Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.

TitleAllosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.
Publication TypeJournal Article
Year of Publication2016
AuthorsStaus, D. P., R. T. Strachan, A. Manglik, B. Pani, A. W. Kahsai, T. Hun Kim, L. M. Wingler, S. Ahn, A. Chatterjee, A. Masoudi, A. C. Kruse, E. Pardon, J. Steyaert, W. I. Weis, S. R Prosser, B. K. Kobilka, T. Costa, and R. J. Lefkowitz
JournalNature
Volume535
Issue7612
Pagination448-52
Date Published2016 Jul 21
ISSN1476-4687
KeywordsAdrenergic beta-2 Receptor Agonists, Allosteric Regulation, Allosteric Site, Crystallography, X-Ray, Drug Partial Agonism, Humans, Isoproterenol, Ligands, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Stability, Receptors, Adrenergic, beta-2, Single-Domain Antibodies
Abstract

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.

DOI10.1038/nature18636
Alternate JournalNature
PubMed ID27409812
PubMed Central IDPMC4961583
Grant ListT32HL007101 / HL / NHLBI NIH HHS / United States
T32 HL007101 / HL / NHLBI NIH HHS / United States
HL16037 / HL / NHLBI NIH HHS / United States
R01 HL016037 / HL / NHLBI NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
R01 HL070631 / HL / NHLBI NIH HHS / United States
HL70631 / HL / NHLBI NIH HHS / United States
R01 NS028471 / NS / NINDS NIH HHS / United States
R37 NS028471 / NS / NINDS NIH HHS / United States
NS028471 / NS / NINDS NIH HHS / United States
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