Allosteric Nanobodies Reveal the Dynamic Range and Diverse Mechanisms of GPCR Activation


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 signaling effectors such as G proteins and β-arrestins. It is widely appreciated that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (i.e., ‘efficacy’)1. Furthermore, mounting biophysical evidence, primarily using the β-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states2–5. 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)6,7. 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 isoproterenol has a 15,000-fold higher affinity for the β2AR in the presence of Nb80 compared to 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. The allosteric behavior of GPCRs is responsible for the complex signaling properties associated with these important regulators of human physiology. GPCR allostery, defined here as a linkage between the extracellular orthosteric ligand pocket and the intracellular Gprotein binding pocket, has long been analyzed by pharmacological methods8–10 (see supplemental material for additional information). Conformational changes within a GPCR induced by agonist binding can enhance the affinity and binding of intracellular signaling transducers, such as G-proteins and β-arrestins. Conversely, transducer coupling further enhances agonist affinity, resulting in the formation of the ternary complex of receptor, transducer, and ligand (Fig. 1a). The conceptual framework of the ternary complex model (TCM) equates the magnitude of these affinity changes with the strength of transducer activation in cells11,12, as demonstrated for several GPCR systems13–16. However, the structural basis underlying these allosteric relationships and how they relate to ligand efficacy is not well understood. Ligand-dependent GPCR activation has traditionally been conceptualized as a conversion between a single inactive and a single active receptor state. However, recent studies utilizing various spectroscopic techniques have identified multiple inactive and active receptor states, Staus et al. Page 2 Nature. Author manuscript; available in PMC 2017 January 21. A uhor M anscript

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@inproceedings{Staus2016AllostericNR, title={Allosteric Nanobodies Reveal the Dynamic Range and Diverse Mechanisms of GPCR Activation}, author={Dean P. Staus and Ryan T. Strachan and Aashish Manglik and B. S. Pani and Alem W. Kahsai and Tae Hun Kim and Laura M. Wingler and Seungkirl Ahn and Arnab K Chatterjee and Ali Masoudi and Andrew C Kruse and Els Pardon and Jan Steyaert and William I Weis and Robert Scott Prosser and Brian K. Kobilka and Tommaso Costa and Robert J . Lefkowitz}, year={2016} }