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• W2066841732 abstract "The nuclear receptor Nurr1 is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates. Recent crystal structures of the Nurr1 ligand binding domain (LBD) and the Drosophila orthologue dHR38 revealed that, although these receptors share the classical LBD architecture, they lack a ligand binding cavity. This volume is instead filled with bulky hydrophobic side chains. Furthermore the “canonical” non-polar co-regulator binding groove is filled with polar side chains; thus, the regulation of transcription by this sub-family of nuclear receptor LBDs may be mediated by some other interaction surface on the LBD. We report here the identification of a novel co-regulator interface on the LBD of Nurr1. We used an NMR footprinting strategy that facilitates the identification of an interaction surface without the need of a full assignment. We found that non-polar peptides derived from the co-repressors SMRT and NCoR bind to a hydrophobic patch on the LBD of Nurr1. This binding surface involves a groove between helices 11 and 12. Mutations in this site abolish activation by the Nurr1 LBD. These findings give insight into the unique mechanism of action of this class of nuclear receptors. The nuclear receptor Nurr1 is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates. Recent crystal structures of the Nurr1 ligand binding domain (LBD) and the Drosophila orthologue dHR38 revealed that, although these receptors share the classical LBD architecture, they lack a ligand binding cavity. This volume is instead filled with bulky hydrophobic side chains. Furthermore the “canonical” non-polar co-regulator binding groove is filled with polar side chains; thus, the regulation of transcription by this sub-family of nuclear receptor LBDs may be mediated by some other interaction surface on the LBD. We report here the identification of a novel co-regulator interface on the LBD of Nurr1. We used an NMR footprinting strategy that facilitates the identification of an interaction surface without the need of a full assignment. We found that non-polar peptides derived from the co-repressors SMRT and NCoR bind to a hydrophobic patch on the LBD of Nurr1. This binding surface involves a groove between helices 11 and 12. Mutations in this site abolish activation by the Nurr1 LBD. These findings give insight into the unique mechanism of action of this class of nuclear receptors. Nuclear receptors comprise a large and ancient family of intra-cellular transcription factors that regulate many aspects of development and metabolism in metazoans. The activity of many nuclear receptors, especially those in vertebrates, is controlled by the binding of fat-soluble hormones and metabolites within a non-polar cavity in the ligand binding domain (LBD) 1The abbreviations used are: LBD, ligand binding domain; HSQC, heteronuclear single quantum coherence; TR, thyroid hormone receptor. of the receptor. The mechanisms through which these small molecules switch the activity of the receptor have been established (reviewed in Ref. 1Nagy L. Schwabe J.W.R. Trends Biochem. Sci. 2004; 29: 317-324Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Ligand binding stabilizes the global structure of the LBD (2Johnson B.A. Wilson E.M. Li Y. Moller D.E. Smith R.G. Zhou G. J. Mol. Biol. 2000; 298: 187-194Crossref PubMed Scopus (135) Google Scholar, 3Pissios P. Tzameli I. Kushner P. Moore D.D. Mol. Cell. 2000; 6: 245-253Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and causes the C-terminal helix (helix 12), which is independently mobile in the absence of a ligand, to adopt a stable conformation packed against the surface of the LBD (4Kallenberger B.C. Love J.D. Chatterjee V.K. Schwabe J.W. Nat. Struct. Biol. 2003; 10: 136-140Crossref PubMed Scopus (145) Google Scholar). This results in the displacement of co-repressor proteins and the recruitment of co-activator proteins. Both co-repressor and co-activator proteins bind to the same non-polar groove on the surface of the LBD through related but distinct interaction motifs that adopt an amphipathic helical conformation (5Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1707) Google Scholar, 6Xu H.E. Stanley T.B. Montana V.G. Lambert M.H. Shearer B.G. Cobb J.E. McKee D.D. Galardi C.M. Plunket K.D. Nolte R.T. Parks D.J. Moore J.T. Kliewer S.A. Willson T.M. Stimmel J.B. Nature. 