G protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β2-adrenergic receptor (β2AR), a prototypical GPCR. We labeled β2AR with 13CH3ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β2AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β2AR’s ability to engage multiple signaling and regulatory proteins.

SUMMARY G protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using 19F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium towards a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate and active states. Complete transition to the active conformation requires subsequent interaction with a G-protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease.

G protein coupled receptors (GPCRs) are seven transmembrane proteins that mediate the majority of cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs1,2,3,4,5 reveal structural conservation extending from the orthosteric ligand binding site in the transmembrane core to the cytoplasmic G protein coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse, and therefore represents an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops (ECLs) 2 and 3. Small molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G protein activation (agonist, neutral antagonist, and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide new insight into the dynamic behavior of GPCRs not addressable by static, inactive-state crystal structures.

b S Supporting Information ' INTRODUCTIONColloidal nanomaterials show unique properties and are widely explored for a variety of applications. 1À4 Lanthanidebased nanomaterials have versatile utility in biological applications, as they can be made either as luminescent, magnetic, or as dual probe by selective doping of lanthanide ions. 5 In particular, paramagnetic Gd 3+ -doped NPs show tremendous potential as contrast agents (CAs) for magnetic resonance imaging (MRI). 6,7 MRI is a powerful medical diagnostic tool, where the relaxation of water protons exposed to an external magnetic field is used to obtain morphological and anatomical information with unlimited tissue penetration and yet high spatial resolution. 8 CAs are used to improve the sensitivity, because they interact with the surrounding water protons and shorten their relaxation time to provide better contrast. Two types of CAs are clinically prevalent: (i) paramagnetic Gd 3+ chelates, which affect the longitudinal relaxivity (r 1 ), and are termed positive (T 1 ) CAs, because they enhance the contrast; 9 and (ii) superparamagnetic iron oxide (SPIO) NPs, which affect transverse relaxivity (r 2 ) and are referred to as negative (T 2 ) CAs, because they diminish the signal intensity at the region of interest. 7,10 T 1 contrast agents are preferred over the T 2 agents as their enhanced brightening effect can easily be used to differentiate the signal from other pathogenic or biological conditions. 7 Gd 3+ chelates that are used clinically have very low body circulation time, because of their low molecular weight and show limitations as molecular probes for long-term tracking. 6 They also provide very low local contrast, because each chelate has only one Gd 3+ ion. To increase the local contrast and relaxivity, second-generation agents have been developed by covalently anchoring Gd 3+ chelates to different nanostructure frameworks, 11 or bundling multiple Gd 3+ chelates together using polymers, dendrimers, liposomes, and viral capsids. 12 These structures have been shown to have high relaxivity and increased local contrast as multiple Gd 3+ ions are coupled to a single nanostructure. The main disadvantage of this class of agents concerns their functionalization, which is tedious, expensive, and the number of ions that can be loaded to a NP is further limited by the number of anchoring sites available. Moreover, some of these aggregates are too large to be clinically useful. 6,7 Recently,