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    • A biased allosteric modulator is a molecular glue for β2AR dimerization.

      Shen, J., Peddada, T. N., Komolov, K. E., De Pascali, F., Garces, A. M., Wang, H., Ehsan, M., Chae, P. S., Lerch, M. T., Benovic, J. L., Xu, J., Kobilka, B. K.

      Nature

      ABSTRACT
      Family A G-protein-coupled receptors (GPCRs) are typically described as monomers, yet growing evidence suggests that they can form dimers with distinct signalling properties1-3. However, the mechanisms and therapeutic potential of such dimerization remain poorly understood. Here we show that AP-7-168, an optimized derivative of a β-arrestin-biased negative allosteric modulator of the β2-adrenergic receptor (β2AR) that sustains bronchorelaxation in cell and tissue models4, functions as a molecular glue to stabilize β2AR homodimerization. Cryogenic electron microscopy structures reveal a unique binding mode in which two AP-7-168 molecules pack within a pocket formed by transmembrane helices 3, 4 and 5 of two protomers, stabilizing a dimeric conformation that selectively prevents β-arrestin coupling. In cells, AP-7-168 robustly stabilizes β2AR dimerization and drives enlarged nanocluster formation. Combined with extensive functional studies, our findings identify an allosteric mechanism by which a small molecule biases β2AR signalling through dimerization, highlighting ligand-stabilized dimerization as a strategy for GPCR modulation.
      https://www.ncbi.nlm.nih.gov/pubmed/42618785 https://doi.org/10.1038/s41586-026-10892-y
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    • Factors modulating the assembly of human β2-adrenergic receptor-β-arrestin complexes.

      Wilhelm, F. M., Pluhackova, K., Janetzko, J., Masureel, M., Marsh, E., Hudspeth, J., Gaßner, W., Schertler, G. F., Kobilka, B. K., Müller, D. J.

      Nature structural & molecular biology

      ABSTRACT
      β-arrestins, pivotal regulators of G protein-coupled receptor (GPCR) signaling, assemble with hundreds of GPCRs. How this assembly rises to functionally distinct complexes in which β-arrestin engages the GPCR tail, core or both, remains a central question. Here employing single-molecule force spectroscopy and molecular dynamics simulations, we monitor assembly of β2-adrenergic receptor (β2AR)-β-arrestin2 (βarr2) tail, core and tail-core complexes in phospholipid membranes and dissect their mechanical and kinetic stabilities. We show that βarr2 engages the phosphorylated β2AR carboxy-terminus (C-tail) within milliseconds, much faster than the active receptor core. In addition, the phospholipid membrane contributes substantially to complex stability, with phosphatidylinositol 4,5-bisphosphate (PIP2) modulating stability and conformation. While PIP2 stabilizes the β2AR-βarr2 core, it precludes βarr2 from concomitantly binding the phosphorylated β2AR C-tail. βarr2 activation and PIP2 strengthen βarr2-membrane association through insertion of the C-edge and finger loop. These findings establish PIP2, alongside ligand binding and receptor phosphorylation, as a central determinant of β2AR-βarr2 complex assembly, offering mechanistic insight into the regulation of GPCR signaling.
      https://www.ncbi.nlm.nih.gov/pubmed/42538433 https://doi.org/10.1038/s41594-026-01842-3
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    • Efficacy Modulation and Structural Insights of Mu Opioid-Sparing Agents

      Paul, B., Huang, P., Ramos-gonzalez, N., Wang, J., Wells, E., Hammond, H., Sortman, B., Tanguturi, P., Polley, A., Appourchaux, K., Seekins, C., Warren, B., Streicher, J., Kobilka, B., Chen, C., Mclaughlin, J., O'brien, E., Majumdar, S.

      https://doi.org/10.1016/j.jpet.2026.103957
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    • The dynamic basis of G-protein recognition and activation by a GPCR.

      Kobayashi, K., Kawakami, K., Matsui, T. E., Yokoi, S., Fukuda, M., Narita, T. J., Arai, H., Tambo, M., Sumikama, T., Tatsumi, M., Yamashita, K., Koyanagi, J., Kugawa, M., Ikeda, H., Sumino, A., Mitsutake, A., Kobilka, B. K., Inoue, A., Kato, H. E.