2002; 415: 813-817Crossref PubMed Scopus (524) Google Scholar, 7Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1785) Google Scholar, 8Nagy L. Kao H.Y. Love J.D. Li C. Banayo E. Gooch J.T. Krishna V. Chatterjee K. Evans R.M. Schwabe J.W. Genes Dev. 1999; 13: 3209-3216Crossref PubMed Scopus (346) Google Scholar, 9Perissi V. Staszewski L.M. McInerney E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; 13: 3198-3208Crossref PubMed Scopus (425) Google Scholar, 10Hu X. Lazar M.A. Nature. 1999; 402: 93-96Crossref PubMed Scopus (530) Google Scholar). Discrimination between co-activators and co-repressors is achieved because the active position of helix 12 does not allow binding of the longer co-repressor helix but stabilizes the binding of the shorter co-activator helix. Although this mechanism is relevant for ligand-regulated receptors, it has become clear that in vertebrates a significant number of nuclear receptors are probably not regulated by a ligand (11Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2849) Google Scholar). This is a reflection of the evolutionarily adaptable nature of the nuclear receptor ligand binding domain that has resulted in many instances of loss (and gain) of ligand regulation (12Escriva H. Delaunay F. Laudet V. BioEssays. 2000; 22: 717-727Crossref PubMed Scopus (253) Google Scholar, 13Schwabe J.W. Teichmann S.A. Science's STKE. 2004; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/217/pe4)PubMed Google Scholar, 14Thornton J.W. Need E. Crews D. Science. 2003; 301: 1714-1717Crossref PubMed Scopus (512) Google Scholar). Such receptors are true orphan receptors that are either active with an unoccupied ligand binding cavity (15Sablin E.P. Krylova I.N. Fletterick R.J. Ingraham H.A. Mol. Cell. 2003; 11: 1575-1585Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), have a constitutive structural co-factor in place of a regulatory ligand (16Wisely G.B. Miller A.B. Davis R.G. Thornquest Jr., A.D. Johnson R. Spitzer T. Sefler A. Shearer B. Moore J.T. Willson T.M. Williams S.P. Structure (Lond.). 2002; 10: 1225-1234Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), or simply lack a ligand binding cavity (17Wang Z. Benoit G. Liu J. Prasad S. Aarnisalo P. Liu X. Xu H. Walker N.P. Perlmann T. Nature. 2003; 423: 555-560Crossref PubMed Scopus (470) Google Scholar, 18Baker K.D. Shewchuk L.M. Kozlova T. Makishima M. Hassell A. Wisely B. Caravella J.A. Lambert M.H. Reinking J.L. Krause H. Thummel C.S. Willson T.M. Mangelsdorf D.J. Cell. 2003; 113: 731-742Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). In vertebrates three closely related receptors, Nurr1 (NR4A2), Nur77 (NGFI-B/NR4A1), and Nor1 (NR4A3), belong to this last group of orphans (19Maruyama K. Tsukada T. Ohkura N. Bandoh S. Hosono T. Yamaguchi K. Int. J. Oncol. 1998; 12: 1237-1243PubMed Google Scholar). All three play a role in the central nervous system as well as in other tissues. Much interest has, in particular, focused on Nurr1 because it plays an important role in the development of dopaminergic neurons in the midbrain and may represent a therapeutic target to treat Parkinson's disease (20Buervenich S. Carmine A. Arvidsson M. Xiang F. Zhang Z. Sydow O. Jonsson E.G. Sedvall G.C. Leonard S. Ross R.G. Freedman R. Chowdari K.V. Nimgaonkar V.L. Perlmann T. Anvret M. Olson L. Am. J. Med. Genet. 2000; 96: 808-813Crossref PubMed Google Scholar). We know that this group of receptors lacks a ligand binding cavity, because crystal structures of the ligand binding domain from Nurr1 (17Wang Z. Benoit G. Liu J. Prasad S. Aarnisalo P. Liu X. Xu H. Walker N.P. Perlmann T. Nature. 