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

Staus, Dean P.; Strachan, Ryan T.; Manglik, Aashish; Pani, Biswaranjan; Kahsai, Alem W.; Kim, Tae Hun; Wingler, Laura M.; Ahn, Seungkirl; Chatterjee, Arnab; Masoudi, Ali; Kruse, Andrew C.; Pardon, Els; Steyaert, Jan; Weis, William I.; Prosser, R. Scott; Kobilka, Brian K.; Costa, Tommaso; Lefkowitz, Robert J.

doi:10.1038/nature18636

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

Journal Article · Wed Jul 13 00:00:00 EDT 2016 · Nature (London)

DOI:https://doi.org/10.1038/nature18636· OSTI ID:1368225

Staus, Dean P. ^[1]; Strachan, Ryan T. ^[2]; Manglik, Aashish ^[3]; Pani, Biswaranjan ^[1]; Kahsai, Alem W. ^[1]; Kim, Tae Hun ^[4]; Wingler, Laura M. ^[1]; Ahn, Seungkirl ^[1]; Chatterjee, Arnab ^[1]; Masoudi, Ali ^[1]; Kruse, Andrew C. ^[5]; Pardon, Els ^[6]; Steyaert, Jan ^[6]; Weis, William I. ^[3]; Prosser, R. Scott ^[4]; Kobilka, Brian K. ^[3]; Costa, Tommaso ^[7]; Lefkowitz, Robert J. ^[8]

Duke Univ. Medical Center, Durham, NC (United States)
Univ. of North Carolina, Chapel Hill, NC (United States)
Stanford Univ. School of Medicine, Stanford, CA (United States)
Univ. of Toronto, ON (Canada)
Harvard Medical School, Boston, MA (United States)
Vrije Univ. Brussel, Brussels (Belgium); VIB, Brussels (Belgium). Structural Biology Research Center
Istituto Superiore di Sanità, Rome (Italy)
Duke Univ. Medical Center, Durham, NC (United States); Howard Hughes Medical Inst., Chevy Chase, MD (United States)

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. Furthermore, 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 βA₂R 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.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: American Heart Association (AHA); Italian Ministry of Health; Mathers Foundation; National Institutes of Health (NIH); Stanford Medical Scientist Training Program

OSTI ID:: 1368225

Journal Information:: Nature (London), Journal Name: Nature (London) Journal Issue: 7612 Vol. 535; ISSN 0028-0836

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Structural basis for ion selectivity in TMEM175 K+ channels Brunner, Janine D.; Jakob, Roman P.; Schulze, Tobias eLife, Vol. 9 https://doi.org/10.7554/elife.53683	journal	April 2020

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59 BASIC BIOLOGICAL SCIENCES
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Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation

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