      Nature

      ABSTRACT
      G-protein-coupled receptor (GPCR) signalling occurs through heterotrimeric G proteins, whose selective activation leads to distinct cellular outcomes1. Although more than 200 GPCR-G protein complex structures have been determined2, these static snapshots provide limited insight into the dynamics of G-protein association and dissociation. Here we present cryo-electron microscopy structures of human neurotensin receptor type 1 (NTSR1) with minimally modified Go and Gq, showing how the receptor's intracellular surface dynamically rearranges to accommodate each G-protein subtype. Furthermore, time-resolved cryo-electron microscopy analyses of NTSR1-Gi visualized G-protein dissociation processes on GDP/GTP binding. Characterization of more than 20 intermediates, complemented by mutational and computational analyses, identifies four key mechanistic features. First, GDP/GTP induces Gi release from both canonical and non-canonical active conformations with distinct kinetics. Second, NTSR1 uses common intracellular rearrangements to recognize different G-protein subtypes and to promote activation of a single subtype. Third, separation from Gβγ involves stepwise remodelling of the Gα switches I-III. Finally, Gi dissociates from the receptor through a pathway that is distinct from that of Gs, and the canonical and non-canonical NTSR1-Gi complexes further diverge in their dissociation trajectories. These findings provide a comprehensive framework for understanding GPCR signalling dynamics and guiding signal-targeted therapeutic development.
      https://www.ncbi.nlm.nih.gov/pubmed/41813902 https://doi.org/10.1038/s41586-026-10228-w
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    • Discovery and dynamic pharmacology of μ-opioid receptor positive allosteric modulators.

      O'Brien, E. S., Wang, J., Tanguturi, P., Li, M., White, E., Shiimura, Y., Paul, B., Appourchaux, K., Kumar, K. K., Huang, W., Majumdar, S., Traynor, J. R., Streicher, J. M., Chen, C., Kobilka, B. K.

      bioRxiv : the preprint server for biology

      ABSTRACT
      Opioid agonists such as morphine and fentanyl exert analgesic effects by binding and activating the μ-opioid receptor (μOR), yet agonism of the μOR causes a slate of serious side effects. μOR-mediated addiction and respiratory depression are the major causes of the current opioid overdose crisis, largely driven by the explosion in illicit use of fentanyl, a potent opioid receptor full agonist. Given these serious side effects (and high resulting societal cost), molecules that act as analgesics with distinct mechanisms of action are of great interest. Positive allosteric modulators (PAMs) of the μOR have the potential to avoid many off-target side effects of conventional opioid orthosteric agonists by enhancing the signaling properties of natural opioid peptide systems. We used a DNA-encoded chemical library screening approach to selectively discover active-state-specific μOR PAMs. Two out of 3 selected prospective PAMs displayed the anticipated enhancement in agonist activity. The most effective of these compounds enhanced the activity of all orthosteric opioid agonists tested, including the native opioid peptide met-enkephalin. Little is known about the underlying dynamic basis of allosteric modulation of Family A GPCRs like the μOR. To that end, we used single-molecule fluorescence resonance energy transfer experiments to detail the impact that our novel μOR PAM has on the dynamic activation behavior of a key region on the intracellular face of the receptor. Our results here provide both a new chemical scaffold that acts as a μOR PAM and detailed pharmacological and dynamic insights into its mechanism of action.
      https://www.ncbi.nlm.nih.gov/pubmed/41757031 https://doi.org/10.64898/2026.02.20.707058
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    • A cryptic pocket in CB1 drives peripheral and functional selectivity

      Rangari, V., O'Brien, E. S., Powers, A. S., Slivicki, R. A., Bertels, Z., Appourchaux, K., Aydin, D., Ramos-Gonzalez, N., Mwirigi, J., Lin, L., Mangutov, E., Sobecks, B. L., Awad-Agbaria, Y., Uphade, M. B., Aguilar, J., Peddada, T., Shiimura, Y., Huang, X., Folarin-Hines, J., Payne, M., Kalathil, A., Varga, B. R., Kobilka, B. K., Pradhan, A. A., Cameron, M. D., Kumar, K., Dror, R. O., Gereau, R. W., Majumdar, S.

