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In prion diseases, the cellular prion protein PrPCis converted into aggregates of PrPSc, leading to profound neurotoxicity through largely unknown mechanisms. Here we report that the cellular prion protein PrPCacts as an antagonist of the adhesion G protein-coupled receptor (GPCR) Adgrd1. When overexpressed in cultured cells, Adgrd1 recruited the G-protein Gαs, inducing excessive cytosolic cAMP, growth arrest and cytotoxicity, all of which were suppressed by FT25-50, a 26-meric peptide from the N-terminal flexible tail (FT) of PrPC. We found that FT25-50forms a complex with Adgrd1 and suppresses its intrinsic activation by the Stachel peptide. Adgrd1 ablation attenuated the neurodegeneration of prion-infected cerebellar organotypic slice cultures and prolonged the healthspan of prion-infected mice. Interaction studies with mutated proteins, computational modeling and docking studies revealed that suppression of Adgrd1 signaling requires the polybasic domain of the FT and the N-terminal fragment of Adgrd1. In the absence of PrPC, the cAMP spike caused by Adgrd1 was suppressed by co-expression of a functionally dead Adgrd1-Adgrg6 chimeric receptor, suggesting that Adgrd1 activation requires an unidentified agonistic ligand displaced by FT25-50. These results identify Adgrd1 as a mediator of prion toxicity and suggest that Adgrd1 modulators may be beneficial against prion-related neurodegeneration.

G protein-coupled receptors (GPCRs) can engage distinct subsets of signaling pathways, but the structural determinants of this functional selectivity remain elusive. The naturally occurring genetic variants of GPCRs, selectively affecting different pathways, offer an opportunity to explore this phenomenon. We previously identified 40 coding variants of the MTNR1B gene encoding the melatonin MT2 receptor (MT2). These mutations differently impact the β-arrestin 2 recruitment, ERK activation, cAMP production, and Gαi1 and Gαz activation. In this study, we combined functional clustering and structural modeling to delineate the molecular features controlling the MT2 functional selectivity. Using non-negative matrix factorization, we analyzed the signaling signatures of the 40 MT2 variants yielding eight clusters defined by unique signaling features and localized in distinct domains of MT2. Using computational homology modeling, we describe how specific mutations can selectively affect the subsets of signaling pathways and offer a proof of principle that natural variants can be used to explore and understand the GPCR functional selectivity.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation., 視覚に関わるタンパク質の超高速分子動画 --薄暗いところで光を感じる仕組み--. 京都大学プレスリリース. 2023-03-23.

Tumor targeting with bivalent radiolabeled ligands for GPCRs is an attractive means for cancer imaging and therapy. Here, we studied and compared the distance dependence of homobivalent ligands for the human gastrin-releasing peptide receptor (hGRP-R) and the somatostatin receptor subtype II (hSstR2a). Oligoprolines were utilized as molecular scaffolds to enable distances of 10, 20, or 30 Å between two identical, agonistic recognition motifs. In vitro internalization assays revealed that ligands with a distance of 20 Å between the recognition motifs exhibit the highest cellular uptake in both ligand series. Structural modeling and molecular dynamics simulations support an optimal distance of 20 Å for accommodating ligand binding to both binding sites of a GPCR dimer. Translation of these findings to the significantly higher complexity in vivo proved difficult and showed only for the hGRP-R increased tumor uptake of the bivalent ligand. © 2020 American Chemical Society. ISSN:1043-1802 ISSN:1520-4812

MG11 is a truncated analog of minigastrin, a peptide with high affinity and specificity toward the cholecystokinin-2 receptor (CCK2R), which is overexpressed by different tumors. Thus, radiolabeled MG11 derivatives have great potential for use in cancer diagnosis and therapy. A drawback of MG11 is its fast degradation by proteases, leading to moderate tumor uptake in vivo. We introduced 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres to replace labile amide bonds of the peptide. The "triazole scan" yielded peptidomimetics with improved resistance to enzymatic degradation and/or enhanced affinity toward the CCK2R. Remarkably, our lead compound achieved a 10-fold increase in receptor affinity, resulting in a 2.6-fold improved tumor uptake in vivo. Modeling of the ligand-CCK2R complex suggests that an additional cation-π interaction of the aromatic triazole moiety with the Arg356 residue of the receptor is accountable for these observations. We show for the first time that the amide-to-triazole substitution strategy offers new opportunities in drug development that go beyond the metabolic stabilization of bioactive peptides.

