Single-particle cryo-EM structural studies of the β2AR–Gs complex bound with a full agonist formoterol

Dear Editor, G-protein-coupled receptors (GPCRs) modulate cytoplasmic signaling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Differential ligands binding to receptor promote different conformations of GPCR–G-protein complex, which can adopt diverse active states. Such liganddirected biased agonism is now an important focus in drug discovery. Therefore, structure determination of GPCR–G-protein complexes in variable activation states is important to elucidate the mechanisms of signal transduction, and to facilitate drug discovery. The β2-adrenergic receptor (β2AR), a canonical class A GPCR, is activated by adrenaline and norepinephrine. Recent years, many agonists have been synthesized to stimulate the activation of β2AR, and some of these ligands have been developed for the clinical treatment of asthma and chronic obstructive pulmonary diseases. Since the first crystal structure of β2AR bound with the inverse agonist carazolol was reported, several crystal structures of the β2AR bound with different agonists have been determined. However, only structure of the BI167107-bound β2AR–Gs complex was determined to date, which represented the real active-state of β2AR . Whether the observed β2AR–Gs interactions in the complex upon BI167107 binding provide a general rule for signal transductions from the binding of different agonists to cyclic adenosine monophosphate (cAMP) accumulation requires further validation, and also remains a major concern for the pharmacological understanding of β2AR and further drug development. Formoterol is a selective, long-acting agonist of β2AR, which is unique as it both has a long-acting bronchodilator effect (> 12 h) and exhibits a fast onset of action (1–3min from inhalation), suggesting that it is effective both as maintenance and reliever medication. Herein, the cryo-EM structure of the formoterol-bound β2AR–Gs complex was determined with an overall resolution of 3.8 Å. Formoterol was reported to have a weaker affinity than BI167107 in β2AR binding, and also has lower β2AR activation potency than BI167107 (Fig. 1a). Therefore, comparisons between the structure of the formoterol– β2AR–Gs complex and the previously reported structure of the BI167107–β2AR–Gs complex will provide insights into the conformational responses of the β2AR upon binding to agonists with different potency. First, we optimized the previously reported β2AR construct and obtained an engineered construct with improved expression in the sf9 insect expression system (Supplementary Fig. S1). The formoterol–β2AR–Gs complex in lauryl maltose neopentyl glycol (LMNG) detergent micelles was visualized using a Titan Krios microscope. After imaging and initial two-dimensional classification, three-dimensional classification yielded a final map at a global resolution of 3.8 Å (Fig. 1b; Supplementary Figs. S2, S3 and Table S1). The cryo-EM density map of the formoterol–β2AR–Gs complex exhibits well-resolved side chains, allowing rotamer placements for most amino acids (Fig. 1b; Supplementary Fig. S4). As revealed in Fig. 1c, the agonist formoterol is clearly identified in the orthosteric-binding site on the

Bacteria were grown at 37°C in LB medium containing 100mg/mL ampicillin to a density of

β2AR-Gs complex preparation
The purified β2AR and Gs complex were mixed at a molar ratio 1:1.

Functional analysis by cAMP assay
The function data of β2AR was measured based on intracellular cAMP assay using cAMP-Gs dynamic kit 3 (Cisbio). HEK293T cells were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum, 50 µg/mL penicillin and 50 μg/mL streptomycin using 37°C incubator supplied with 5% CO2. Cells were seeded onto 6-well cell culture plates before transfection.
After overnight culture, the cells were transfected with β2AR-pcDNA3.1 plasmid (or β2AR mutation plasmid) using Lipofectamine 3000 transfection reagent (Invitrogen). About 12 h after transfection, the cells were collected, and suspended in DMEM containing 500 µM 3-isobutyl-1methylxanthine (IBMX) (Sigma). Then, the cells were seeded onto 384-well plates at a density of 7500 cells per well. The transfected cells were incubated for 45 min with gradient concentrations of formoterol at incubator. Finally, the cells were incubated with cAMP-D2 and anti-cAMP Crypate for 1 h at room temperature and time-resolved FRET signals were measured at 620 nm and 650 nm using CLARIOstar plate reader (BMG LabTech, Germany). Data were mean ± s.e.m. of three independent experiments performed in triplicate.

Cryo-EM images were recorded on a Gatan K3 Summit direct electron detector in an FEI
Titan Krios electron microscope at 300 kV. SerialEM was used for automated data collection 4 .
Movies were collected at a nominal magnification of 29000 × in counting mode, corresponding to a pixel size of 0.53 Å. The defocus range was set to -1.5 to -2.5 µm . Image stacks were record with a total dose of ~60 e -/Å. The electron dose rate is 20 e -/Å/s.

Image processing
A total of 2102 micrograph stacks were collected and subjected for motion correction using motioncor2 5 . Contrast transfer function parameters were estimated with Gctf 6 . After manually removal of bad micrographs, a total of 1,781,163 particles were automatically picked from 1733 micrographs. These particles were then subjected to reference-free 2D classification and particles of the best classes were re-extracted for further data processing. A 3D initial model was generated using stochastic gradient descent (SGD) algorithm in Relion-3.0 7 . Next, 776,825 picked particles were performed for global angular searching 3D classification. 218,011 particles from the best-looking class were selected for 3D auto-refinement, which generated an EM map with an overall resolution of 4.06 Å. By post-processing and particle polishing, the final resolution of was improved to 3.8 Å. Map resolution was estimated with the gold-standard Fourier shell correction 0.143 criterion. Local resolution was estimated using Resmap 8 .

Model building and refinement
The initial model for the formoterol-bound β2AR-Gs complex was derived from BI167107-bound β2AR-Gs complex (3SN6) followed by extensive remodeling using COOT 9 .
The N-terminal residues 1-29, residues 240-264 and C-terminal residues 341-413 of β2AR were not built due to the lack of corresponding densities. Structure refinements were carried out by PHENIX in real space with secondary structure and geometry restraints to prevent structure overfitting 10,11 . Overfitting of the model was monitored by reefing the model in one or two independent maps from the gold-standard refinement approach and testing the refined model Supplementary Table S1. Statistics of cryo-EM data collection, 3D reconstruction and model refinement.