      NATURE

      ABSTRACT
      The current opioid overdose epidemic highlights the urgent need to develop safer and more effective treatments for chronic pain1. Cannabinoid receptor type 1 (CB1) is a promising non-opioid target for pain relief, but its clinical use has been limited by centrally mediated psychoactivity and tolerance. We overcame both issues by designing peripherally restricted CB1 agonists that minimize arrestin recruitment. We achieved these goals by computationally designing positively charged derivatives of the potent CB1 agonist MDMB-Fubinaca2. We designed these ligands to occupy a cryptic pocket identified through molecular dynamics simulations-an extended binding pocket that opens rarely and leads to the conserved signalling residue D2.50 (ref. 3). We used structure determination, pharmacological assays and molecular dynamics simulations to verify the binding modes of these ligands and to determine the molecular mechanism by which they achieve this dampening of arrestin recruitment. Our lead ligand, VIP36, is highly peripherally restricted and demonstrates notable efficacy in three mouse pain models, with 100-fold dose separation between analgesic efficacy and centrally mediated side effects. VIP36 exerts analgesic efficacy through peripheral CB1 receptors and shows limited analgesic tolerance. These results show how targeting a cryptic pocket in a G-protein-coupled receptor can lead to enhanced peripheral selectivity, biased signalling, desired in vivo pharmacology and reduced adverse effects. This has substantial implications for chronic pain treatment but could also revolutionize the design of drugs targeting other G-protein-coupled receptors.
      https://www.ncbi.nlm.nih.gov/pubmed/40044849 https://doi.org/10.1038/s41586-025-08618-7
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    • The story of a structural sage.

      Kobilka, B., Greed, S.

      Nature reviews. Chemistry

      https://www.ncbi.nlm.nih.gov/pubmed/40442299 https://doi.org/10.1038/s41570-025-00731-5
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    • Structure and dynamics determine G protein coupling specificity at a class A GPCR.

      Casiraghi, M., Wang, H., Brennan, P. C., Habrian, C., Hübner, H., Schmidt, M. F., Maul, L., Pani, B., Bahriz, S. M., Xu, B., Staffen, N., Assafa, T. E., Chen, B., White, E., Sunahara, R. K., Inoue, A., Xiang, Y. K., Lefkowitz, R. J., Isacoff, E. Y., Nucci, N., Gmeiner, P., Lerch, M. T., Kobilka, B. K.

      Science advances

      ABSTRACT
      G protein-coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The β2-adrenergic receptor is an example of GPCR with high selectivity for Gαs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gαi family of G proteins inhibiting adenylyl cyclase. By developing a Gαi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gαs and Gαi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.
      https://www.ncbi.nlm.nih.gov/pubmed/40106559 https://doi.org/10.1126/sciadv.adq3971
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    • The Role of Intrinsically Disordered Domains in Regulating G Protein-Coupled Receptor Signaling.

      Xu, J., Qiu, R., Garces, A. M., Hübner, H., Xu, X., Hao, C., Weikert, D., Gmeiner, P., Lerch, M. T., Kobilka, B. K.

      Journal of the American Chemical Society

      ABSTRACT
      The α2A adrenergic receptor (α2AAR) is a clinically important target for various diseases including hypertension, diabetes, and chronic pain. Here, using single-molecule fluorescence resonance energy transfer imaging, we show how agonist-specific activation dynamics in both structured transmembrane domain (TMD) and intrinsic disorder regions (IDRs) of α2AAR lead to diverse signaling profiles. Through seven pairs of strategically designed fluorophore labels, we systematically investigate the real-time conformational changes of α2AAR. Our study reveals unique TM6 dynamics in α2AAR, featured by a high energy barrier for agonist-induced outward movements essential for activation. In contrast, we identify agonist-specific conformational dynamics of a partially disordered extracellular loop (ECL2), highlighting its role as a dynamic regulatory module that controls receptor function. Moreover, we characterize the conformational landscapes of the long third intracellular loop (ICL3), revealing its compact structural features and membrane-proximal localization in the basal state, where it acts as a negative allosteric regulator in transducer coupling. Furthermore, we identify multiple functional substates of ICL3 that are dynamically modulated by both kinase phosphorylation and drug efficacy. These findings offer previously underappreciated structural and dynamic insights into α2AAR function governed by both TMD and IDRs and may open up new avenues for the development of better therapeutics.
      https://www.ncbi.nlm.nih.gov/pubmed/41454868 https://doi.org/10.1021/jacs.5c17796
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    • Ligand-dependent G protein dynamics underlying opioid signaling efficacy.