Advances in singe-particle cryo-electron microscopy (cryo-EM) have made possible to solve the structures of numerous Family A and Family B G protein coupled receptors (GPCRs) in complex with G proteins and arrestins, as well as several Family C GPCRs. Determination of these structures has been facilitated by the presence of large extra-membrane components (such as G protein, arrestin, or Venus flytrap domains) in these complexes that aid in particle alignment during processing of the cryo-EM data. In contrast, determination of the inactive state structure of Family A GPCRs is more challenging due to the relatively small size of the seven transmembrane domain (7TM) and to the surrounding detergent micelle that, in the absence of other features, make particle alignment impossible. Here we describe an alternative protein engineering strategy where the heterodimeric protein calcineurin is fused to a GPCR by three points of attachment, the cytoplasmic ends of TM5, TM6 and TM7. This three-point attachment provides a more rigid link with the GPCR transmembrane domain that facilitates particle alignment during data processing, allowing us to determine the structures of the β2 adrenergic receptor (β2AR) in the apo, antagonist-bound, and agonist-bound states. We expect that this fusion strategy may have broad application in cryo-EM structural determination of other Family A GPCRs.

G protein-coupled receptors (GPCRs) are one of the most important drug-target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-A distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-angstrom distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-angstrom distance have different potencies toward beta-arrestin-1 and beta-arrestin-2, and 3) the divalent agonistic ligand with 10-angstrom distance specifically reduces Gq potency without affecting beta-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling., Proceedings of the National Academy of Sciences of the United States of America, 118 (48), ISSN:0027-8424, ISSN:1091-6490

Significance G protein–coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Upon ligand binding, GPCRs initiate intracellular signaling pathways by interacting with partner proteins. Assays that quantify the interplay between ligand binding and initiation of downstream signaling cascades are critical in the early stages of drug development. We have developed a high-throughput mass spectrometry method to unravel GPCR–protein complex interplay and demonstrated its use with three GPCRs to provide quantitative information about ligand-modulated coupling selectivity. This method provides insights into the molecular details of GPCR interactions and could serve as an approach for discovery of drugs that initiate specific cell-signaling pathways., G protein–coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR–G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand–GPCR–partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [β1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using β1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαiβγ and β-arrestin-1 and showed that carvedilol induces an increase in coupling of β-arrestin-1 and Gαiβγ to β1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.

G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gα

International audience; Melatonin is a hormone mainly produced by the pineal gland and MT1 is one of the two G protein-coupled receptors (GPCRs) mediating its action. Despite an increasing number of available GPCR crystal structures, the molecular mechanism of activation of a large number of receptors, including MT1, remains poorly understood. The purpose of this study is to elucidate the structural elements involved in the process of MT1’s activation using naturally occurring variants affecting its function. Thirty-six nonsynonymous variants, including 34 rare ones, were identified in MTNR1A (encoding MT1) from a cohort of 8687 individuals and their signaling profiles were characterized using Bioluminescence Resonance Energy Transfer-based sensors probing 11 different signaling pathways. Computational analysis of the experimental data allowed us to group the variants in clusters according to their signaling profiles and to analyze the position of each variant in the context of the three-dimensional structure of MT1 to link functional selectivity to structure. MT1 variant signaling profiles revealed three clusters characterized by (1) wild-type-like variants, (2) variants with selective defect of βarrestin-2 recruitment, and (3) severely defective variants on all pathways. Our structural analysis allows us to identify important regions for βarrestin-2 recruitment as well as for Gα12 and Gα15 activation. In addition to identifying MT1 domains differentially controlling the activation of the various signaling effectors, this study illustrates how natural variants can be used as tools to study the molecular mechanisms of receptor activation.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in the pathology of cancer, HIV, and COVID-19. Despite its significance as a drug target, the activation mechanism of CCR5, i.e. how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N-terminus of agonist chemokines pushes onto an aromatic connector that transmits activation to the canonical GPCR microswitch network. This activation mechanism differs significantly from other CC chemokine receptors that bind shorter chemokines in a shallow binding mode and have unique sequence signatures and a specialized activation mechanism.One-sentence summaryThe structure of CCR5 in complex with the chemokine agonist [6P4]CCL5 and the heterotrimeric Gi protein reveals its activation mechanism

SummaryChimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible, which makes them unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH)1, can be seamlessly fused2to terminal helices of proteins, forming an extended and partially free-standing rigid helix. Through the intrinsic stability of the SAH, two domains can be connected with a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano, and we show that a single SAH allows the connection of two separate structural domains with sufficient rigidity to form ordered crystals. The analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact in construct flexibility (for the study of protein dynamics), and to design novel biomaterials.

G protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new 3D molecular structures of GPCRs (3D-GPCRome) during the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. While experimentally-resolved structures undoubtedly provide valuable snapshots of specific GPCR conformational states, they give only limited information on their flexibility and dynamics associated with function. Molecular dynamics (MD) simulations have become a widely established technique to explore the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations requires efficient storage resources and specialized software, hence limiting the dissemination of these data to specialists in the field. Here we present the GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyse and share GPCR MD data. GPCRmd originates from a community-driven effort to create the first open, interactive, and standardized database of GPCR MD simulations. We demonstrate the power of this resource by performing comparative analyses of multiple GPCR simulations on two mechanisms critical to receptor function: internal water networks and sodium ion interaction.

G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.

Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible and therefore unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH), can be seamlessly fused to terminal helices of proteins, forming an extended, partially free-standing rigid helix. This enables the connection of two domains at a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano. Analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example, to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact on construct flexibility (for the study of protein dynamics), and to design novel biomaterials.

Box jellyfish and vertebrates are separated by >500 million years of evolution yet have structurally analogous lens eyes that employ rhodopsin photopigments for vision. All opsins possess a negatively charged residue—the counterion—to maintain visible-light sensitivity and facilitate photoisomerization of their retinaldehyde chromophore. In vertebrate rhodopsins, the molecular evolution of the counterion position—from a highly conserved distal location in the second extracellular loop (E181) to a proximal location in the third transmembrane helix (E113)—is established as a key driver of higher fidelity photoreception. Here, we use computational biology and heterologous action spectroscopy to determine whether the appearance of the advanced visual apparatus in box jellyfish was also accompanied by changes in the opsin tertiary structure. We found that the counterion in an opsin from the lens eye of the box jellyfish Carybdea rastonii (JellyOp) has also moved to a unique proximal location within the transmembrane bundle—E94 in TM2. Furthermore, we reveal that this Schiff base/counterion system includes an additional positive charge—R186—that has coevolved with E94 to functionally separate E94 and E181 in the chromophore-binding pocket of JellyOp. By engineering this pocket—neutralizing R186 and E94, or swapping E94 with the vertebrate counterion E113—we can recreate versions of the invertebrate and vertebrate counterion systems, respectively, supporting a relatively similar overall architecture in this region of animal opsins. In summary, our data establish the third only counterion site in animal opsins and reveal convergent evolution of tertiary structure in opsins from distantly related species with advanced visual systems., Proceedings of the National Academy of Sciences of the United States of America, 115 (24), ISSN:0027-8424, ISSN:1091-6490

G proteins represent intracellular switches that transduce signals relayed from G protein-coupled receptors. The structurally related macrocyclic depsipeptides FR900359 (FR) and YM-254890 (YM) are potent, selective inhibitors of the Gαq protein family. We recently discovered that radiolabeled FR and YM display strongly divergent residence times, which translates into significantly longer antiasthmatic effects of FR. The present study is aimed at investigating the molecular basis for this observed disparity. Based on docking studies, we mutated amino acid residues of the Gαq protein predicted to interact with FR or YM, and recombinantly expressed the mutated Gαq proteins in cells in which the native Gαq proteins had been knocked out by CRISPR-Cas9. Both radioligands showed similar association kinetics, and their binding followed a conformational selection mechanism, which was rationalized by molecular dynamics simulation studies. Several mutations of amino acid residues near the putative binding site of the “lipophilic anchors” of FR, especially those predicted to interact with the isopropyl group present in FR but not in YM, led to dramatically accelerated dissociation kinetics. Our data indicate that the long residence time of FR depends on lipophilic interactions within its binding site. The observed structure-kinetic relationships point to a complex binding mechanism of FR, which likely involves snap-lock- or dowel-like conformational changes of either ligand or protein, or both. These experimental data will be useful for the design of compounds with a desired residence time, a parameter that has now been recognized to be of utmost importance in drug development.

