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Structural Insights Into Binding Specificity ARTICLES https://doi.org/10.1038/s41589-018-0145-x Corrected: Publisher Correction Structural insights into binding specificity, efficacy and bias of a β2AR partial agonist Matthieu Masureel1,17, Yaozhong Zou1,2,17, Louis-Philippe Picard3, Emma van der Westhuizen 3,13, Jacob P. Mahoney4,14, João P. G. L. M. Rodrigues1,5,6, Thomas J. Mildorf7,15, Ron O. Dror7,16, David E. Shaw7,8, Michel Bouvier 3, Els Pardon9,10, Jan Steyaert9,10, Roger K. Sunahara 11, William I. Weis1,6, Cheng Zhang 12* and Brian K. Kobilka1* Salmeterol is a partial agonist for the β 2 adrenergic receptor (β2 AR) and the first long-acting β 2AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol’s safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound β2 AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between β 1AR and β 2AR explain the high receptor-subtype selectivity. A structural comparison with the β2 AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser2045.43 and Asn2936.55. Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited β -arrestin recruitment for salmeterol. -protein-coupled receptors (GPCRs) are able to respond to mortality in asthmatics in several clinical trials. This liability is miti- a large variety of ligands with different efficacy profiles for gated when LABAs are combined with an inhaled corticosteroid9–11. Gspecific signaling pathways1: full agonists that maximally The high selectivity and long-acting properties of salmeterol have stimulate; partial agonists that produce submaximal stimulation been attributed to its unusual bitopic structure. In addition to the even at fully saturating concentrations; inverse agonists that sup- saligenin ethanolamine pharmacophore that replaces the catechol- press basal signaling; and neutral antagonists that bind the receptor amine structure of the endogenous β2 AR ligand epinephrine (also without stimulating or inhibiting basal signaling. Among these cat- known as adrenaline), salmeterol contains an additional moiety of egories, partial agonists are often better tolerated than full agonists an aryloxyalkyl tail consisting of a phenol ring with an 11-atom ether as therapeutics2. chain (Fig. 1a). While the pharmacophore binds the orthosteric site, Salmeterol is a partial agonist for the human β2 AR. As a potent which is responsible for receptor activation, the aryloxyalkyl tail has bronchodilator, it is among the most prescribed drugs for the been proposed to bind to an additional site (exosite), thus allowing treatment of asthma and chronic obstructive pulmonary disease for additional interactions responsible for the high receptor-subtype 3 (COPD) . Compared with some other β2 AR agonists such as iso- selectivity and the slow dissociation rate contributing to its long proterenol and salbutamol, salmeterol has two desirable pharmaco- duration of action12. Previous mutagenesis and biochemical stud- 4,12–16 logical properties. First, salmeterol is able to distinguish β2 AR from ies seeking to locate the exosite have yielded conflicting results . 4 β1 AR for selective stimulation (1,400- to 3,000-fold selectivity) , Salmeterol is also of interest because it is a functionally selective 5 thereby minimizing cardiac toxicity . Second, salmeterol belongs to β2 AR partial agonist with a 5- to 20-fold bias toward Gs over arres- 17,18 the class of long-acting β2 AR agonists (LABAs), which have a dura- tin . Previous studies have revealed that activation of the β2 AR tion of action up to 12 h, in contrast to the short-acting β2 AR ago- by salmeterol, compared with isoproterenol, leads to slower initial nists, such as salbutamol, which have only a 4- to 6-h duration of rates of G-protein-coupled receptor kinase (GRK) phosphorylation action6–8. Those pharmacological properties of salmeterol have con- and similar maximal degrees of phosphorylation19,20, but strongly tributed to its successful use in treating asthma and COPD for more decreased arrestin-mediated receptor internalization and desensiti- than two decades. However, LABAs, especially salmeterol, when zation17,21,22, thus contributing to the prolonged therapeutic effect of used alone, have been suggested to be associated with increased salmeterol in bronchial dilation as a result of β2 AR stimulation. This 1Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. 2Geneus Technologies, Ltd, Chengdu, Sichuan, People’s Republic of China. 