2003; 423: 555-560Crossref PubMed Scopus (470) Google Scholar) and its orthologue in Drosophila, dHR38 (18Baker K.D. Shewchuk L.M. Kozlova T. Makishima M. Hassell A. Wisely B. Caravella J.A. Lambert M.H. Reinking J.L. Krause H. Thummel C.S. Willson T.M. Mangelsdorf D.J. Cell. 2003; 113: 731-742Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), revealed that the ligand binding cavity is almost entirely filled with bulky hydrophobic side chains. This implies that this family of receptors cannot bind and is not regulated by ligands in the conventional fashion. These structures also revealed that the canonical co-regulator binding groove observed in other receptors is filled with polar side chains and is therefore not likely to support conventional non-polar interactions with co-regulators. This would appear to suggest that the LBD of Nurr1 and related receptors might, at first sight, be inert and play no direct role in the regulation of transcription. However, this is clearly not the case, because the Nurr1 LBD exhibits a potent activation function, albeit dependent upon cell type. Thus, the question is how can the Nurr1 LBD activate transcription when the lack of a conventional co-regulator binding groove would appear to preclude interaction with known co-regulator proteins. The simplest answer to this is that there is another, as yet unidentified co-regulator interaction surface. These issues thus raise two questions: what are the co-regulators that interact with the Nurr1 LBD, and where on the surface of the LBD do they interact? Various interaction studies have shown that the co-repressor proteins SMRT (21Sohn Y.C. Kwak E. Na Y. Lee J.W. Lee S.K. J. Biol. Chem. 2001; 276: 43734-43739Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and PIASγ (22Galleguillos D. Vecchiola A. Fuentealba J.A. Ojeda V. Alvarez K. Gomez A. Andres M.E. J. Biol. Chem. 2004; 279: 2005-2011Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) are able to bind to the Nur77 and Nurr1 LBDs, respectively. An interaction was also inferred with an ASC-2 adaptor protein (21Sohn Y.C. Kwak E. Na Y. Lee J.W. Lee S.K. J. Biol. Chem. 2001; 276: 43734-43739Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Finally, it was also shown that the N-terminal AF1 domains of Nur77 and Nor1 interact directly with their respective LBDs and that this interaction is stabilized by the co-activator protein SRC-2 (23Wansa K.D. Harris J.M. Muscat G.E. J. Biol. Chem. 2002; 277: 33001-33011Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 24Wansa K.D. Harris J.M. Yan G. Ordentlich P. Muscat G.E. J. Biol. Chem. 2003; 278: 24776-24790Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). To identify the surface or surfaces on the Nurr1 LBD that might mediate interaction with co-regulators and the N-terminal AF1 domain, we explored its interactions with peptides from conserved regions of the N-terminal domain of Nurr1 and from the co-repressors SMRT and NCoR (a SMRT homologue). None of these peptides have been reported previously to interact with the Nurr1 LBD, but such interactions seemed to be a possibility given the homology between Nurr1 and both Nur77 and Nor1. The receptor interaction domains from SMRT and NCoR were found to interact with the Nurr1 LBD in pull-down, fluorescence quenching, and NMR binding assays. Calculation of the hydrophobic potential of the surface of the Nurr1 LBD led to the identification of a highly hydrophobic patch/groove between helices 11 and 12 suggestive of an interaction surface. Mutations in this region confirmed its role in co-repressor interaction using both NMR footprinting and pull-down assays. Preparation of Recombinant Nurr1—The uniformly 15N-labeled Nurr1 LBD (amino acids 353–598 preceded by a non-native glycine; molecular mass of 27.8kDa) was expressed with a hexahistidine tag and a tobacco etch virus protease cleavage site at the N terminus using the pET-13a expression vector in the Escherichia coli strain BL21 (DE3). Cells were grown in M9 medium supplemented with 1 g/liter 15NH4Cl, 2 g/liter glucose, nitrogen bases, vitamins, and oligoelements as described previously (25Codina A. Gairi M. Tarrago T. Viguera A.R. Feliz M. Ludevid D. Giralt E. J. Biomol. NMR. 2002; 22: 295-296Crossref PubMed Scopus (6) Google Scholar). Cells were grown at 37 °C to an A600 of 0.6–0.8 and induced with 0.4 mm isopropyl β-d-thiogalactopyranoside. Three hours after induction, cells were harvested and resuspended in lysis buffer (20 mm β-mercaptoethanol, 50 mm NaCl, 20 mm imidazole, 20 mm Tris-HCl, pH 7.4, 0.5 mm 4-(2-aminoethyl)benzenesulphonyl fluoride-HCl, and the complete EDTA-free protease inhibitor tablet (Roche Applied Science)). The lysate