Hélène Irwin Fagan Chair of Cardiology


  • Structural basis of α1A-adrenergic receptor activation and recognition by an extracellular nanobody. Nature communications Toyoda, Y., Zhu, A., Kong, F., Shan, S., Zhao, J., Wang, N., Sun, X., Zhang, L., Yan, C., Kobilka, B. K., Liu, X. 2023; 14 (1): 3655


    The α1A-adrenergic receptor (α1AAR) belongs to the family of G protein-coupled receptors that respond to adrenaline and noradrenaline. α1AAR is involved in smooth muscle contraction and cognitive function. Here, we present three cryo-electron microscopy structures of human α1AAR bound to the endogenous agonist noradrenaline, its selective agonist oxymetazoline, and the antagonist tamsulosin, with resolutions range from 2.9 Å to 3.5 Å. Our active and inactive α1AAR structures reveal the activation mechanism and distinct ligand binding modes for noradrenaline compared with other adrenergic receptor subtypes. In addition, we identified a nanobody that preferentially binds to the extracellular vestibule of α1AAR when bound to the selective agonist oxymetazoline. These results should facilitate the design of more selective therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

    View details for DOI 10.1038/s41467-023-39310-x

    View details for PubMedID 37339967

    View details for PubMedCentralID 8185284

  • Structure based approaches on fentanyl template to design novel mu opioid modulators Ople, R., Wang, H., Li, Q., Polacco, B., Bernhard, S., Appourchaux, K., Sribhashyam, S., Eans, S., Huttenhain, R., McLaughlin, J., Kobilka, B., Majumdar, S. AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS. 2023
  • Structural basis for activation of CB1 by an endocannabinoid analog. Nature communications Krishna Kumar, K., Robertson, M. J., Thadhani, E., Wang, H., Suomivuori, C. M., Powers, A. S., Ji, L., Nikas, S. P., Dror, R. O., Inoue, A., Makriyannis, A., Skiniotis, G., Kobilka, B. 2023; 14 (1): 2672


    Endocannabinoids (eCBs) are endogenous ligands of the cannabinoid receptor 1 (CB1), a G protein-coupled receptor that regulates a number of therapeutically relevant physiological responses. Hence, understanding the structural and functional consequences of eCB-CB1 interactions has important implications for designing effective drugs targeting this receptor. To characterize the molecular details of eCB interaction with CB1, we utilized AMG315, an analog of the eCB anandamide to determine the structure of the AMG315-bound CB1 signaling complex. Compared to previous structures, the ligand binding pocket shows some differences. Using docking, molecular dynamics simulations, and signaling assays we investigated the functional consequences of ligand interactions with the "toggle switch" residues F2003.36 and W3566.48. Further, we show that ligand-TM2 interactions drive changes to residues on the intracellular side of TM2 and are a determinant of efficacy in activating G protein. These intracellular TM2 rearrangements are unique to CB1 and are exploited by a CB1-specific allosteric modulator.

    View details for DOI 10.1038/s41467-023-37864-4

    View details for PubMedID 37160876

    View details for PubMedCentralID PMC10169858

  • Constrained catecholamines gain β2AR selectivity through allosteric effects on pocket dynamics. Nature communications Xu, X., Shonberg, J., Kaindl, J., Clark, M. J., Stößel, A., Maul, L., Mayer, D., Hübner, H., Hirata, K., Venkatakrishnan, A. J., Dror, R. O., Kobilka, B. K., Sunahara, R. K., Liu, X., Gmeiner, P. 2023; 14 (1): 2138


    G protein-coupled receptors (GPCRs) within the same subfamily often share high homology in their orthosteric pocket and therefore pose challenges to drug development. The amino acids that form the orthosteric binding pocket for epinephrine and norepinephrine in the β1 and β2 adrenergic receptors (β1AR and β2AR) are identical. Here, to examine the effect of conformational restriction on ligand binding kinetics, we synthesized a constrained form of epinephrine. Surprisingly, the constrained epinephrine exhibits over 100-fold selectivity for the β2AR over the β1AR. We provide evidence that the selectivity may be due to reduced ligand flexibility that enhances the association rate for the β2AR, as well as a less stable binding pocket for constrained epinephrine in the β1AR. The differences in the amino acid sequence of the extracellular vestibule of the β1AR allosterically alter the shape and stability of the binding pocket, resulting in a marked difference in affinity compared to the β2AR. These studies suggest that for receptors containing identical binding pocket residues, the binding selectivity may be influenced in an allosteric manner by surrounding residues, like those of the extracellular loops (ECLs) that form the vestibule. Exploiting these allosteric influences may facilitate the development of more subtype-selective ligands for GPCRs.

