Many drugs that target G-protein-coupled receptors (GPCRs) induce or inhibit their signal transduction with different strengths, which affect their therapeutic properties. However, the mechanism underlying the differences in the signalling levels is still not clear, although several structures of GPCRs complexed with ligands determined by X-ray crystallography are available. Here we utilized NMR to monitor the signals from the methionine residue at position 82 in neutral antagonist- and partial agonist-bound states of β2-adrenergic receptor (β2AR), which are correlated with the conformational changes of the transmembrane regions upon activation. We show that this residue exists in a conformational equilibrium between the inverse agonist-bound states and the full agonist-bound state, and the population of the latter reflects the signal transduction level in each ligand-bound state. These findings provide insights into the multi-level signalling of β2AR and other GPCRs, including the basal activity, and the mechanism of signal transduction mediated by GPCRs.

The 826 G protein-coupled receptors (GPCRs) in the human proteome regulate key physiological processes, and thus have long been attractive as drug targets. With crystal structure determinations of more than 50 different human GPCRs during the last decade, an initial platform for structure-based rational design has been established for drugs that target GPCRs, which is currently being augmented with cryo-EM structures of higher-order GPCR complexes. Nuclear magnetic resonance (NMR) spectroscopy in solution is one of the key approaches for expanding this platform with dynamic features, which can be accessed at physiological temperature and with minimal modification of the wild-type GPCR covalent structures. Here, we review strategies for the use of advanced biochemistry and NMR techniques with GPCRs, survey projects where crystal or cryo-EM structures have been complemented with NMR investigations, and discuss the impact of this integrative approach on GPCR biology and drug discovery. More than 30% of all drugs approved by the US Food and Drug Administration target G protein-coupled receptors (GPCRs)1–3, and these drugs are utilized in a wide range of therapeutic areas, including inflammation and diseases of the central nervous system as well as the cardiovascular, respiratory and gastrointestinal systems2,4. Currently more than 300 agents are in clinical trials, of which around 60 target novel GPCRs for which no drug has as yet been approved2. The novel GPCR targets also include orphan GPCRs, for which endogenous ligands have not yet been discovered2. Overall, the drugs approved so far target only 27% of the human non-olfactory GPCRs2, indicating that much excitement still lies ahead. Identifying new GPCR drugs will need additional detailed knowledge of GPCR biology, especially knowledge from structural biology, given the complex structure–function relationships involved in GPCR signaling. Along this line, recent reviews on GPCRs have covered studies with antibodies5 and nanobodies6, allosteric modulation7–10 biased signaling11–15, methods in GPCR structural biology16–19, GPCR crystal structures20–27 and drug development2,4,28,29, with some reviews addressing specific GPCR families30,31. Complementing the substantial number of GPCR crystal structures that have become available in the past decade, as well as the recent demonstrations of the potential for cryo-EM to provide information on higher-order GPCR complexes32–36, dynamic studies of GPCRs are important for providing new insights into GPCR biology that can assist drug discovery. In this respect, nuclear magnetic resonance (NMR) spectroscopy in solution is a key tool for analysing function-related conformational equilibria in GPCRs as they relate to allosteric coupling, variable efficacies and biased signaling of GPCR ligands, which are of particular interest for their potential as drugs. Furthermore, NMR spectroscopy is also a useful tool for fragment-based lead discovery with GPCR targets. In this article, we first overview the structural biology of GPCRs ba...

The chemokine stromal cell-derived factor-1 (SDF-1/ CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 ␤-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the "fly-casting" mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.Chemokines are a number of small (8 -10 kDa) secreted proteins that direct cell migration in immune systems by activating their receptors expressed on the cell surface (1, 2). The chemokine, stromal cell-derived factor-1 (SDF-1, 2 also known as CXCL12) (3, 4), and its receptor, CXCR4 (5-7), play many essential physiological roles, such as homeostatic regulation of leukocyte traffic, hematopoiesis, and embryonic development (8 -11). The interaction between SDF-1 and CXCR4 also controls cancer metastasis (12, 13), and CXCR4 is a co-receptor for T-tropic strains of human immunodeficiency virus, type 1 (5, 14).The most abundant splice variant of SDF-1 (SDF-1␣) is composed of 68 amino acids, and its NMR (15, 16) and crystal structures (17, 18) demonstrated that SDF-1␣ assumes a typical chemokine fold as follows: an unstructured N terminus (Lys 1 -Tyr 7 ) followed by a long flexible loop (N-loop), a three-stranded anti-parallel ␤-sheet, and an ␣-helix. The mutational analyses revealed that although the SDF-1␣ N terminus is critical for the CXCR4-mediated signaling (15), both the N terminus and the N-loop residues are implicated in the receptor binding (15,18,19). In addition, recent mutational analysis suggested that the residues on the SDF-1␣ ␤-sheet are also important for receptor binding (20).CXCR4, composed of 352 amino acids, belongs to the class A G-protein-coupled receptor (GPCR) family, with the seven transmembrane (TM) helices. Whereas GPCR activation is mediated by the conformation...

