R. K., and J. A. K. conducted the experiments. P. M. M., B. R. K., J. A. K., I. I., and E. A. O. performed data analysis. P. M. M., B. R. K., J. A. K., I. I., and E. A. O. wrote or contributed to the writing of the manuscript.

      Acknowledgments

      Computational resources were provided in part by National Science Foundation XSEDE Allocation CHE110042 and Allocation m1254 at the National Energy Research Scientific Computing Center supported by the United States Department of Energy Office of Science Contract DE-AC02-05CH11231.

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