The DOI matches its Crossref record; 38 of the 38 references it cites were found in a registry or an index.
Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation
Open accessAbstract
G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.
Overview
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Verification
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
In the citing paper
“The absence of the L chain means that the nanobody’s CDRs are distinct from those of antibodies in both sequence and structure, and as a result nanobodies are able to bind antibody-inaccessible epitopes in enzyme active sites, viral capsids and G protein coupled receptors [233, 234].”
From Computational approaches to therapeutic antibody design: established methods and emerging trends (2020) · cites this paper as [233]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Citations
Cites
[20] Processing of X-ray diffraction data collected in oscillation mode
Otwinowski, Z., & Minor, W. (1997) · Methods in Enzymology · doi:10.1016/s0076-6879(97)76066-x · Cited by 33,475
Coot : model-building tools for molecular graphics
Emsley, P., & Cowtan, K. (2004) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444904019158 · Cited by 30,571
Colowick, S. P., Joshi, M. D., Jagannathan, & KAPLAN, N. O. (1966) · Cited by 26,137
PHENIX : a comprehensive Python-based system for macromolecular structure solution
Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J., Grosse-Kunstleve, R. W., et al. (2010) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444909052925 · Cited by 23,716
Phaser crystallographic software
McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., & Read, R. J. (2007) · Journal of Applied Crystallography · doi:10.1107/s0021889807021206 · Cited by 20,612
MolProbity : all-atom structure validation for macromolecular crystallography
Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., & Richardson, D. C. (2010) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444909042073 · Cited by 14,115
Modified Spin-Echo Method for Measuring Nuclear Relaxation Times
Meiboom, S., & Gill, D. (1958) · Review of Scientific Instruments · doi:10.1063/1.1716296 · Cited by 6,001
Crystal structure of the β2 adrenergic receptor–Gs protein complex
Rasmussen, S. G. F., DeVree, B. T., Zou, Y., Kruse, A. C., Chung, K. Y., Kobilka, T. S., Thian, F. S., Chae, P. S., Pardon, E., Calinski, D., et al. (2011) · Nature · doi:10.1038/nature10361 · Cited by 3,009
Self-citationBallesteros, J. A., & Weinstein, H. (1995) · Methods in neurosciences · doi:10.1016/s1043-9471(05)80049-7 · Cited by 3,001
Structure of a nanobody-stabilized active state of the β2 adrenoceptor
Rasmussen, S. G. F., Choi, H., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S., DeVree, B. T., Rosenbaum, D. M., Thian, F. S., Kobilka, T. S., et al. (2011) · Nature · doi:10.1038/nature09648 · Cited by 1,709
Self-citationLéan, A. D., Stadel, J. M., & Lefkowitz, R. J. (1980) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(20)79672-9 · Cited by 1,606
Self-citationSamama, P., Cotecchia, S., Costa, T., & Lefkowitz, R. J. (1993) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)53442-6 · Cited by 1,256
Self-citationColquhoun, D. M. (1998) · British Journal of Pharmacology · doi:10.1038/sj.bjp.0702164 · Cited by 893
The Dynamic Process of β2-Adrenergic Receptor Activation
Nygaard, R., Zou, Y., Dror, R. O., Mildorf, T. J., Arlow, D. H., Manglik, A., Pan, A. C., Liu, C. W., Fung, J. J., Bokoch, M. P., et al. (2013) · Cell · doi:10.1016/j.cell.2013.01.008 · Cited by 815
Self-citationBiased Signaling Pathways in β 2 -Adrenergic Receptor Characterized by 19 F-NMR
Liu, J. J., Horst, R., Katritch, V., Stevens, R. C., & Wüthrich, K. (2012) · Science · doi:10.1126/science.1215802 · Cited by 706