G-protein-coupled receptors (GPCRs) exist in conformational equilibrium between active and inactive states, and the former population determines the efficacy of signaling. However, the conformational equilibrium of GPCRs in lipid bilayers is unknown owing to the low sensitivities of their NMR signals. To increase the signal intensities, a deuteration method was developed for GPCRs expressed in an insect cell/baculovirus expression system. The NMR sensitivities of the methionine methyl resonances from the β2 -adrenergic receptor (β2 AR) in lipid bilayers of reconstituted high-density lipoprotein (rHDL) increased by approximately 5-fold upon deuteration. NMR analyses revealed that the exchange rates for the conformational equilibrium of β2 AR in rHDLs were remarkably different from those measured in detergents. The timescales of GPCR signaling, calculated from the exchange rates, are faster than those of receptor tyrosine kinases and thus enable rapid neurotransmission and sensory perception.

The C-terminal region of G-protein-coupled receptors (GPCRs), stimulated by agonist binding, is phosphorylated by GPCR kinases, and the phosphorylated GPCRs bind to arrestin, leading to the cellular responses. To understand the mechanism underlying the formation of the phosphorylated GPCR-arrestin complex, we performed NMR analyses of the phosphorylated β2-adrenoceptor (β2AR) and the phosphorylated β2AR–β-arrestin 1 complex, in the lipid bilayers of nanodisc. Here we show that the phosphorylated C-terminal region adheres to either the intracellular side of the transmembrane region or lipids, and that the phosphorylation of the C-terminal region allosterically alters the conformation around M2155.54 and M2796.41, located on transemembrane helices 5 and 6, respectively. In addition, we found that the conformation induced by the phosphorylation is similar to that corresponding to the β-arrestin-bound state. The phosphorylation-induced structures revealed in this study propose a conserved structural motif of GPCRs that enables β-arrestin to recognize dozens of GPCRs.

G-protein-coupled receptor (GPCR) ligands impart differing degrees of signaling in the G-protein and arrestin pathways, in phenomena called “biased signaling”. However, the mechanism underlying the biased signaling of GPCRs is still unclear, although crystal structures of GPCRs bound to the G protein or arrestin are available. In this study, we observed the NMR signals from methionine residues of the μ-opioid receptor (μOR) in the balanced- and biased-ligand-bound states. We found that the intracellular cavity of μOR exists in an equilibrium between closed and multiple open conformations with coupled conformational changes on the transmembrane helices 3, 5, 6, and 7, and that the population of each open conformation determines the G-protein- and arrestin-mediated signaling levels in each ligand-bound state. These findings provide insight into the biased signaling of GPCRs and will be helpful for development of analgesics that stimulate μOR with reduced tolerance and dependence.

G-protein-coupled receptor (GPCR) ligands impart differing degrees of signaling in the G-protein and arrestin pathways, in phenomena called "biased signaling". However, the mechanism underlying the biased signaling of GPCRs is still unclear,although crystal structures of GPCRs bound to the Gprotein or arrestin are available.I nt his study,w eo bserved the NMR signals from methionine residues of the m-opioid receptor (mOR) in the balanced-and biased-ligand-bound states.Wefound that the intracellular cavity of mOR exists in an equilibrium between closed and multiple open conformations with coupled conformational changes on the transmembrane helices 3, 5, 6, and 7, and that the population of eacho pen conformation determines the G-protein-and arrestin-mediated signaling levels in each ligand-bound state.T hese findings provideinsight into the biased signaling of GPCRs and will be helpful for development of analgesics that stimulate mOR with reduced tolerance and dependence.

CC-chemokine receptor 5 (CCR5) belongs to the G protein-coupled receptor (GPCR) family and plays important roles in the inflammatory response. In addition, its ligands inhibit the HIV infection. Structural analyses of CCR5 have been hampered by its instability in the detergent-solubilized form. Here, CCR5 was reconstituted into high density lipoprotein (rHDL), which enabled CCR5 to maintain its functions for >24 h and to be suitable for structural analyses. By applying the methyl-directed transferred cross-saturation (TCS) method to the complex between rHDL-reconstituted CCR5 and its ligand MIP-1alpha, we demonstrated that valine 59 and valine 63 of MIP-1alpha are in close proximity to CCR5 in the complex. Furthermore, these results suggest that the protective influence on HIV-1 infection of a SNP of MIP-1alpha is due to its change of affinity for CCR5. This method will be useful for investigating the various and complex signaling mediated by GPCR, and will also provide structural information about the interactions of other GPCRs with lipids, ligands, G-proteins, and effector molecules.