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, 5-HT) 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) (5-HT4R) 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 intra-molecular 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.
This paper was published online in Molecular and Cellular Proteomics and is free to access.