Light-sensitive G protein-coupled receptors (GPCRs)-rhodopsins-absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that "reisomerize" upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9-cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more "activation-ready" conformation often observed in other nonphotosensitive class A GPCRs. These similarities suggest the role of jumping spider rhodopsin-1 as a potential model system in the study of the structure-function relationship of both photosensitive and nonphotosensitive class A GPCRs., Proceedings of the National Academy of Sciences of the United States of America, 116 (29), ISSN:0027-8424, ISSN:1091-6490

G protein-coupled receptors (GPCRs) are the largest class of integral membrane proteins and represent key targets for pharmacological research. GPCRs modulate cell physiology by engaging and activating a diversity of intracellular transducers, prominently heterotrimeric G proteins, but also G protein-receptor kinases (GRKs) and arrestins. The recent surge in the number of structures of GPCR-G protein complexes has expanded our understanding of G protein recognition and GPCR-mediated signal transduction. However, many aspects of these mechanisms, including the existence of transient interactions with transducers, have remained elusive.Here, we present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. In contrast to all reported structures, our density map reveals the receptor C-terminal tail bound to the Gβ subunit of the G protein heterotrimer. This observation provides a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as scaffold for recruiting Gα subunits and GRKs. By comparing all available complex structures, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.

Animals sense light using photosensitive proteins—rhodopsins—containing a chromophore—retinal—that intrinsically absorbs in the ultraviolet. Visible light-sensitivity depends primarily on protonation of the retinylidene Schiff base (SB), which requires a negatively-charged amino acid residue—counterion—for stabilization. Little is known about how the most common counterion among varied rhodopsins, Glu181, functions. Here, we demonstrate that in a spider visual rhodopsin, orthologue of mammal melanopsins relevant to circadian rhythms, the Glu181 counterion functions likely by forming a hydrogen-bonding network, where Ser186 is a key mediator of the Glu181–SB interaction. We also suggest that upon light activation, the Glu181–SB interaction rearranges while Ser186 changes its contribution. This is in contrast to how the counterion of vertebrate visual rhodopsins, Glu113, functions, which forms a salt bridge with the SB. Our results shed light on the molecular mechanisms of visible light-sensitivity relevant to invertebrate vision and vertebrate non-visual photoreception., Takashi Nagata et al. use UV-visible spectroscopic analysis to show that Ser186 increases the retinylidene Schiff base (SB) pKa of a spider rhodopsin in the inactive state but not after light activation. The change in contribution of Ser186 to the SB pKa suggests that the counterion (Glu181)–SB interaction rearranges upon light activation.

Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore—attached to the protein through a protonated Schiff base—and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography. ISSN:0006-3495 ISSN:1542-0086

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.

Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin−arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC50) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin–arrestin-1 complex. The achieved stabilization of the active rhodopsin–arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs)., Scientific Reports, 9, ISSN:2045-2322

Bioconjugate Chemistry, 30 (3), ISSN:1043-1802, ISSN:1520-4812

Heterotrimeric G protein subunits Gαq and Gα11 are inhibited by two cyclic depsipeptides, FR900359 (FR) and YM-254890 (YM), both of which are being used widely to implicate Gq/11 proteins in the regulation of diverse biological processes. An emerging major research question therefore is whether the cellular effects of both inhibitors are on-target, that is, mediated via specific inhibition of Gq/11 proteins, or off-target, that is, the result of nonspecific interactions with other proteins. Here we introduce a versatile experimental strategy to discriminate between these possibilities. We developed a Gαq variant with preserved catalytic activity, but refractory to FR/YM inhibition. A minimum of two amino acid changes were required and sufficient to achieve complete inhibitor resistance. We characterized the novel mutant in HEK293 cells depleted by CRISPR–Cas9 of endogenous Gαq and Gα11 to ensure precise control over the Gα-dependent cellular signaling route. Using a battery of cellular outcomes with known and concealed Gq contribution, we found that FR/YM specifically inhibited cellular signals after Gαq introduction via transient transfection. Conversely, both inhibitors were inert across all assays in cells expressing the drug-resistant variant. These findings eliminate the possibility that inhibition of non-Gq proteins contributes to the cellular effects of the two depsipeptides. We conclude that combined application of FR or YM along with the drug-resistant Gαq variant is a powerful in vitro strategy to discern on-target Gq against off-target non-Gq action. Consequently, it should be of high value for uncovering Gq input to complex biological processes with high accuracy and the requisite specificity.