was clarified by centrifugation (40,000 × g for 30 min at 4 °C), and the His tag protein was bound to nickel-agarose resin (Qiagen). The resin was washed extensively with wash buffer (50 mm Tris-HCl pH 7.4, 100 mm NaCl, 20 mm imidazole, and 10% glycerol), and the protein was eluted and cleaved from the tag by on-resin His-tagged tobacco etch virus digestion overnight at 4 °C. Cleaved protein was concentrated and purified by size exclusion chromatography using a Sephadex S200 column. The gel filtration buffer (buffer X) was 50 mm sodium phosphate, pH 6.9, 50 mm NaCl, 0.02% NaN3, and protease inhibitors. Nurr1 was further concentrated to 0.3–0.7 mm, and 7% (v/v) D20 was added to the NMR sample. 15N labeled Nurr1 mutants were expressed, purified, and concentrated as described for the wild-type protein. Cell-free Protein Preparation and Peptide Interaction Assays—Proteins were transcribed and translated in vitro in the presence of [35S]methionine using a T7 polymerase/S30 (EcoProRmix T7) extract-coupled transcription/translation system according to the manufacturer's instructions (Novagen). The quality of translation and labeling was monitored using SDS-PAGE and autoradiography. The Nurr1 mutants were prepared using the QuikChange site-directed mutagenesis protocol from Novagen. Vent DNA polymerase was used instead of PfuTurbo and SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, and 20 mM glucose) instead of NZY+ in the transformation step. The mutations were confirmed by DNA sequencing. The peptides were synthesized chemically (Southampton PolyPeptides Ltd). Lyophilized peptides were resuspended in buffer X or in H2O/buffer X mixtures depending on their solubility. The primary amino groups (amino termini and/or lysine side chains) of the synthetic peptides were coupled with an N-hydroxysuccinimideactivated affinity resin according to the manufacturer's instructions (Amersham Biosciences). Peptide interaction assays were carried out by adding the 35S-labeled Nurr1 to the peptide affinity resin in the binding buffer (50 mm sodium phosphate, 50 mm NaCl, 0.02% NaN3, protease inhibitors, and 0.5% Triton X-100) at 4 °C for 1 h. After extensive washing with the same buffer, the protein was eluted from the resin with sample buffer, analyzed by SDS-PAGE, and visualized by autoradiography. NMR—NMR spectra were recorded on Bruker Avance 800 and DMX 600 spectrometers equipped with 5-mm triple resonance (1H-15N-13C) single-axis gradient probes. Data were processed using the program NMRPipe (26Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2741) Google Scholar) and analyzed with the program nmrView (27Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2692) Google Scholar). All NMR data were acquired at 300 K. Titrations were made by adding successive amounts of peptide (CR1, CR1b, and N1-N5) to a solution containing 0.3 mm protein and acquiring HSQC spectra at peptide to protein ratios of 0.5:1, 1:1, 2:1, 4:1, 6:1, and 8:1. The Nurr1 concentration was derived from A280 measurements using an extinction coefficient calibrated by determining the concentration by amino acid analysis on an Amersham Biosciences Biochrom 20-amino acid analyzer after 18 h in 6 m HCl at 110 °C. Peptide concentrations were calculated from dry weight. Fluorescence Quenching Assays—Fluorescence spectra were recorded in a Luminescence Spectrometer LS 50 B (Perkin Elmer) controlled by a personal computer equipped with FL Winlab software (PerkinElmer Life Sciences). Tryptophan quenching was measured at 20 °C. The excitation wavelength was 295 nm, and fluorescence emission scans were recorded between 260 and 450 nm at 200 nm/min. The excitation and emission slit widths were at 5 and 10 nm, respectively. Titrations were made by adding successive amounts of the CR1 peptide to a 500-μl solution containing 11.3 μm Nurr1 in buffer X (the A280 of the solution was between 0.5 and 1 so that the quenching process is linear). The peptide was dissolved in 50 μl of the same protein solution. A three-scan spectrum was recorded after each peptide addition (0–27 μl of the peptide solution). Data were analyzed assuming static quenching using the program Prism® (GraphPad). Peptide Interaction with the Nurr1 LBD—To identify potential