Whorton, M. R., Bokoch, M. P., Rasmussen, S. G. F., Huang, B., Zare, R. N., Kobilka, B. K., & Sunahara, R. K. (2007) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0611448104 · Cited by 686
Self-citationStructural Insights into the Dynamic Process of β2-Adrenergic Receptor Signaling
Manglik, A., Kim, T. H., Masureel, M., Altenbach, C., Yang, Z., Hilger, D., Lerch, M. T., Kobilka, T. S., Thian, F. S., Hubbell, W. L., et al. (2015) · Cell · doi:10.1016/j.cell.2015.04.043 · Cited by 671
Self-citationBallesteros, J. A., Jensen, A. D., Liapakis, G., Rasmussen, S. G. F., Shi, L., Gether, U., & Javitch, J. A. (2001) · Journal of Biological Chemistry · doi:10.1074/jbc.m103747200 · Cited by 619
Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody
Ring, A. M., Manglik, A., Kruse, A. C., Enos, M. D., Weis, W. I., García, K. C., & Kobilka, B. K. (2013) · Nature · doi:10.1038/nature12572 · Cited by 500
Self-citationQuantifying Ligand Bias at Seven-Transmembrane Receptors
Rajagopal, S., Ahn, S., Rominger, D. H., Gowen-MacDonald, W., Lam, C. M., DeWire, S. M., Violin, J. D., & Lefkowitz, R. J. (2011) · Molecular Pharmacology · doi:10.1124/mol.111.072801 · Cited by 391
Self-citationDror, R. O., Arlow, D. H., Borhani, D. W., Jensen, M. Ø., Piana, S., & Shaw, D. E. (2009) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0811065106 · Cited by 307
G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody
Hino, T., Arakawa, T., Iwanari, H., Yurugi-Kobayashi, T., Ikeda-Suno, C., Nakada-Nakura, Y., Kusano‐Arai, O., Weyand, S. N., Shimamura, T., Nomura, N., et al. (2012) · Nature · doi:10.1038/nature10750 · Cited by 302
Multiple ligand-specific conformations of the β2-adrenergic receptor
Kahsai, A. W., Xiao, K., Rajagopal, S., Ahn, S., Shukla, A. K., Sun, J., Oas, T. G., & Lefkowitz, R. J. (2011) · Nature Chemical Biology · doi:10.1038/nchembio.634 · Cited by 252
Self-citationKofuku, Y., Ueda, T., Okude, J., Shiraishi, Y., Kondo, K., Maeda, M., Tsujishita, H., & Shimada, I. (2012) · Nature Communications · doi:10.1038/ncomms2046 · Cited by 199
Moukhametzianov, R., Warne, T., Edwards, P. C., Serrano‐Vega, M. J., Leslie, A. G. W., Tate, C. G., & Schertler, G. F. X. (2011) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1100185108 · Cited by 179
Ehlert, F. J. (1985) · Molecular Pharmacology · doi:10.1016/s0026-895x(25)14182-5 · Cited by 168
Kobilka, B. K. (1995) · Analytical Biochemistry · doi:10.1006/abio.1995.1533 · Cited by 166
Self-citationWREGGETT, K. A., & Léan, A. D. (1984) · Molecular Pharmacology · doi:10.1016/s0026-895x(25)15078-5 · Cited by 151
The Dynamic Process of b 2 -Adrenergic Receptor Activation
Nygaard, R., Zou, Y., Dror, R. O., Mildorf, T. J., Arlow, D. H., Manglik, A., Pan, A. C., Liu, C. W., Bokoch, M. P., Thian, F. S., et al. (2013) · Cited by 146
Self-citationStaus, D. P., Wingler, L. M., Strachan, R. T., Rasmussen, S. G. F., Pardon, E., Ahn, S., Steyaert, J., Kobilka, B. K., & Lefkowitz, R. J. (2013) · Molecular Pharmacology · doi:10.1124/mol.113.089516 · Cited by 145
Self-citationA three-stage biophysical screening cascade for fragment-based drug discovery
Mashalidis, E. H., Sledz, P., Lang, S., & Abell, C. (2013) · Nature Protocols · doi:10.1038/nprot.2013.130 · Cited by 144
Strachan, R. T., Sun, J., Rominger, D. H., Violin, J. D., Ahn, S., Thomsen, A. R. B., Zhu, X., Kleist, A. B., Costa, T., & Lefkowitz, R. J. (2014) · Journal of Biological Chemistry · doi:10.1074/jbc.m114.548131 · Cited by 135
Self-citationThe quantitative analysis of drug–receptor interactions: a short history
Colquhoun, D. M. (2006) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2006.01.008 · Cited by 130
Tracing an allosteric pathway regulating the activity of the HslV protease
Shi, L., & Kay, L. E. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1318476111 · Cited by 82
Onaran, H. O., Rajagopal, S., & Costa, T. (2014) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2014.09.010 · Cited by 43
Self-citationAllosteric Coupling and Conformational Fluctuations in Proteins
Onaran, H. O., & Costa, T. (2009) · Current Protein and Peptide Science · doi:10.2174/138920309787847644 · Cited by 21
Self-citationVezzi, V., Onaran, H. O., Molinari, P., Guerrini, R., Balboni, G., Calò, G., & Costa, T. (2013) · Journal of Biological Chemistry · doi:10.1074/jbc.m113.474452 · Cited by 19
Self-citationComplex ligand-protein systems: a globally convergent iterative method for the n × m case
Pradines, J., Hasty, J., & Pakdaman, K. (2001) · Journal of Mathematical Biology · doi:10.1007/s002850100086 · Cited by 8
Flagged first, then by citations
Cited by
Sorted by citations
The Molecular Basis of G Protein–Coupled Receptor Activation
Weis, W. I., & Kobilka, B. K. (2018) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-060614-033910 · Cited by 1,227
Self-citationGPCR Dynamics: Structures in Motion
Latorraca, N. R., Venkatakrishnan, A., & Dror, R. O. (2016) · Chemical Reviews · doi:10.1021/acs.chemrev.6b00177 · Cited by 861
Biased signalling: from simple switches to allosteric microprocessors
Smith, J. S., Lefkowitz, R. J., & Rajagopal, S. (2018) · Nature Reviews Drug Discovery · doi:10.1038/nrd.2017.229 · Cited by 777
Self-citationCommon activation mechanism of class A GPCRs
Zhou, Q., Yang, D., Wu, M., Guo, Y., Guo, W., Li, Z., Cai, X., Dai, A., Jang, W., Shakhnovich, E. I., et al. (2019) · eLife · doi:10.7554/elife.50279 · Cited by 648
How Ligands Illuminate GPCR Molecular Pharmacology
Wacker, D., Stevens, R. C., & Roth, B. L. (2017) · Cell · doi:10.1016/j.cell.2017.07.009 · Cited by 560
Metadata
- Authors
- Staus, D. P., Strachan, R. T., Manglik, A., Pani, B., Kahsai, A. W., Kim, T. H., Wingler, L. M., Ahn, S., Chatterjee, A., Masoudi, A., Kruse, A. C., Pardon, E., Steyaert, J., Weis, W. I., Prosser, R. S., Kobilka, B. K., Costa, T., & Lefkowitz, R. J.
- Affiliations
- Duke Medical Center (US)
- University of North Carolina at Chapel Hill (US)
- Stanford Medicine (US)
- University of Toronto (CA)
- Harvard University (US)
- Vrije Universiteit Brussel (BE)
- Istituto Superiore di Sanità (IT)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Kobilka, Brian K.
- Costa, Tommaso
- Lefkowitz, Robert J.
- ORCID
- Venue
- Nature
- Published
- 2016-07
- Volume
- 535
- Issue
- 7612
- Pages
- 448-452
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC4961583
- OpenAlex
- W2471992026
- Cited by
- 330
- References
- 38
- Citations by year
- 2026: 11 · 2025: 18 · 2024: 23 · 2023: 28 · 2022: 26 · 2021: 46 · 2020: 36 · 2019: 47 · 2018: 43 · 2017: 39 · 2016: 13
- FWCI
- 25.13
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- eScholarship (California Digital Library) (submittedVersion)
- VUBIR (Vrije Universiteit Brussel) (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- American Heart Association
- Ministero della Salute (02351158, RF-201102351158)
- National Heart, Lung, and Blood Institute (HL 16037, R01HL070631, HL-70631, T32HL007101, R01HL016037)
- National Institute of General Medical Sciences (T32GM008294)
- National Institute of Diabetes and Digestive and Kidney Diseases (T32DK007731)
- National Institute of Neurological Disorders and Stroke (NS-028471, R01NS028471, R37NS028471)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- beta2-adrenergic receptor, G protein-coupled receptor activation, receptor conformational states, three-state model, partial agonists, full agonists, ligand efficacy, receptor stabilization, orthosteric binding pocket, receptor dynamics
- MeSH terms
- Allosteric Regulation, Allosteric Site, Humans, Isoproterenol, Ligands, Models, Molecular, Protein Conformation, Receptors, Adrenergic, beta-2, Crystallography, X-Ray, Nuclear Magnetic Resonance, Biomolecular, Drug Partial Agonism, Protein Stability, Adrenergic beta-2 Receptor Agonists, Single-Domain Antibodies
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