      Deutsch, J., Hilger, D., Janetzko, J., Schroeder, C. M., Chu, S., Kobilka, B. K., Shivnaraine, R. V.

      bioRxiv : the preprint server for biology

      ABSTRACT
      Activation of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) requires large-scale opening of the Gα α-helical domain (AHD) to expose the nucleotide-binding site and facilitate GDP-GTP exchange. While orthosteric ligands are known to modulate GPCR conformation and signaling efficacy, how these effects propagate to the G protein itself remains unclear. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, we monitored AHD motions in Gi proteins coupled to the μ-opioid receptor (μOR) across a spectrum of ligand- and nucleotide-bound states. We find that receptor ligands differentially modulate these dynamics from over 70 Å away, with higher-efficacy agonists more effectively promoting transitions to an open, low-nucleotide-affinity conformation. These data also capture transient μOR-Gi intermediates during nucleotide binding and suggest that μ-opioid ligand efficacy arises in part from allosteric control over G protein conformational equilibria that kinetically gate activation.
      https://www.ncbi.nlm.nih.gov/pubmed/40631176 https://doi.org/10.1101/2025.06.30.662233
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    • A biased allosteric modulator functions as a molecular glue to induce β2AR dimerization.

      Shen, J., Peddada, T. N., Komolov, K. E., De Pascali, F., Garces, A. M., Wang, H., Lerch, M. T., Benovic, J. L., Xu, J., Kobilka, B. K.

      bioRxiv : the preprint server for biology

      ABSTRACT
      Family A G-protein coupled receptors (GPCRs) are typically described as monomers, yet growing evidence suggests they can form dimers with distinct signaling properties1-3. The mechanisms and therapeutic potential of such dimerization, however, remain poorly understood. Here, we show that AP-7-168, an optimized derivative of a β-arrestin-biased negative allosteric modulator of the β2-adrenergic receptor (β2AR) that sustains bronchorelaxation in cell and tissue models4, functions as a molecular glue to promote β2AR homodimerization. Cryo-EM structures reveal a unique binding mode in which two AP-7-168 molecules pack within a pocket formed by transmembrane helices 3, 4, and 5 of two protomers, stabilizing a dimeric conformation that selectively prevents β-arrestin coupling. In cells, AP-7-168 robustly induces β2AR dimerization and drives enlarged nanocluster formation. Combined with extensive functional studies, our findings unveil a novel allosteric mechanism by which a small molecule biases β2AR signaling through dimerization, highlighting ligand-induced dimerization as a strategy for GPCR modulation.
      https://www.ncbi.nlm.nih.gov/pubmed/41279134 https://doi.org/10.1101/2025.10.21.683802
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    • A biased allosteric modulator functions as a molecular glue to induce β2AR dimerization.

      Kobilka, B., Shen, J., Peddada, T., Komolov, K., De Pascali, F., Garces, A., Wang, H., Lerch, M., Benovic, J., Xu, J.

      Research square

      ABSTRACT
      Family A G-protein coupled receptors (GPCRs) are typically described as monomers, yet growing evidence suggests they can form dimers with distinct signaling properties1-3. The mechanisms and therapeutic potential of such dimerization, however, remain poorly understood. Here, we show that AP-7-168, an optimized derivative of a β-arrestin-biased negative allosteric modulator of the β2-adrenergic receptor (β2AR) that sustains bronchorelaxation in cell and tissue models4, functions as a molecular glue to promote β2AR homodimerization. Cryo-EM structures reveal a unique binding mode in which two AP-7-168 molecules pack within a pocket formed by transmembrane helices 3, 4, and 5 of two protomers, stabilizing a dimeric conformation that selectively prevents β-arrestin coupling. In cells, AP-7-168 robustly induces β2AR dimerization and drives enlarged nanocluster formation. Combined with extensive functional studies, our findings unveil a novel allosteric mechanism by which a small molecule biases β2AR signaling through dimerization, highlighting ligand-induced dimerization as a strategy for GPCR modulation.
      https://www.ncbi.nlm.nih.gov/pubmed/41356349 https://doi.org/10.21203/rs.3.rs-8001844/v1
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    • Membrane phosphoinositides allosterically tune β-arrestin dynamics to facilitate GPCR core engagement.

