Explore the vast range of titles available.

Many approved drugs bind to G protein–coupled receptors (GPCRs). A challenge in targeting GPCRs is that different ligands preferentially activate different signaling pathways. Two papers show how biased signaling arises for the angiotensin II type 1 receptor that couples to two signaling partners (G proteins and arrestins). Suomivuori et al. used large-scale atomistic simulations to show that coupling to the two pathways is through two distinct GPCR conformations and that extracellular ligands favor one or the other conformation. Wingler et al. present crystal structures of the same receptor bound to ligands with different bias profiles. These structures show conformational changes in and around the binding pocket that match those observed in simulations. This work could provide a framework for the rational design of drugs that are more effective and have fewer side effects. Science , this issue p. 881 , p. 888 Atomic-level molecular dynamics reveals how arrestin bias and G protein bias arise at the angiotensin II type 1 receptor. Biased signaling, in which different ligands that bind to the same G protein–coupled receptor preferentially trigger distinct signaling pathways, holds great promise for the design of safer and more effective drugs. Its structural mechanism remains unclear, however, hampering efforts to design drugs with desired signaling profiles. Here, we use extensive atomic-level molecular dynamics simulations to determine how arrestin bias and G protein bias arise at the angiotensin II type 1 receptor. The receptor adopts two major signaling conformations, one of which couples almost exclusively to arrestin, whereas the other also couples effectively to a G protein. A long-range allosteric network allows ligands in the extracellular binding pocket to favor either of the two intracellular conformations. Guided by this computationally determined mechanism, we designed ligands with desired signaling profiles.

Many approved drugs bind to G protein–coupled receptors (GPCRs). A challenge in targeting GPCRs is that different ligands preferentially activate different signaling pathways. Two papers show how biased signaling arises for the angiotensin II type 1 receptor that couples to two signaling partners (G proteins and arrestins). Suomivuori et al. used large-scale atomistic simulations to show that coupling to the two pathways is through two distinct GPCR conformations and that extracellular ligands favor one or the other conformation. Wingler et al. present crystal structures of the same receptor bound to ligands with different bias profiles. These structures show conformational changes in and around the binding pocket that match those observed in simulations. This work could provide a framework for the rational design of drugs that are more effective and have fewer side effects. Science , this issue p. 881 , p. 888 Crystal structures elucidate how ligands select among signaling conformations of a G protein–coupled receptor. Biased agonists of G protein–coupled receptors (GPCRs) preferentially activate a subset of downstream signaling pathways. In this work, we present crystal structures of angiotensin II type 1 receptor (AT1R) (2.7 to 2.9 angstroms) bound to three ligands with divergent bias profiles: the balanced endogenous agonist angiotensin II (AngII) and two strongly β-arrestin–biased analogs. Compared with other ligands, AngII promotes more-substantial rearrangements not only at the bottom of the ligand-binding pocket but also in a key polar network in the receptor core, which forms a sodium-binding site in most GPCRs. Divergences from the family consensus in this region, which appears to act as a biased signaling switch, may predispose the AT1R and certain other GPCRs (such as chemokine receptors) to adopt conformations that are capable of activating β-arrestin but not heterotrimeric G q protein signaling.

G protein-coupled receptors (GPCRs) have evolved to recognize incredibly diverse extracellular ligands while sharing a common architecture and structurally conserved intracellular signaling partners. It remains unclear how binding of diverse ligands brings about GPCR activation, the common structural change that enables intracellular signaling. Here, we identify highly conserved networks of water-mediated interactions that play a central role in activation. Using atomic-level simulations of diverse GPCRs, we show that most of the water molecules in GPCR crystal structures are highly mobile. Several water molecules near the G protein-coupling interface, however, are stable. These water molecules form two kinds of polar networks that are conserved across diverse GPCRs: (i) a network that is maintained across the inactive and the active states and (ii) a network that rearranges upon activation. Comparative analysis of GPCR crystal structures independently confirms the striking conservation of water-mediated interaction networks. These conserved water-mediated interactions near the G protein-coupling region, along with diverse water-mediated interactions with extracellular ligands, have direct implications for structure-based drug design and GPCR engineering.

