title: Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.
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.
authors.forename: Dean P
authors.initials: DP
authors.lastname: Staus
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Strachan
authors.forename: Aashish
authors.initials: A
authors.lastname: Manglik
authors.forename: Biswaranjan
authors.initials: B
authors.lastname: Pani
authors.forename: Alem W
authors.initials: AW
authors.lastname: Kahsai
authors.forename: Tae Hun
authors.initials: TH
authors.lastname: Kim
authors.forename: Laura M
authors.initials: LM
authors.lastname: Wingler
authors.forename: Seungkirl
authors.initials: S
authors.lastname: Ahn
authors.forename: Arnab
authors.initials: A
authors.lastname: Chatterjee
authors.forename: Ali
authors.initials: A
authors.lastname: Masoudi
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Kruse
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.forename: William I
authors.initials: WI
authors.lastname: Weis
authors.forename: R Scott
authors.initials: RS
authors.lastname: Prosser
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kobilka
authors.forename: Tommaso
authors.initials: T
authors.lastname: Costa
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology*; D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site / Q000187:drug effects; D018360:Crystallography, X-Ray; D054333:Drug Partial Agonism; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000378:metabolism*; D061905:Single-Domain Antibodies / Q000494:pharmacology*
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D061905:Single-Domain Antibodies; D007545:Isoproterenol