interaction surfaces on the Nurr1 LBD, we first sought to identify, using a pull-down assay, short peptides that are able to interact with the Nurr1 LBD. Candidate peptides of a diverse character were derived from the nuclear receptor co-repressors SMRT and NCoR and the N-terminal AF1 domain of Nurr1 (Fig. 1), which have previously been reported to interact with the LBD of this class of receptors (21Sohn Y.C. Kwak E. Na Y. Lee J.W. Lee S.K. J. Biol. Chem. 2001; 276: 43734-43739Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 23Wansa K.D. Harris J.M. Muscat G.E. J. Biol. Chem. 2002; 277: 33001-33011Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). A peptide from the co-activator SRC-1, which is not thought to interact with the Nurr1 LBD (28Castro D.S. Arvidsson M. Bondesson Bolin M. Perlmann T. J. Biol. Chem. 1999; 274: 37483-37490Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), was also included in the assay. The Nurr1 AF1 domain peptides were chosen on the basis of a region of high homology with Nur77 and Nor1. The co-regulator peptides were chosen on the basis of minimal regions required for interaction with other nuclear receptors. Peptides were coupled chemically to N-hydroxysuccinimide-activated Sepharose and challenged with in vitro translated and [35S]methionine-labeled Nurr1 LBD (Fig. 1). The two co-repressor peptides (CR1 and CR2) recruited the Nurr1 LBD more strongly than either of the two controls (resin alone and resin coupled to bacterially expressed Nurr1 LBD). Furthermore, as has been previously reported for Nur77 (21Sohn Y.C. Kwak E. Na Y. Lee J.W. Lee S.K. J. Biol. Chem. 2001; 276: 43734-43739Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), mutation of the four conserved hydrophobic residues in the co-repressor peptide (CR1m) abolishes interaction with the Nurr1 LBD (Fig. 1). This finding suggests that although Nurr1 lacks the canonical non-polar co-regulator interaction groove, the nature of the interaction with the co-repressor peptides might be rather similar to that with other nuclear receptors. As expected, the co-activator peptide CA did not recruit the Nurr1 LBD. Of the peptides derived from the N-terminal domain of Nurr1, only N1 (comprising the 13 N-terminal residues of the receptor) was observed to recruit the LBD. Significantly, this N-terminal peptide bears no sequence similarity to the co-repressor peptides, suggesting that the interaction between this peptide and the Nurr1 LBD might be very different in nature. To further investigate the interaction between the Nurr1 LBD and the various peptides, we examined the effect of adding increasing amounts of a peptide to a 15N-Nurr1 LBD sample and recording a 1H-15N HSQC NMR spectrum at each step of the titration. Although we observed an apparently tight interaction between the N1 peptide and the Nurr1 LBD in the pull-down assay, titrations with this peptide resulted in no discernible perturbations in the 1H-15N HSQC spectra, even at ratios of peptide to protein as high as 8:1. We conclude from this result that the pull-down assay with this peptide is misleading, possibly due to oxidation of the cysteine residue. Several of the other N-terminal peptides did cause some limited spectral perturbations, but because these were only seen at high peptide/LBD ratios and interaction was not observed in the pull-down assay, they were not investigated further. In contrast to the N-terminal peptides, the addition of the CR1 peptide to the 15N-Nurr1 LBD resulted in numerous changes in the 1H-15N HSQC spectrum even at a 1:1 ratio. The 1H-15N HSQC spectra of the Nurr1 LBD contain 191 sharp, well resolved backbone amide resonances as well as a few resolved side chain amide protons. The CR1 peptide caused significant chemical shift perturbations and/or broadening of 56 of these resonances (Fig. 2). The observed changes are reproducible and are essentially identical using peptides derived from either SMRT (CR1) or NCoR (CR1b; inset in Fig. 2). The addition of more peptide induced further changes in the spectrum, suggesting that the binding is not fully saturated at 1:1 and, therefore, that the binding is rather weak (saturation is reached around 8:1). It is also possible that the peptide binds to multiple sites in fast exchange. However, the number of observed spectral changes and the titration profile suggest that the co-repressor peptide is probably interacting with a single site on the surface of the Nurr1 LBD. Notably, many of the spectral changes on peptide addition involve broadening as well as chemical shift perturbations. This broadening suggests that the exchange may be in the intermediate rate regime (29Zuiderweg E.R. Biochemistry. 