Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Structural Insights into the Process of GPCR-G Protein Complex Formation.

The crystal structure of the ß2-adrenergic receptor (ß2AR) bound to the G protein adenylyl cyclase stimulatory G protein (Gs) captured the complex in a nucleotide-free state (ß2AR-Gsempty). Unfortunately, the ß2AR-Gsempty complex does not provide a clear explanation for G protein coupling specificity. Evidence from several sources suggests the existence of a transient complex between the ß2AR and GDP-bound Gs protein (ß2AR-GsGDP) that may represent an intermediate on the way to the formation of ß2AR-Gsempty and may contribute to coupling specificity. Here we present a structure of the ß2AR in complex with the carboxyl terminal 14 amino acids from Gαs along with the structure of the GDP-bound Gs heterotrimer. These structures provide evidence for an alternate interaction between the ß2AR and Gs that may represent an intermediate that contributes to Gs coupling specificity.

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

Assembly of a GPCR-G Protein Complex.

The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.

Structure of a Signaling Cannabinoid Receptor 1-G Protein Complex.

Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Activation of CB1-Gi signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of Gi activation by CB1. Here, we present the structure of the CB1-Gi signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in Gi. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.

Structural Insights into the Dynamic Process of ß2-Adrenergic Receptor Signaling.

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 ß2-adrenergic receptor (ß2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound ß2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward 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.

Time-resolved cryo-EM of G-protein activation by a GPCR.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the ß2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.

Conformational transitions of a neurotensin receptor 1-Gi1 complex.

Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.

Structural mechanisms of selectivity and gating in anion channelrhodopsins.

Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anion channels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.

Structure of the µ-opioid receptor-Gi protein complex.

The µ-opioid receptor (µOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by µOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the µOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the µOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR.

A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides. The library was created based on the general idea of peptide synthesis, that is, amino acid mimics were reacted in silico to form the dipeptide mimics, yielding 2,036,819 unique compounds. After docking calculations, two compounds from the library were synthesized and tested against WD repeat-containing protein 5 (WDR5) and histamine receptors H1-H4 to evaluate whether these molecules are viable in assays. The compounds showed the highest potency at the histamine H3 receptor, with Ki values in the two-digit micromolar range.

Single-molecule analysis of ligand efficacy in ß2AR-G-protein activation.

G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-molecule fluorescence resonance energy transfer imaging, we examine TM6 movements in the ß2 adrenergic receptor (ß2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound ß2AR-Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound ß2AR-Gs species that are distinct from known structures, and provide single-molecule perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.

Viewing rare conformations of the ß2 adrenergic receptor with pressure-resolved DEER spectroscopy.

The ß2 adrenergic receptor (ß2AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The ß2AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free ß2AR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in ß2AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

Structure of active ß-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ß-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ß-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ß-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ß-arrestin-1. The structure of the ß-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ß-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of ß-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ß-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

Initial steps of photosystem II de novo assembly and preloading with manganese take place in biogenesis centers in Synechocystis.

In the cyanobacterium Synechocystis sp PCC 6803, early steps in thylakoid membrane (TM) biogenesis are considered to take place in specialized membrane fractions resembling an interface between the plasma membrane (PM) and TM. This region (the PratA-defined membrane) is defined by the presence of the photosystem II (PSII) assembly factor PratA (for processing-associated TPR protein) and the precursor of the D1 protein (pD1). Here, we show that PratA is a Mn(2+) binding protein that contains a high affinity Mn(2+) binding site (K(d) = 73 µM) and that PratA is required for efficient delivery of Mn(2+) to PSII in vivo, as Mn(2+) transport is retarded in pratA(-). Furthermore, ultrastructural analyses of pratA(-) depict changes in membrane organization in comparison to the wild type, especially a semicircle-shaped structure, which appears to connect PM and TM, is lacking in pratA(-). Immunogold labeling located PratA and pD1 to these distinct regions at the cell periphery. Thus, PratA is necessary for efficient delivery of Mn(2+) to PSII, leading to Mn(2+) preloading of PSII in the periplasm. We propose an extended model for the spatial organization of Mn(2+) transport to PSII, which is suggested to take place concomitantly with early steps of PSII assembly in biogenesis centers at the cell periphery.

Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment.

Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H2R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. Structural comparison with the inactive conformation of H2R and the inactive and Gq-coupled active state of H1R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H2R, its agonist-dependent internalization and its interaction with endogenously synthesized H1R and H2R in HEK293 cells by applying a recently developed nanotransfer technique.

