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Two-thirds of human hormones and one-third of clinical drugs activate ~350 G-protein-coupled receptors (GPCR) belonging to four classes: A, B1, C and F. Whereas a model of activation has been described for class A, very little is known about the activation of the other classes, which differ by being activated by endogenous ligands bound mainly or entirely extracellularly. Here we show that, although they use the same structural scaffold and share several ‘helix macroswitches’, the GPCR classes differ in their ‘residue microswitch’ positions and contacts. We present molecular mechanistic maps of activation for each GPCR class and methods for contact analysis applicable for any functional determinants. This provides a superfamily residue-level rationale for conformational selection and allosteric communication by ligands and G proteins, laying the foundation for receptor-function studies and drugs with the desired modality. Comparative analysis of inactive/active-state structures reveals molecular mechanistic maps of activation of the major GPCR classes. The findings and new approaches lay the foundation for targeted receptor-function studies and drugs with desired modalities.

A highly conserved rearrangement of residue contacts functions as a common step in the activation pathways of diverse G-protein-coupled receptors. Structural convergence in GPCRs A comprehensive structural analysis of 27 class A G-protein-coupled receptors (GPCRs) reveals that, despite the extensive diversity in the activation pathways between receptors, the pathways converge near the G-protein-coupling region. The convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices. These findings may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins, and will have implications for the modelling and engineering of GPCRs for structure-based drug discovery. Class A G-protein-coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions, including vision, neurotransmission and immune responses 1 , 2 , 3 , 4 . They are the targets of nearly one-third of all prescribed medicinal drugs 5 such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins 3 , 6 . Structural rearrangements of residue contacts in the transmembrane domain serve as ‘activation pathways’ that connect the ligand-binding pocket to the G-protein-coupling region within the receptor. In order to investigate the similarities in activation pathways across class A GPCRs, we analysed 27 GPCRs from diverse subgroups for which structures of active, inactive or both states were available. Here we show that, despite the diversity in activation pathways between receptors, the pathways converge near the G-protein-coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins.

Although several X-ray crystal structures of G protein-coupled receptors (GPCRs) have been reported, relatively little is known about the conformational dynamics of these important membrane proteins; here, the authors used NMR spectroscopy to monitor the conformational changes that occur in the turkey β1-adrenergic receptor in the presence of antagonists, partial agonists, and full agonists. Flexibility in G protein-coupled receptors G protein-coupled receptors (GPCRs) are membrane proteins that are involved in many biological processes and are important drug targets. Although several X-ray crystal structures of GPCRs have been reported in the past few years, relatively little is known about their conformational dynamics. In this manuscript, the authors used NMR spectroscopy to monitor the conformational changes that occur in the turkey β 1 -adrenergic receptor in the presence of antagonists, partial agonists and full agonists. The authors identified several allosteric signalling pathways and determined that agonist binding to this GPCR initiates the bending of a key transmembrane helix towards the 'active' conformation observed in the 2011 X-ray crystal structure of the β 2 -adrenergic receptor/G protein complex. G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography 1 , 2 , 3 , the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR 4 , but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained 5 , 6 , 7 , 8 , 9 . Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey β 1 -adrenergic receptor (β 1 AR) 10 , 11 , 12 . Labelling with [ 15 N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental method to delineate signal transmission networks at high resolution in GPCRs.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways. Beta-arrestins play central roles in the mechanisms regulating GPCR signalling and trafficking. Here the authors identify a selective inhibitor of the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP-2, which they use to dissect the role of the β-arrestin/β2-adaptin interaction in GPCR signalling.

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: ‘key sites’ required for arrestin binding and activation, an ‘inhibitory site’ that abrogates arrestin binding, and ‘modulator sites’ that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell. The cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Here, authors use a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods.

Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures.