2002; 41: 1-7Crossref PubMed Scopus (479) Google Scholar). To gain a measure of the binding affinity of the CR1 peptide, we monitored the quenching of tryptophan fluorescence upon the addition of peptide to the Nurr1 LBD in solution (Fig. 3). There are two tryptophan residues in the LBD, residues 420 and 482, both of which are partially exposed to solvent. The titration of bacterially expressed Nurr1 LBD with increasing amounts of the CR1 peptide showed a saturable 30% quench in the total tryptophan fluorescence (Fig. 3a). This finding is most likely due to static (rather than collisional) quenching of one of the tryptophans as a result of peptide binding to the Nurr1 LBD. These data suggest that the peptide is probably interacting with a single site on the surface of the LBD, although it is not possible to rule out binding to multiple sites with similar affinity. The observed Kq (Fig. 3b) indicates a dissociation constant of ∼50 μm, assuming static quenching and single site binding. This dissociation constant is apparently two orders of magnitude higher than that of other measured co-regulator-LBD interactions (30Iannone M.A. Consler T.G. Pearce K.H. Stimmel J.B. Parks D.J. Gray J.G. Cytometry. 2001; 44: 326-337Crossref PubMed Scopus (54) Google Scholar). This finding fits with the observed behavior in the NMR experiments but may indicate that this short peptide does not recapitulate full physiological interaction. It is also possible that, as has been suggested for co-activators, the co-repressor interaction could require other stabilizing factors. However it should be noted that the pull-down assays, which include a stringent washing procedure, show a clear recruitment of the Nurr1 LBD by this peptide. Prediction of a Peptide Binding Surface—Given that the Nurr1 LBD is able to interact with the receptor interaction domains from co-repressor proteins but lacks a conventional co-regulator interaction surface, we wished to understand where on the surface of the LBD the co-repressor peptides are binding. Because we and others have found that the interaction requires the conserved hydrophobic residues in the CoR peptide, we might expect this surface to be largely non-polar. Hydrophobic potential mapping has proven to be a useful way of identifying non-polar interaction surfaces on proteins of known structure (for an example, see Ref. 31Owen D.J. Vallis Y. Noble M.E. Hunter J.B. Dafforn T.R. Evans P.R. McMahon H.T. Cell. 1999; 97: 805-815Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). For such mapping, the energy of a hydrophobic probe is calculated using the program GRID (32Goodford P. J. Chemometrics. 1996; 10: 107-117Crossref Scopus (60) Google Scholar) and displayed as a potential map. Such analysis of the surface of the Nurr1 LBD compared with that of the TR LBD is quite revealing (Fig. 4, a and b). As reported previously (17Wang Z. Benoit G. Liu J. Prasad S. Aarnisalo P. Liu X. Xu H. Walker N.P. Perlmann T. Nature. 2003; 423: 555-560Crossref PubMed Scopus (470) Google Scholar, 18Baker K.D. Shewchuk L.M. Kozlova T. Makishima M. Hassell A. Wisely B. Caravella J.A. Lambert M.H. Reinking J.L. Krause H. Thummel C.S. Willson T.M. Mangelsdorf D.J. Cell. 2003; 113: 731-742Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), the conventional co-regulator interaction surface (labeled Co-reg in Fig. 4a) is quite different between the two receptors; Nurr1 lacks the hydrophobic groove seen in the TR. However, Nurr1 has an alternative groove adjacent to helix 12 that has a strikingly high hydrophobic potential (colored yellow/orange and labeled Nurr1 specific in Fig. 4b). This surface is formed from the side chains of residues Leu-570 and Phe-574 in helix 10/11 and those of residues Phe-592, Leu-593, Leu-596, and Phe-598 in and just