    View details for DOI 10.1038/s41467-023-37808-y

    View details for PubMedID 37059717

    View details for PubMedCentralID PMC10104803

  • Function and dynamics of the intrinsically disordered carboxyl terminus of beta2 adrenergic receptor. Nature communications Heng, J., Hu, Y., Perez-Hernandez, G., Inoue, A., Zhao, J., Ma, X., Sun, X., Kawakami, K., Ikuta, T., Ding, J., Yang, Y., Zhang, L., Peng, S., Niu, X., Li, H., Guixa-Gonzalez, R., Jin, C., Hildebrand, P. W., Chen, C., Kobilka, B. K. 2023; 14 (1): 2005


    Advances in structural biology have provided important mechanistic insights into signaling by the transmembrane core of G-protein coupled receptors (GPCRs); however, much less is known about intrinsically disordered regions such as the carboxyl terminus (CT), which is highly flexible and not visible in GPCR structures. The beta2 adrenergic receptor's (beta2AR) 71 amino acid CT is a substrate for GPCR kinases and binds beta-arrestins to regulate signaling. Here we show that the beta2AR CT directly inhibits basal and agonist-stimulated signaling in cell lines lacking beta-arrestins. Combining single-molecule fluorescence resonance energy transfer (FRET), NMR spectroscopy, and molecular dynamics simulations, we reveal that the negatively charged beta2AR-CT serves as an autoinhibitory factor via interacting with the positively charged cytoplasmic surface of the receptor to limit access to G-proteins. The stability of this interaction is influenced by agonists and allosteric modulators, emphasizing that the CT plays important role in allosterically regulating GPCR activation.

    View details for DOI 10.1038/s41467-023-37233-1

    View details for PubMedID 37037825

  • Negative allosteric modulation of the glucagon receptor by RAMP2. Cell Krishna Kumar, K., O'Brien, E. S., Habrian, C. H., Latorraca, N. R., Wang, H., Tuneew, I., Montabana, E., Marqusee, S., Hilger, D., Isacoff, E. Y., Mathiesen, J. M., Kobilka, B. K. 2023; 186 (7): 1465-1477.e18


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

    View details for DOI 10.1016/j.cell.2023.02.028

    View details for PubMedID 37001505

  • Negative allosteric modulation of the glucagon receptor by RAMP2 O'Brien, E. S., Kumar, K., Habrian, C., Latorraca, N. R., Wang, H., Tuneew, I., Montabana, E., Marqusee, S., Hilger, D., Isacoff, E. Y., Mathiesen, J. M., Kobilka, B. K. CELL PRESS. 2023: 161A
  • Structural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature communications Xu, J., Wang, Q., Hübner, H., Hu, Y., Niu, X., Wang, H., Maeda, S., Inoue, A., Tao, Y., Gmeiner, P., Du, Y., Jin, C., Kobilka, B. K. 2023; 14 (1): 376


    The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance β-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.

    View details for DOI 10.1038/s41467-022-35726-z

    View details for PubMedID 36690613

    View details for PubMedCentralID PMC9870890

  • Structure-based design of bitopic ligands for the µ-opioid receptor. Nature Faouzi, A., Wang, H., Zaidi, S. A., DiBerto, J. F., Che, T., Qu, Q., Robertson, M. J., Madasu, M. K., El Daibani, A., Varga, B. R., Zhang, T., Ruiz, C., Liu, S., Xu, J., Appourchaux, K., Slocum, S. T., Eans, S. O., Cameron, M. D., Al-Hasani, R., Pan, Y. X., Roth, B. L., McLaughlin, J. P., Skiniotis, G., Katritch, V., Kobilka, B. K., Majumdar, S. 2022


    Mu opioid receptor (µOR) agonists like fentanyl have long been used for pain management, but are considered a major public health concern due to their adverse side effects, including lethal overdose.1 To design safer therapeutics, we report a conceptually novel approach targeting conserved sodium (Na+) binding site2, observed in µOR3 and many other class A GPCRs, by bitopic fentanyl derivatives functionalized via a linker with a positively charged guanidino group. Cryo-EM structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the ligand's guanidine and the key Asp2.50 residue in the Na+ site. While the lead bitopics maintain nanomolar potency and high efficacy at Gi subtypes, they show strongly reduced arrestin recruitment, one also shows the lowest Gz-efficacy among the panel of µOR agonists, including partial and biased, morphinan and fentanyl analogs. In mice, the best bitopic ligand displayed µOR dependent antinociception with attenuated adverse effects supporting the µOR Na+ site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands engaging the Na+ pocket in class A GPCRs can be designed to control their efficacy and functional selectivity profiles for Gi/o/z subtypes and arrestins, thus modulating their in vivo pharmacology.