      Janetzko, J., Deutsch, J., Shi, Y., Siepe, D. H., Masureel, M., Liu, W., Viner, R., Inoue, A., Kobilka, B. K., Shivnaraine, R. V.

      bioRxiv : the preprint server for biology

      ABSTRACT
      Arrestin proteins bind active G protein-coupled receptors (GPCRs) through coordinated protein-protein, protein-phosphate, and protein-lipid interactions to attenuate G protein signaling and promote GPCR internalization and trafficking. While there are hundreds of diverse GPCRs, only two β-arrestin isoforms (βarrs) must recognize and engage this wide range of receptors with varied phosphorylation patterns. Traditional models suggest that βarr activation requires displacement of its autoinhibitory C-tail by a phosphorylated GPCR C-terminus; however, this paradigm fails to explain how minimally phosphorylated GPCRs still complex with βarrs. Using single-molecule Förster resonance energy transfer imaging and hydrogen-deuterium exchange mass spectrometry, we observe basal dynamics in which the βarr1 C-tail spontaneously releases from the N-domain, transiently adopting an active conformation that can facilitate binding of the phosphorylated GPCR C-terminus. We further demonstrate the importance of an intermediate state of βarr1 arising from spontaneous C-tail release stabilized by the membrane phosphoinositide PI(4,5)P2. Both PI(4,5)P2 and mutations in the proximal or middle regions of the C-tail shift βarr1 towards a partially released state, revealing an allosteric connection that informs a refined model for βarr activation. In this model, membrane engagement conformationally primes βarrs prior to receptor binding, thereby explaining how βarrs are recruited by diverse GPCRs, even those with limited C-terminal phosphorylation.
      https://www.ncbi.nlm.nih.gov/pubmed/40501946 https://doi.org/10.1101/2025.06.06.658200
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    • The role of intrinsically disordered domains in regulating G protein coupled receptor signaling.

      Xu, J., Qiu, R., Garces, A. M., Hübner, H., Xu, X., Weikert, D., Gmeiner, P., Lerch, M. T., Kobilka, B. K.

      bioRxiv : the preprint server for biology

      ABSTRACT
      The α2A adrenergic receptor (α2AAR) is a clinically important target for various diseases including hypertension, diabetes and chronic pain. Here, using single-molecule fluorescence resonance energy transfer imaging, we show how agonist-specific activation dynamics in both structured transmembrane domain (TMD) and intrinsically disorders regions (IDRs) of α2AAR lead to diverse signaling profiles. Through seven pairs of strategically designed fluorophore labels, we systematically investigate the real-time conformational changes of α2AAR. Our study reveals unique TM6 dynamics in α2AAR, featured by a high energy barrier for agonist-induced outward movements essential for activation. In contrast, we identify agonist-specific conformational dynamics of a partially disordered extracellular loop (ECL2), highlighting its role as a dynamic regulatory module that controls receptor function. Moreover, we characterize the conformational landscapes of the long third intracellular loop (ICL3), revealing its compact structural features and membrane-proximal localization in the basal state, where it acts as a negative allosteric regulator in transducer coupling. Furthermore, we identify multiful functional sub-states of ICL3 that are dynamically modulated by both kinase phosphorylation and drug efficacy. These findings offer previously underappreciated structural and dynamic insights into α2AAR function governed by both TMD and IDRs, and may open up new avenues for the development of better therapeutics.
      https://www.ncbi.nlm.nih.gov/pubmed/40964316 https://doi.org/10.1101/2025.09.10.675278
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    • Calcineurin-fusion facilitates cryo-EM structure determination of a Family A GPCR.

      Xu, J., Chen, G., Wang, H., Cao, S., Heng, J., Deupi, X., Du, Y., Kobilka, B. K.

      Proceedings of the National Academy of Sciences of the United States of America

      ABSTRACT
      Advances in singe-particle cryo-electron microscopy (cryo-EM) have made it 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 extramembrane components (such as G protein, arrestin, or Venus flytrap domains) in these complexes that aid in particle alignment during the 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.
      https://www.ncbi.nlm.nih.gov/pubmed/39565314 https://doi.org/10.1073/pnas.2414544121
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    • Author Correction: Stepwise activation of a metabotropic glutamate receptor.

