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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.

Antibiotic resistance mediated by constantly evolving beta-lactamases is a serious threat to human health. The mechanism of inhibition of these enzymes by therapeutic beta-lactamase inhibitors is probed using a novel approach involving Raman microscopy and x-ray crystallography. We have presented here the high resolution crystal structures of the beta-lactamase inhibitors sulbactam and clavulanic acid bound to the deacylation-deficient E166A variant of SHV-1 beta-lactamase. Our previous Raman measurements have identified the trans-enamine species for both inhibitors and were used to guide the soaking time and concentration to achieve full occupancy of the active sites. The two inhibitor-bound x-ray structures revealed a linear trans-enamine intermediate covalently attached to the active site Ser-70 residue. This intermediate was thought to play a key role in the transient inhibition of class A beta-lactamases. Both the Raman and x-ray data indicated that the clavulanic acid intermediate is decarboxylated. When compared with our previously determined tazobactam-bound inhibitor structure, our new inhibitor-bound structures revealed an increased disorder in the tail region of the inhibitors as well as in the enamine skeleton. The x-ray crystallographic observations correlated with the broadening of the O-C=C-N (enamine) symmetric stretch Raman band near 1595 cm(-1). Band broadening in the sulbactam and clavulanic acid inter-mediates reflected a heterogeneous conformational population that results from variations of torsional angles in the O-(C=O)-C=C=NH-C skeleton. These observations led us to conclude that the conformational stability of the trans-enamine form is critical for their transient inhibitory efficacy.