Extracellular ligand binding to G protein-coupled receptors (GPCRs) modulates G-protein and β-arrestin signaling by changing the conformational states of the cytoplasmic region of the receptor. Using site-specific 19F-NMR labels in the β2-adrenergic receptor (β2AR) in complexes with various ligands, we observed that the cytoplasmic ends of helices VI and VII adopt two major conformational states. Changes in the NMR signals reveal that agonist binding primarily shifts the equilibrium towards the G protein specific active state of helix VI. In contrast, β-arrestin-biased ligands predominantly impact the conformational states of helix VII. The selective effects of different ligands on the conformational equilibria involving helices VI and VII provide insights into the long-range structural plasticity of β2AR in partial and biased agonist signaling.

Odorants are transmitted by small hydrophobic molecules that cross the aqueous sensillar lymph surrounding the dendrites of the olfactory neurons to stimulate the olfactory receptors. In insects, the transport of pheromones, which are a special class of odorants, is mediated by pheromone-binding proteins (PBPs), which occur at high concentrations in the sensillar lymph. The PBP from the silk moth Bombyx mori (BmPBP) undergoes a pH-dependent conformational transition between the forms BmPBP A present at pH 4.5 and BmPBP B present at pH 6.5. Here, we describe the NMR structure of BmPBP A , which consists of a tightly packed arrangement of seven ␣-helices linked by well defined peptide segments and knitted together by three disulfide bridges. A scaffold of four ␣-helices that forms the ligand binding site in the crystal structure of a BmPBP-pheromone complex is preserved in BmPBP A . The C-terminal dodecapeptide segment, which is in an extended conformation and located on the protein surface in the pheromone complex, forms a regular helix, ␣7, which is located in the pheromone-binding site in the core of the unliganded BmPBP A . Because investigations by others indicate that the pH value near the membrane surface is reduced with respect to the bulk sensillar lymph, the pH-dependent conformational transition of BmPBP suggests a novel physiological mechanism of intramolecular regulation of protein function, with the formation of ␣7 triggering the release of the pheromone from BmPBP to the membrane-standing receptor. Male moths have an exquisitely sensitive olfactory system capable of detecting over great distances single molecules of pheromones emitted by the female, and they are capable of distinguishing highly selectively between closely similar compounds or isomers (1). These capabilities of male moths are because of olfactory sensory organs that consist of large branched antennae and can readily be isolated for biochemical characterization or electrophysiological recordings (2). The surface of the antennae is covered with hairlike protrusions composed of cuticle, which form a sheath (sensillum) surrounding one or several dendrite endings from olfactory neurons. The dendrites in each sensillum are bathed in a special fluid, the sensillum lymph. Odorant molecules gain access to the dendritic membrane through pores penetrating the sensillum wall and then crossing the sensillar lymph to interact with G proteincoupled olfactory receptors, which form a large eukaryotic protein family. Upon interaction with the olfactory receptors, a cascade of events leads to a change of the membrane potential (3), resulting in opening of ion channels and depolarization.Common odorants are hydrophobic molecules that are poorly soluble in aqueous media, such as the sensillar lymph. Odorants overcome this barrier by binding to odorant-binding proteins (OBPs). OBPs are present at high concentrations in the lymph (up to 10 mM), bind the odorant, and transport it from the sensillum pore wall to the receptor at the dendritic membrane. It is ...

Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A adenosine receptor (AAR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone N-H NMR signals in AAR were individually assigned. These NMR probes provided insight into the role of Asp52 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 246, and delineated the structural response to variable efficacy of bound drugs across AAR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp52.