    View details for DOI 10.1038/s41586-022-05588-y

    View details for PubMedID 36450356

  • Insights into distinct signaling profiles of the OR activated by diverse agonists. Nature chemical biology Qu, Q., Huang, W., Aydin, D., Paggi, J. M., Seven, A. B., Wang, H., Chakraborty, S., Che, T., DiBerto, J. F., Robertson, M. J., Inoue, A., Suomivuori, C., Roth, B. L., Majumdar, S., Dror, R. O., Kobilka, B. K., Skiniotis, G. 2022


    Drugs targeting the mu-opioid receptor (muOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two muOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and beta-arrestin recruitment. Cryo-EM structures of muOR-Gi1 complex with MP (2.5A) and LFT (3.2A) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and beta-arrestins bind. These observations highlight how drugs engaging different parts of the muOR orthosteric pocket can lead to distinct signaling outcomes.

    View details for DOI 10.1038/s41589-022-01208-y

    View details for PubMedID 36411392

  • Membrane phosphoinositides regulate GPCR-beta-arrestin complex assembly and dynamics. Cell Janetzko, J., Kise, R., Barsi-Rhyne, B., Siepe, D. H., Heydenreich, F. M., Kawakami, K., Masureel, M., Maeda, S., Garcia, K. C., von Zastrow, M., Inoue, A., Kobilka, B. K. 2022


    Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with beta-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and invitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in beta-arrestin recruitment and GPCR-beta-arrestin complexdynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for beta-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of beta-arrestin and stabilize GPCR-beta-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-beta-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for beta-arrestin recruitment, this provides a mechanism for beta-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.

    View details for DOI 10.1016/j.cell.2022.10.018

    View details for PubMedID 36368322

  • A cholesterol analog stabilizes the human beta2-adrenergic receptor nonlinearly with temperature. Science signaling Serdiuk, T., Manna, M., Zhang, C., Mari, S. A., Kulig, W., Pluhackova, K., Kobilka, B. K., Vattulainen, I., Muller, D. J. 2022; 15 (737): eabi7031


    In cell membranes, G protein-coupled receptors (GPCRs) interact with cholesterol, which modulates their assembly, stability, and conformation. Previous studies have shown how cholesterol modulates the structural properties of GPCRs at ambient temperature. Here, we characterized the mechanical, kinetic, and energetic properties of the human beta2-adrenergic receptor (beta2AR) in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) at room temperature (25°C), at physiological temperature (37°C), and at high temperature (42°C). We found that CHS stabilized various structural regions of beta2AR differentially, which changed nonlinearly with temperature. Thereby, the strongest effects were observed for structural regions that are important for receptor signaling. Moreover, at 37°C, but not at 25° or 42°C, CHS caused beta2AR to increase and stabilize conformational substates to adopt to basal activity. These findings indicate that the nonlinear, temperature-dependent action of CHS in modulating the structural and functional properties of this GPCR is optimized for 37°C.

    View details for DOI 10.1126/scisignal.abi7031

    View details for PubMedID 35671340

  • Structure-based Evolution of G protein-biased mu-opioid Receptor Agonists. Angewandte Chemie (International ed. in English) Gmeiner, P., Wang, H., Hetzer, F., Huang, W., Qu, Q., Meyerowitz, J., Kaindl, J., Hubner, H., Skiniotis, G., Kobilka, B. K. 2022


    The mu-opioid receptor (muOR) is the major target for opioid analgesics. Activation of muOR initiates signaling through G protein pathways as well as through beta-arrestin recruitment. muOR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased muOR agonist. Here we report the cryoEM structure of PZM21 bound muOR in complex with G i protein. Structure-based evolution led to multiple PZM21 analogs with more pronounced G i protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to muOR in complex with G i protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce beta-arrestin recruitment by the muOR, and hold promise for developing next-generation analgesics with fewer adverse effects.