3Department of Biochemistry, Institute for Research in Immunology and Cancer, Université de Montreal, Montreal, Québec, Canada. 4Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA. 5Department of Computer Science, Stanford University, Stanford, CA, USA. 6Department of Structural Biology, Stanford University, Stanford, CA, USA. 7D. E. Shaw Research, New York, NY, USA. 8Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA. 9Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium. 10Structural Biology Research Center, VIB, Brussels, Belgium. 11Department of Pharmacology, University of California San Diego School of Medicine, La Jolla, CA, USA. 12Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 13Present address: Monash Institute for Pharmaceutical Sciences, Monash University, Victoria, Australia. 14Present address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. 15Present address: Dropbox, New York, NY, USA. 16Present address: Department of Computer Science and Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA, USA. 17These authors contributed equally: Matthieu Masureel, Yaozhong Zou. *e-mail: [email protected]; [email protected] NatURE CHemical Biology | VOL 14 | NOVEMBER 2018 | 1059–1066 | www.nature.com/naturechemicalbiology 1059 ARTICLES NATURE CHEMICAL BIOLOGY a OH b H T4L N HO O Salmeterol HO OH AR H β2 N Salmeterol HO HO Salbutamol Nb71 OH c H Extracellular HO N Exosite Epinephrine HO O O OH H HN N HO Orthosteric site BI-167107 Fig. 1 | Crystal structure of salmeterol-bound β2AR. a, Chemical structures of the following β2 AR ligands: partial agonists salmeterol and salbutamol; full agonists epinephrine and BI-16707. The respective pharmacophores that bind the orthosteric ligand-binding pocket are highlighted in purple, and the aryloxyalkyl tail of salmeterol, which binds the exosite, is highlighted in blue. b, Overall ribbon representation of the salmeterol-bound β2 AR–Nb71 complex. The T4L lysozyme fusion facilitates crystallization, and Nb71 stabilizes the active, salmeterol-bound (orange spheres) β2 AR. c, Cross-section through the receptor, with the interior in black, highlighting salmeterol (orange spheres) occupying the orthosteric site and the exosite. signaling bias may contribute to the advantageous therapeutic profile the active-state structures of the β 2AR and µ -opioid receptor have of salmeterol by maintaining bronchodilation through Gs-mediated required either a G protein or conformation-specific camelid anti- signaling while minimizing arrestin-mediated β2 AR desensitization body fragments (nanobodies) to stabilize the active states of the and avoiding arrestin-dependent proinflammatory effects23,24. receptors25,26,29. The first active-state structure of a hormone-acti- Recent progress in the structural characterization of β adrenergic vated GPCR was obtained with a nanobody named Nb80, which was receptors, especially the crystal structures of active β2 AR bound to the obtained from a llama immunized with β 2AR bound to the ultra- full agonists BI-167107 or epinephrine, with either a Gs protein or high-affinity full agonist BI-167107 reconstituted into phospholipid 25 active-state-stabilizing nanobody, as well as the crystal structures of vesicles . The structures of β2 AR in the β2 AR–Nb80 complex and 25,26 inactive avian β1 AR bound to a variety of full and partial agonists, have the β2 AR–Gs complex are very similar . However, evidence from greatly advanced understanding of the pharmacology and activation biophysical studies suggests that partial agonists may stabilize dis- of β adrenergic receptors25–29. To further understand the molecular tinct states33; consequently, Nb80 may not be the best candidate to basis for the unusual pharmacological properties of salmeterol and stabilize salmeterol-bound β2 AR. its partial agonism, we obtained the crystal structure of salmeterol- Whereas Nb80 preferentially binds agonist-occupied β2 AR rather 25 bound human β2 AR in an active state stabilized by a conformation- than antagonist-occupied β2 AR , Octet RED studies indicated that specific nanobody, Nb71. The structure reveals the location of the Nb80 also preferentially binds β2 AR bound to BI-167107 and epi- exosite and provides a structural explanation for the high receptor- nephrine rather than β 2AR bound to salmeterol (Supplementary subtype selectivity and distinct signaling behavior of salmeterol. Table 1). Moreover,
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