C-terminal to helix 12 (Fig. 4, b and c). For comparison, the largely non-polar region of the LBD surface that mediates heterodimerization with the retinoid X receptor is also indicated (labeled Dim in Fig. 4b). If these residues did contribute to a co-regulator interaction surface, it would explain the finding that mutations in or deletion of helix 12 abolishes the activation activity of the Nurr1 LBD (28Castro D.S. Arvidsson M. Bondesson Bolin M. Perlmann T. J. Biol. Chem. 1999; 274: 37483-37490Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In particular, the mutation of residues Asp-589, Phe-592, and Leu-593, which are largely exposed to solvent on this surface, abolish the activity of the LBD. Together, these findings suggest that this surface might constitute a co-regulator binding surface on the Nurr1 LBD. We wished to determine whether this might be the site of interaction of the CoR peptide. Because the peptide binding was found to quench tryptophan fluorescence, we asked whether either of the two tryptophans in Nurr1 is exposed near this surface. Significantly, Try-482 is partially exposed on the surface of the LBD and is within 7.2 and 10.5 Å of residues Leu-570 and Phe-592, respectively. Mapping the Peptide Binding Surface—To test directly whether this highly hydrophobic patch acts as the binding surface for the CoR peptides, we made mutations (F592A, L593A, and F598A) in this region of Nurr1. We then compared the 1H-15N HSQC spectra of each of these mutants with that of the wild-type LBD and finally correlated the observed perturbations due to the site-directed mutations with those resulting from the binding of the CoR peptide. The HSQC NMR spectra of the 15N-labeled mutants indicate that the mutations perturb the backbone amide signals of between 19 and 48 residues. These perturbations are mostly rather small and are not consistent with any significant disruption of the secondary or tertiary structure of the domain. Thus, we can conclude that the structure and stability of the Nurr1 LBD is unaffected by these predominantly surface mutations. Given the close spatial proximity of the mutated residues to one another, it is not surprising that many signals in the spectra are perturbed by more than one mutation. Furthermore, there is a striking overlap between the signals perturbed by the mutations and those perturbed in the 1:1 complex of the wild-type Nurr1 LBD with the CoR-ID1 peptide. This observation is illustrated for a few specific cases by the expansions of HSQC spectra shown in Fig. 5 and, in a more general way, by the statistics summarized in Fig. 6.Fig. 6Perturbations in the NMR spectra due to mutations in the hydrophobic patch overlap perturbations resulting from peptide binding. For the F592A mutant, 14 resonances are perturbed by the mutation but not by peptide binding (blue), 34 resonances are perturbed by both the mutation and peptide binding (blue/red), and two resonances are perturbed by peptide binding but not by the mutation (red). 124 resonances are not perturbed by either peptide binding or by the mutation. There is very significant overlap between the sets of perturbed residues, supporting the conclusion that the mutated residues are very close to or form part of the peptide binding surface.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In Fig. 5, the colored arrowheads indicate some of the cross-peaks that are perturbed on binding peptide and by at least two of the mutations. For instance, the cross-peak labeled by the orange arrowhead (Fig. 5) is greatly broadened on the binding peptide. The 1H frequency of this cross-peak is shifted upf" @default.
• W2066841732 created "2016-06-24" @default.
• W2066841732 creator A5027822011 @default.
• W2066841732 creator A5038630683 @default.
• W2066841732 creator A5043269848 @default.
• W2066841732 creator A5078062860 @default.
• W2066841732 creator A5085340246 @default.
• W2066841732 creator A5090876009 @default.
• W2066841732 date "2004-12-01" @default.
• W2066841732 modified "2023-10-10" @default.
• W2066841732 title "Identification of a Novel Co-regulator Interaction Surface on the Ligand Binding Domain of Nurr1 Using NMR Footprinting" @default.
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