    View details for DOI 10.1002/anie.202200269

    View details for PubMedID 35385593

  • Atypical structural snapshots of human cytomegalovirus GPCR interactions with host G proteins. Science advances Tsutsumi, N., Maeda, S., Qu, Q., Vogele, M., Jude, K. M., Suomivuori, C., Panova, O., Waghray, D., Kato, H. E., Velasco, A., Dror, R. O., Skiniotis, G., Kobilka, B. K., Garcia, K. C. 1800; 8 (3): eabl5442


    Human cytomegalovirus (HCMV) encodes G protein-coupled receptors (GPCRs) US28 and US27, which facilitate viral pathogenesis through engagement of host G proteins. Here we report cryo-electron microscopy structures of US28 and US27 forming nonproductive and productive complexes with Gi and Gq, respectively, exhibiting unusual features with functional implications. The "orphan" GPCR US27 lacks a ligand-binding pocket and has captured a guanosine diphosphate-bound inactive Gi through a tenuous interaction. The docking modes of CX3CL1-US28 and US27 to Gi favor localization to endosome-like curved membranes, where US28 and US27 can function as nonproductive Gi sinks to attenuate host chemokine-dependent Gi signaling. The CX3CL1-US28-Gq/11 complex likely represents a trapped intermediate during productive signaling, providing a view of a transition state in GPCR-G protein coupling for signaling. Our collective results shed new insight into unique G protein-mediated HCMV GPCR structural mechanisms, compared to mammalian GPCR counterparts, for subversion of host immunity.

    View details for DOI 10.1126/sciadv.abl5442

    View details for PubMedID 35061538

  • Translating science to medicine: The case for physician-scientists. Science translational medicine Utz, P. J., Jain, M. K., Cheung, V. G., Kobilka, B. K., Lefkowitz, R., Yamada, T., Dzau, V. J. 2022; 14 (632): eabg7852


    As the number of physician-scientists continues to decline, action must be taken to support them as they embark on their careers.

    View details for DOI 10.1126/scitranslmed.abg7852

    View details for PubMedID 35171650

  • Structures of active melanocortin-4 receptor-Gs-protein complexes with NDP-alpha-MSH and setmelanotide. Cell research Heyder, N. A., Kleinau, G., Speck, D., Schmidt, A., Paisdzior, S., Szczepek, M., Bauer, B., Koch, A., Gallandi, M., Kwiatkowski, D., Burger, J., Mielke, T., Beck-Sickinger, A. G., Hildebrand, P. W., Spahn, C. M., Hilger, D., Schacherl, M., Biebermann, H., Hilal, T., Kuhnen, P., Kobilka, B. K., Scheerer, P. 2021


    The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R-Gs-protein complexes with two drugs recently approved by theFDA, the peptide agonists NDP-alpha-MSH and setmelanotide, with 2.9A and 2.6A resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-alpha-MSH, a high-affinity linear variant of the endogenous agonist alpha-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor-Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.

    View details for DOI 10.1038/s41422-021-00569-8

    View details for PubMedID 34561620

  • G-protein activation by a metabotropic glutamate receptor. Nature Seven, A. B., Barros-Álvarez, X., de Lapeyrière, M., Papasergi-Scott, M. M., Robertson, M. J., Zhang, C., Nwokonko, R. M., Gao, Y., Meyerowitz, J. G., Rocher, J. P., Schelshorn, D., Kobilka, B. K., Mathiesen, J. M., Skiniotis, G. 2021


    Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6-TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound Gi can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6-TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation.

    View details for DOI 10.1038/s41586-021-03680-3

    View details for PubMedID 34194039

  • Crystal structure of dopamine D1 receptor in complex with G protein and a non-catechol agonist. Nature communications Sun, B., Feng, D., Chu, M. L., Fish, I., Lovera, S., Sands, Z. A., Kelm, S., Valade, A., Wood, M., Ceska, T., Kobilka, T. S., Lebon, F., Kobilka, B. K. 2021; 12 (1): 3305


    Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8A resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.

    View details for DOI 10.1038/s41467-021-23519-9

    View details for PubMedID 34083522

  • Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1. Immunity Tsutsumi, N., Qu, Q., Mavri, M., Baggesen, M. S., Maeda, S., Waghray, D., Berg, C., Kobilka, B. K., Rosenkilde, M. M., Skiniotis, G., Garcia, K. C. 2021


    Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.