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

International audience; 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. They are the targets of nearly one-third of all prescribed medicinal drugs such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins. 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.

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, 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, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey β1-adrenergic receptor (β1AR). 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.

Selective coupling of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) to specific Gα-protein subtypes is critical to transform extracellular signals, carried by natural ligands and clinical drugs, into cellular responses. At the center of this transduction event lies the formation of a signaling complex between the receptor and G protein. We report the crystal structure of light-sensitive GPCR rhodopsin bound to an engineered mini-Go protein. The conformation of the receptor is identical to all previous structures of active rhodopsin, including the complex with arrestin. Thus, rhodopsin seems to adopt predominantly one thermodynamically stable active conformation, effectively acting like a “structural switch,” allowing for maximum efficiency in the visual system. Furthermore, our analysis of the well-defined GPCR–G protein interface suggests that the precise position of the carboxyl-terminal “hook-like” element of the G protein (its four last residues) relative to the TM7/helix 8 (H8) joint of the receptor is a significant determinant in selective G protein activation. ISSN:2375-2548

Selective coupling of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) to specific Gα-protein subtypes is critical to transform extracellular signals, carried by natural ligands and clinical drugs, into cellular responses. At the center of this transduction event lies the formation of a signaling complex between the receptor and G protein. We report the crystal structure of light-sensitive GPCR rhodopsin bound to an engineered mini-G

G protein-coupled receptor (GPCR) structural biology has progressed dramatically in the last decade. There are now over 120 GPCR crystal structures deposited in the Protein Data Bank of 32 different receptors from families scattered across the phylogenetic tree, including class B, C, and Frizzled GPCRs. These structures have been obtained in combination with a wide variety of ligands and captured in a range of conformational states. This surge in structural knowledge has enlightened research into the molecular recognition of biologically active molecules, the mechanisms of receptor activation, the dynamics of functional selectivity, and fueled structure-based drug design efforts for GPCRs. Here we summarize the innovations in both protein engineering/molecular biology and crystallography techniques that have led to these advances in GPCR structural biology and discuss how they may influence the resulting structural models. We also provide a brief molecular pharmacologist's guide to GPCR X-ray crystallography, outlining some key aspects in the process of structure determination, with the goal to encourage noncrystallographers to interrogate structures at the molecular level. Finally, we show how chemogenomics approaches can be used to marry the wealth of existing receptor pharmacology data with the expanding repertoire of structures, providing a deeper understanding of the mechanistic details of GPCR function.

We present comprehensive maps at single-amino acid resolution of the residues stabilizing the human Gαi1 subunit in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of alanine mutations on the stability of Gαi1 and the rhodopsin-Gαi1 complex. We identified stabilization clusters in the GTPase and helical domains responsible for structural integrity and the conformational changes associated with activation. In activation cluster I, helices α1 and α5 pack against strands β1-β3 to stabilize the nucleotide-bound states. In the receptor-bound state, these interactions are replaced by interactions between α5 and strands β4-β6. Key residues in this cluster are Y320, which is crucial for the stabilization of the receptor-bound state, and F336, which stabilizes nucleotide-bound states. Destabilization of helix α1, caused by rearrangement of this activation cluster, leads to the weakening of the interdomain interface and release of GDP.

