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

Author Contributions D.P.S and R.T.S conceived the project. Pharmacological assessment of the interactions between Nb60 and Nb80 with the β 2AR were designed, performed and analyzed by D.P.S., R.T.S, B.P., S.A., and A.C. Formation, purification, and crystallization of the β 2 AR-Nb60-Carazolol complex was conducted by D.P.S. and A.M. Data collection, refinement, and structural analysis was done by A.M., A.C.K., and A. Masoudi, and W.I.W. NMR spectroscopy was executed by A.M., T.H.K, and supervised by R.S.P. Isothermal titration calorimetry was conducted by A.W.K. Nanobody reagents were provided by E.P. and J.S. Detailed allosteric analysis of radioligand binding data was implemented by T.C. Figures were created by D.P.S., A.M., T.C., L.M.W., R.T.S., and A. Masoudi. Manuscript was written by D.P.S, T.C., R.T.S., L.M.W., A.M., A.K.C, and R.J.L. Overall research was supervised by B.K.K. and R.J.L. 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 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 ( β 2 AR) as a model system, supports the existence of multiple active and inactive conformational states 2–5 . 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 β 2 AR 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 β 2 AR 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 β 2 AR 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 (R 80 ). In contrast, for a number of partial agonists, both stabilization of R 80 and destabilization of the inactive, Nb60-bound state (R 60 ) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing