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 β (RORβ) 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 RORβ 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.
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