Time-resolved cryo-EM of G protein activation by a GPCR.

G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating the exchange of guanine nucleotide in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G protein complex. Using variability analysis to monitor the transitions of the stimulatory Gs protein in complex with the ß 2 -adrenergic receptor (ß 2 AR) at short sequential time points after GTP addition, we identified the conformational trajectory underlying G protein activation and functional dissociation from the receptor. Twenty transition structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of events driving G protein activation upon GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα Switch regions and the α5 helix that weaken the G protein-receptor interface. Molecular dynamics (MD) simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP upon closure of the alpha-helical domain (AHD) against the nucleotide-bound Ras-homology domain (RHD) correlates with irreversible α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signaling events.

Allosteric modulation of GPCRs: From structural insights to in silico drug discovery.

G protein-coupled receptors (GPCRs) play critical roles in human physiology and are one of the prime targets for marketed drugs. While traditional drug discovery programs have focused on the development of ligands targeting the binding site of endogenous ligands (orthosteric site), allosteric modulators offer new avenues for the regulation of GPCR function with potential therapeutic benefits. Recent advances in the structure determination of GPCRs bound to different types of allosteric modulators have led to the identification of multiple allosteric sites and significantly enhanced our understanding of how allosteric ligands interact with receptors. These structural insights, together with the plethora of GPCR structures available today, will facilitate structure-based discovery and development of allosteric modulators as novel therapeutic candidates. In this review, we provide a systematic analysis of the currently available GPCR structures in complex with small-molecule allosteric ligands in terms of the location of allosteric pockets, receptor-ligand interactions, and the chemical features of the allosteric modulators. In addition, we summarize current strategies for the identification of allosteric sites as well as ligand-based and structure-based drug discovery and design.

Biochemical Characterization of Cell-free Synthesized Human ß1 Adrenergic Receptor Cotranslationally Inserted into Nanodiscs.

Cell-free expression enables direct cotranslational insertion of G protein coupled receptors (GPCRs) and other membrane proteins into the defined membrane environments of nanodiscs. This technique avoids GPCR contacts with detergents and allows rapid identification of lipid effects on GPCR function as well as fast screening of receptor derivatives. Critical steps of conventional GPCR preparation from cellular membranes followed by detergent-based reconstitution into nanodisc membranes are thus eliminated. We report the efficient cotranslational insertion of full-length human ß1-adrenergic receptor and of a truncated derivative into preformed nanodisc membranes. Their biochemical characterization revealed significant differences in lipid requirements, dimer formation and ligand binding activity. The truncated receptor showed a higher affinity to most tested ligands, in particular in presence of choline-containing lipids. However, introducing the naturally occurring G389R polymorphism in the full-length receptor resulted into an increased affinity to the antagonists alprenolol and carvedilol. Receptor quality was generally improved by coexpression with the agonist isoproterenol and the percentage of the ligand binding active fraction was twofold increased. Specific coupling of full-length and truncated human receptors in nanodisc membranes to Mini-Gαs protein as well as to purified Gs heterotrimer could be demonstrated and homogeneity of purified GPCR/Gs protein complexes in nanodiscs was demonstrated by negative stain single particle analysis.

The role of structural dynamics in GPCR-mediated signaling.

G protein-coupled receptors (GPCRs) play critical roles in the regulation of human physiology in response to a wide array of different extracellular stimuli and thus represent one of the largest groups of therapeutic drug targets. Recent advances in the structural characterization of GPCRs in different conformations and in complex with G proteins and arrestins have provided important insights into the mechanism and function of GPCRs. However, in order to truly understand the molecular basis of the functional versatility of GPCRs, the structural snapshots obtained by X-ray crystallography or cryo-EM need to be complimented with information about the conformational dynamics of receptors and their signaling complexes. In the last decade, a combination of biophysical approaches and computational studies has been utilized to examine the molecular motions of GPCRs and their transducer complexes and how they are regulated by ligands of different efficacy and bias. These studies revealed that GPCRs are highly dynamic allosteric proteins that can sample multiple conformational states. Ligands with distinct signaling profiles not only impact the conformational landscape of GPCRs but also of the receptor-engaged G proteins and arrestins. The conformational dynamics of GPCRs and their signaling complexes and the ligand-dependent bias sampling of distinct functional states are important underlying principles behind the complex signaling behavior of GPCRs.