Abstract
Hybrid structural methods have been used in recent years to understand protein-protein or protein-ligand interactions where high resolution crystallography or NMR data on the protein of interest has been limited. For G protein-coupled receptors (GPCRs), high resolution structures of native structural forms other than rhodopsin have not yet been achieved; gaps in our knowledge have been filled by creative crystallography studies that have developed stable forms of receptors by multiple means. The neurotransmitter serotonin (5-hydroxytryptamine) is a key GPCR-based signaling molecule affecting many physiological manifestations in humans ranging from mood and anxiety to bowel function. However, a high resolution structure of any of the serotonin receptors has not yet been solved. Here, we used structural mass spectrometry along with theoretical computations, modeling, and other biochemical methods to develop a structured model for human serotonin receptor subtype 4(b) in the presence and absence of its antagonist GR125487. Our data confirmed the overall structure predicted by the model and revealed a highly conserved motif in the ligand-binding pocket of serotonin receptors as an important participant in ligand binding. In addition, identification of waters in the transmembrane region provided clues as to likely paths mediating intramolecular signaling. Overall, this study reveals the potential of hybrid structural methods, including mass spectrometry, to probe physiological and functional GPCR-ligand interactions with purified native protein.
Highlights
From ‡Polgenix Inc., Cleveland, Ohio 44106, the ¶Center for Proteomics and Bioinformatics, Center for Synchrotron Biosciences, and **Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, the ‡‡Department of Molecular Structure, Amgen Inc., Thousand Oaks, California 91320-1799, and ¶¶NeoProteomics Inc., Cleveland, Ohio 44016
Functional Characterization of Purified Receptors—Purified 5-HT4 receptors (5-HT4Rs) samples of both ligand-free and antagonist-bound forms used in this study as analyzed by SDS-PAGE are shown in supplemental Fig. S1
The second messenger produced upon activation of 5-HT4R is cAMP, which was assayed after exposing the 5-HT4R-expressing live cells to saturating concentrations (1 mM) of the synthetic agonist cisapride (Tocris Biosciences, Minneapolis, MN) for 15–20 min at room temperature
Summary
From ‡Polgenix Inc., Cleveland, Ohio 44106, the ¶Center for Proteomics and Bioinformatics, Center for Synchrotron Biosciences, and **Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, the ‡‡Department of Molecular Structure, Amgen Inc., Thousand Oaks, California 91320-1799, and ¶¶NeoProteomics Inc., Cleveland, Ohio 44016. In the context of high resolution crystallographic data on this receptor, we identified sites of conformational change as a function of light-induced activation and discovered that radiolysis could identify oxidized sites adjacent to internal water molecules identified by crystallographic data. We extend these structural MS approaches to an examination of the 5-HT4R subtype b GPCR and its ligand interactions. We conducted radiolytic footprinting of this receptor in the presence and absence of ligand to locate internal water molecules and monitor the conformational dynamics of ligand binding. As our homology model explicitly included structural waters, we compared the footprinting results to these proposed sites of water binding
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