    View details for DOI 10.1016/j.immuni.2021.06.001

    View details for PubMedID 34216564

  • How GPCR Phosphorylation Patterns Orchestrate Arrestin-Mediated Signaling. Cell Latorraca, N. R., Masureel, M., Hollingsworth, S. A., Heydenreich, F. M., Suomivuori, C., Brinton, C., Townshend, R. J., Bouvier, M., Kobilka, B. K., Dror, R. O. 2020


    Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary substantially for a given GPCR, and different phosphorylation patterns trigger different arrestin-mediated effects. Here, we determine how GPCR phosphorylation influences arrestin behavior by using atomic-level simulations and site-directed spectroscopy to reveal the effects of phosphorylation patterns on arrestin binding and conformation. We find that patterns favoring binding differ from those favoring activation-associated conformational change. Both binding and conformation depend more on arrangement of phosphates than on their total number, with phosphorylation at different positions sometimes exerting opposite effects. Phosphorylation patterns selectively favor a wide variety of arrestin conformations, differently affecting arrestin sites implicated in scaffolding distinct signaling proteins. We also reveal molecular mechanisms of these phenomena. Our work reveals the structural basis for the long-standing "barcode" hypothesis and has important implications for design of functionally selective GPCR-targeted drugs.

    View details for DOI 10.1016/j.cell.2020.11.014

    View details for PubMedID 33296703

  • Viewing rare conformations of the beta2 adrenergic receptor with pressure-resolved DEER spectroscopy. Proceedings of the National Academy of Sciences of the United States of America Lerch, M. T., Matt, R. A., Masureel, M., Elgeti, M., Kumar, K. K., Hilger, D., Foys, B., Kobilka, B. K., Hubbell, W. L. 2020


    The beta2 adrenergic receptor (beta2AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 A) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The beta2AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free beta2AR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in beta2AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

    View details for DOI 10.1073/pnas.2013904117

    View details for PubMedID 33257561

  • Structural basis for GLP-1 receptor activation by LY3502970, an orally active nonpeptide agonist. Proceedings of the National Academy of Sciences of the United States of America Kawai, T., Sun, B., Yoshino, H., Feng, D., Suzuki, Y., Fukazawa, M., Nagao, S., Wainscott, D. B., Showalter, A. D., Droz, B. A., Kobilka, T. S., Coghlan, M. P., Willard, F. S., Kawabe, Y., Kobilka, B. K., Sloop, K. W. 2020


    Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over beta-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein-coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.

    View details for DOI 10.1073/pnas.2014879117

    View details for PubMedID 33177239

  • Analysis of beta2AR-Gs and beta2AR-Gi complex formation by NMR spectroscopy. Proceedings of the National Academy of Sciences of the United States of America Ma, X., Hu, Y., Batebi, H., Heng, J., Xu, J., Liu, X., Niu, X., Li, H., Hildebrand, P. W., Jin, C., Kobilka, B. K. 2020


    The beta2-adrenergic receptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the beta2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the beta2AR-Gs complex revealed the interaction interface of beta2AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549-555 (2011)], yet, the dynamic process of the beta2AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the beta2AR in response to the full agonist BI-167107 and Gs and Gi1 These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the beta2AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the beta2AR-Gs and the beta2AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs These differences between the beta2AR-Gs and beta2AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.

    View details for DOI 10.1073/pnas.2009786117

    View details for PubMedID 32868434

  • Author Correction: Structural insights into mu-opioid receptor activation. Nature Huang, W., Manglik, A., Venkatakrishnan, A. J., Laeremans, T., Feinberg, E. N., Sanborn, A. L., Kato, H. E., Livingston, K. E., Thorsen, T. S., Kling, R. C., Granier, S., Gmeiner, P., Husbands, S. M., Traynor, J. R., Weis, W. I., Steyaert, J., Dror, R. O., Kobilka, B. K. 2020


    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

    View details for DOI 10.1038/s41586-020-2542-z

    View details for PubMedID 32724208

  • Structural insights into probe-dependent positive allosterism of the GLP-1 receptor. Nature chemical biology Bueno, A. B., Sun, B., Willard, F. S., Feng, D., Ho, J. D., Wainscott, D. B., Showalter, A. D., Vieth, M., Chen, Q., Stutsman, C., Chau, B., Ficorilli, J., Agejas, F. J., Cumming, G. R., Jimenez, A., Rojo, I., Kobilka, T. S., Kobilka, B. K., Sloop, K. W. 2020


    Drugs that promote the association of protein complexes are an emerging therapeutic strategy. We report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a 'molecular glue'. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric Gs. The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Our findings expand protein-protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.

    View details for DOI 10.1038/s41589-020-0589-7

    View details for PubMedID 32690941