Tetrahydrocannabinol (THC) is the principal psychoactive compound derived from the cannabis plant Cannabis sativa and approved for emetic conditions, appetite stimulation and sleep apnea relief. THC’s psychoactive actions are mediated primarily by the cannabinoid receptor CB 1 . Here, we determine the cryo-EM structure of HU210, a THC analog and widely used tool compound, bound to CB 1 and its primary transducer, G i1 . We leverage this structure for docking and 1000 ns molecular dynamics simulations of THC and 10 structural analogs delineating their spatiotemporal interactions at the molecular level. Furthermore, we pharmacologically profile their recruitment of G i and β-arrestins and reversibility of binding from an active complex. By combining detailed CB 1 structural information with molecular models and signaling data we uncover the differential spatiotemporal interactions these ligands make to receptors governing potency, efficacy, bias and kinetics. This may help explain the actions of abused substances, advance fundamental receptor activation studies and design better medicines. Tetrahydrocannabinol (THC) analogs target the cannabinoid receptor CB1 for therapeutic and psychoactive effects. Here, the authors determine spatiotemporal ligand interactions critical for potency, efficacy, off-rates and drug design.

The tumor suppressor p53 is mutationally inactivated in [almost equal to]50% of human cancers. Approximately one-third of the mutations lower the melting temperature of the protein, leading to its rapid denaturation. Small molecules that bind to those mutants and stabilize them could be effective anticancer drugs. The mutation Y220C, which occurs in [almost equal to]75,000 new cancer cases per annum, creates a surface cavity that destabilizes the protein by 4 kcal/mol, at a site that is not functional. We have designed a series of binding molecules from an in silico analysis of the crystal structure using virtual screening and rational drug design. One of them, a carbazole derivative (PhiKan083), binds to the cavity with a dissociation constant of [almost equal to]150 μM. It raises the melting temperature of the mutant and slows down its rate of denaturation. We have solved the crystal structure of the protein-PhiKan083 complex at 1.5-Å resolution. The structure implicates key interactions between the protein and ligand and conformational changes that occur on binding, which will provide a basis for lead optimization. The Y220C mutant is an excellent \"druggable\" target for developing and testing novel anticancer drugs based on protein stabilization. We point out some general principles in relationships between binding constants, raising of melting temperatures, and increase of protein half-lives by stabilizing ligands.

G protein-coupled receptors (GPCRs) are important therapeutic drug targets for a wide range of diseases. Upon activation, GPCRs can initiate several signaling pathways, each with unique therapeutic implications. Therefore, understanding how drugs selectively engage specific signaling pathways becomes paramount. However, achieving this selectivity remains highly challenging. To unravel the underlying multifaceted mechanisms, we integrate systematic mutagenesis of the CB 2 R, comprehensive profiling of G αi2 and β-arrestin1 engagements and computer simulations to track the effects of mutations on receptor dynamics. Our research reveals multiple triggers within a complex allosteric communication network (ACN) that converge to preferential CB 2 R coupling by modulating evolutionarily conserved motifs. Utilizing network path analysis, we find that potent triggers are typically highly connected nodes and are located near regions of high information transmission within the ACN. Our insights highlight the complexity of GPCR signaling and provide a framework for the rational design of drug candidates tailored to evoke specific functional responses, ultimately enhancing the precision and efficacy of therapeutic interventions. Nonselective engagement of GPCR signaling pathways by GPCR-targeting drugs can reduce treatment efficacy and cause side effects. The authors show that signaling selectivity in CB2R can be tuned by reshaping allosteric networks, offering insights for more precise therapies.

A new crystal structure of BtuCD, a bacterial ABC transporter that uses ATP hydrolysis to drive vitamin B12 uptake, bound to an AMP-PNP nucleotide, completes the structural elucidation of intermediates in the transport cycle and reveals how ATP accelerates transport. The reaction mechanism of BtuCD–F–catalyzed vitamin B12 transport into Escherichia coli is currently unclear. Here we present the structure of the last missing state in the form of AMP-PNP–bound BtuCD, trapped by a disulfide cross-link. Our structural and biochemical data allow a consistent mechanism to be formulated, thus rationalizing the roles of substrate, ATP and substrate-binding protein.