      Krishna Kumar, K., Wang, H., Habrian, C., Latorraca, N. R., Xu, J., O'Brien, E. S., Zhang, C., Montabana, E., Koehl, A., Marqusee, S., Isacoff, E. Y., Kobilka, B. K.

      Nature

      https://www.ncbi.nlm.nih.gov/pubmed/38702522 https://doi.org/10.1038/s41586-024-07470-5
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    • Stepwise activation of a metabotropic glutamate receptor.

      Krishna Kumar, K., Wang, H., Habrian, C., Latorraca, N. R., Xu, J., O'Brien, E. S., Zhang, C., Montabana, E., Koehl, A., Marqusee, S., Isacoff, E. Y., Kobilka, B. K.

      Nature

      ABSTRACT
      Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding.
      https://www.ncbi.nlm.nih.gov/pubmed/38632403 https://doi.org/10.1038/s41586-024-07327-x
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    • A µ-opioid receptor modulator that works cooperatively with naloxone.

      O'Brien, E. S., Rangari, V. A., El Daibani, A., Eans, S. O., Hammond, H. R., White, E., Wang, H., Shiimura, Y., Krishna Kumar, K., Jiang, Q., Appourchaux, K., Huang, W., Zhang, C., Kennedy, B. J., Mathiesen, J. M., Che, T., McLaughlin, J. P., Majumdar, S., Kobilka, B. K.

      Nature

      ABSTRACT
      The µ-opioid receptor (µOR) is a well-established target for analgesia1, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl2, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.
      https://www.ncbi.nlm.nih.gov/pubmed/38961287 https://doi.org/10.1038/s41586-024-07587-7
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    • Mechanistic insights into G-protein coupling with an agonist-bound G-protein-coupled receptor.

      Batebi, H., Pérez-Hernández, G., Rahman, S. N., Lan, B., Kamprad, A., Shi, M., Speck, D., Tiemann, J. K., Guixà-González, R., Reinhardt, F., Stadler, P. F., Papasergi-Scott, M. M., Skiniotis, G., Scheerer, P., Kobilka, B. K., Mathiesen, J. M., Liu, X., Hildebrand, P. W.

      Nature structural & molecular biology

      ABSTRACT
      G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting guanine nucleotide exchange. Here, we investigate the coupling of G proteins with GPCRs and describe the events that ultimately lead to the ejection of GDP from its binding pocket in the Gα subunit, the rate-limiting step during G-protein activation. Using molecular dynamics simulations, we investigate the temporal progression of structural rearrangements of GDP-bound Gs protein (Gs·GDP; hereafter GsGDP) upon coupling to the β2-adrenergic receptor (β2AR) in atomic detail. The binding of GsGDP to the β2AR is followed by long-range allosteric effects that significantly reduce the energy needed for GDP release: the opening of α1-αF helices, the displacement of the αG helix and the opening of the α-helical domain. Signal propagation to the Gs occurs through an extended receptor interface, including a lysine-rich motif at the intracellular end of a kinked transmembrane helix 6, which was confirmed by site-directed mutagenesis and functional assays. From this β2AR-GsGDP intermediate, Gs undergoes an in-plane rotation along the receptor axis to approach the β2AR-Gsempty state. The simulations shed light on how the structural elements at the receptor-G-protein interface may interact to transmit the signal over 30 Å to the nucleotide-binding site. Our analysis extends the current limited view of nucleotide-free snapshots to include additional states and structural features responsible for signaling and G-protein coupling specificity.
      https://www.ncbi.nlm.nih.gov/pubmed/38867113 https://doi.org/10.1038/s41594-024-01334-2
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    • Ligand efficacy modulates conformational dynamics of the -opioid receptor.

      Zhao, J., Elgeti, M., O'Brien, E. S., Sar, C. P., Ei Daibani, A., Heng, J., Sun, X., White, E., Che, T., Hubbell, W. L., Kobilka, B. K., Chen, C.

      Nature

      ABSTRACT
      The -opioid receptor (OR) is an important target for pain management1 and molecular understanding of drug action on OR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of OR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of beta-arrestin-1 with the muOR core binding site appears less specific and occurs with much lower affinity than binding of Gi.
      https://www.ncbi.nlm.nih.gov/pubmed/38600384 https://doi.org/10.1038/s41586-024-07295-2
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