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Using nondenaturing mass spectrometry to detect fortuitous ligands in orphan nuclear receptors

¹ Laboratoire de Spectrométrie de Masse Bio-Organique, Ecole Européenne de Chimie, Polymères et Matériaux, CNRS UMR7509, 67087 Strasbourg, France
² Département de Biologie et de Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR7104, 67404 Illkirch, France

Reprint requests to: Alain van Dorsselaer (mass spectrometry), Laboratoire de Spectrométrie de Masse Bio-Organique, Ecole Européenne de Chimie, Polymères et Matériaux, CNRS UMR7509, 25 rue Becquerel, 67087 Strasbourg, France; e-mail: vandors{at}chimie.u-strasbg.fr; fax: 33-3-90-24-2781; or Dino Moras (biology), Département de Biologie et de Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR7104, 1 rue Laurent Fries B.P.10142, 67404 Illkirch, France; e-mail: moras{at}igbmc.u-strasbg.fr; fax: 33-3-88-65-3276.

Nondenaturing electrospray mass spectrometry (ESI-MS) has been used to reveal the presence of potential ligands in the ligand-binding domain (LBD) of orphan nuclear receptors. This new approach, based on supramolecular mass spectrometry, allowed the detection and identification of fortuitous ligands for the retinoic acid-related orphan receptor ß (RORß) and the ultraspiracle protein (USP). These fortuitous ligands were specifically captured from the host cell with the proper stoichiometry. After organic extraction, these molecules have been characterized by classic analytical methods and identified as stearic acid for RORß and a phosphatidylethanolamine (PE) for USP, as confirmed by crystallography. These molecules act as "fillers" and may not be the physiological ligands, but they prove to be essential to stabilize the active conformation of the LBD, enabling its crystallization. The resulting crystal structures provide a detailed picture of the ligand-binding pocket, allowing the design of highly specific synthetic ligands that can be used to characterize the function of orphan nuclear receptors. An additional advantage of this new method is that it is not based on a functional test and that it can detect low-affinity ligands.

Keywords: Nuclear receptor; orphan receptor; ROR; USP; fortuitous ligand; electrospray mass spectrometry, noncovalent interactions

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