Rhodopsin the dim light photoreceptor present in the rod cells of the retina is both a retinal binding protein and a G protein coupled receptor (GPCR). Due to this conjunction it benefits from an arsenal of spectroscopy techniques that can be used for its characterization while being a model system for the important family of Class A (also referred to as "rhodopsin like") GPCRs. For instance rhodopsin has been a crucial player in the field of GPCR structural biology. Until 2007 it was the only GPCR for which a high resolution crystal structure was available so all structure activity analyses on GPCRs from structure based drug discovery to studies of structural changes upon activation were based on rhodopsin. At present about a third of currently available GPCR structures are still from rhodopsin. In this review I show some examples of how these structures can still be used to gain insight into general aspects of GPCR activation. First the analysis of the third intracellular loop in rhodopsin structures allows us to gain an understanding of the structural and dynamic properties of this region which is absent (due to protein engineering or poor electron density) in most of the currently available GPCR structures. Second a detailed analysis of the structure of the transmembrane domains in inactive intermediate and active rhodopsin structures allows us to detect early conformational changes in the process of ligand induced GPCR activation. Finally the analysis of a conserved ligand activated transmission switch in the transmembrane bundle of GPCRs in the context of the rhodopsin activation cycle allows us to suggest that the structures of many of the currently available agonist bound GPCRs may correspond to intermediate active states. While the focus in GPCR structural biology is inevitably moving away from rhodopsin in other aspects rhodopsin is still at the forefront. For instance the first studies of the structural basis of disease mutants in GPCRs or the most detailed analysis of cellular GPCR signal transduction networks using a systems biology approach have been carried out in rhodopsin. Finally due again to its unique properties among GPCRs rhodopsin will likely play an important role in the application of X ray free electron laser crystallography to time resolved structural biology in membrane proteins. Rhodopsin thus still remains relevant as a model system to study the molecular mechanisms of GPCR activation. This article is part of a Special Issue entitled: Retinal Proteins You can teach an old dog new tricks. © 2013 Elsevier B.V. All rights reserved.

Arrestins function as adapter proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and additional rounds of signaling. Here we have compared binding of the GPCR rhodopsin to 403 mutants of arrestin-1 covering its complete sequence. This comprehensive and unbiased mutagenesis approach provides a functional dimension to the crystal structures of inactive, preactivated p44 and phosphopeptide-bound arrestins and will guide our understanding of arrestin-GPCR complexes. The presented functional map quantitatively connects critical interactions in the polar core and along the C tail of arrestin. A series of amino acids (Phe375, Phe377, Phe380, and Arg382) anchor the C tail in a position that blocks binding of the receptor. Interaction of phosphates in the rhodopsin C terminus with Arg29 controls a C-tail exchange mechanism in which the C tail of arrestin is released and exposes several charged amino acids (Lys14, Lys15, Arg18, Lys20, Lys110, and Lys300) for binding of the phosphorylated receptor C terminus. In addition to this arrestin phosphosensor, our data reveal several patches of amino acids in the finger (Gln69 and Asp73-Met75) and the lariat loops (L249-S252 and Y254) that can act as direct binding interfaces. A stretch of amino acids at the edge of the C domain (Trp194-Ser199, Gly337-Gly340, Thr343, and Thr345) could act as membrane anchor, binding interface for a second rhodopsin, or rearrange closer to the central loops upon complex formation. We discuss these interfaces in the context of experimentally guided docking between the crystal structures of arrestin and light-activated rhodopsin.

SummaryTransmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase activity, suggesting that precise TMD orientation is mandatory for kinase activation. Nuclear magnetic resonance spectroscopy revealed that TMD helices in activated constructs were rotated by 180° relative to the interface of the wild-type conformation, confirming that ligand-mediated receptor activation indeed results from transmembrane helix rearrangement. A molecular dynamics simulation confirmed the transmembrane helix arrangement of wild-type and mutant TMDs revealed by nuclear magnetic resonance spectroscopy.

Congenital stationary night blindness (CSNB) is an inherited and non progressive retinal dysfunction. Here we present the crystal structure of CSNB causing T94I(2.61) rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin II state by establishing a direct van der Waals contact with K296(7.43) the site of retinal attachment. This is in stark contrast to the light activated state of the CSNB causing G90D(2.57) mutation where the charged mutation forms a salt bridge with K296(7.43) To find the common denominator between these two functional modifications we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D(2.57) and the hydrophobic T94I(2.61) mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E113(3.28) We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients.

G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y 6.40 constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all- trans -retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11- cis -retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y223 5.58 in helix 5, Y306 7.53 of the NPxxY motif and R135 3.50 of the E(D)RY motif, highly conserved residues of the G protein binding site.