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 G i1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR–G i/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. Cryo-electron microscopy structures of human neurotensin receptor 1 in complex with G i1 protein and the agonist JMV449 reveal a non-canonical state that may represent an intermediate form in G-protein activation.

Despite intense interest in discovering drugs that cause G-protein-coupled receptors (GPCRs) to selectively stimulate or block arrestin signalling, the structural mechanism of receptor-mediated arrestin activation remains unclear 1 , 2 . Here we reveal this mechanism through extensive atomic-level simulations of arrestin. We find that the receptor’s transmembrane core and cytoplasmic tail—which bind distinct surfaces on arrestin—can each independently stimulate arrestin activation. We confirm this unanticipated role of the receptor core, and the allosteric coupling between these distant surfaces of arrestin, using site-directed fluorescence spectroscopy. The effect of the receptor core on arrestin conformation is mediated primarily by interactions of the intracellular loops of the receptor with the arrestin body, rather than the marked finger-loop rearrangement that is observed upon receptor binding. In the absence of a receptor, arrestin frequently adopts active conformations when its own C-terminal tail is disengaged, which may explain why certain arrestins remain active long after receptor dissociation. Our results, which suggest that diverse receptor binding modes can activate arrestin, provide a structural foundation for the design of functionally selective (‘biased’) GPCR-targeted ligands with desired effects on arrestin signalling. Molecular dynamics simulations and site-directed fluorescence spectroscopy show that the transmembrane core and cytoplasmic tail of G-protein-coupled receptors independently and cooperatively activate arrestin.

The >800 human G protein–coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state- and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here, we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G protein signal transduction. We tested 7800 of 7828 possible single amino acid substitutions to the beta-2 adrenergic receptor (β 2 AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for β 2 AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we identify residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.

More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand–receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR–G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin–G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin–Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR–Gs structures, providing insight into specificity determinants.

The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate the signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists—ligands that trigger GPCR activation—function by causing rearrangement of the GPCR’s transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations that disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate the signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets. Ligands that activate GPCRs generally do so by stabilizing a particular conformation of the transmembrane helices. Here, the authors reveal a distinct activation mechanism where a ligand instead stabilizes a particular intracellular loop conformation.

Volume-regulated anion channels (VRACs) are ubiquitously expressed vertebrate ion channels that open in response to hypotonic swelling. VRACs assemble as heteromers of LRRC8A and LRRC8B-E subunits, with different subunit combinations resulting in channels with different properties. Recent studies have described the structures of LRRC8A:C VRACs, but how other VRACs assemble, and which structural features are conserved or variant across channel assemblies remains unknown. Herein, we used cryo-EM to determine structures of a LRRC8A:D VRAC with a 4:2 subunit stoichiometry, which we captured in two conformations. The presence of LRRC8D subunits widens and increases hydrophobicity of the selectivity filter, which may contribute to the unique substrate selectivity of LRRC8D-containing VRACs. The structures reveal lipids bound inside the channel pore, similar to those observed in LRRC8A:C VRACs. We observe that LRRC8D subunit incorporation disrupts packing of the cytoplasmic LRR domains, increasing channel dynamics and opening lateral intersubunit gaps, which we speculate are necessary for pore lipid evacuation and channel activation. Molecular dynamics simulations show that lipids can reside stably within the pore to close the channel. Using electrophysiological experiments, we confirmed that pore lipids block conduction in the closed state, demonstrating that lipid-gating is a general property of VRACs. VRACs are ubiquitously expressed osmosensitive ion channels assembled from LRRC8A-E subunits. Here, the authors determine the structures of a LRRC8A:D VRAC using cryo-EM and identified that these channels are gated by lipids inside the channel pore.

Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.