Structural equilibrium underlying ligand-dependent activation of β2-adrenoreceptor

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins mediating cellular signals in response to extracellular stimuli. Although three-dimensional structures showcase snapshots

that can be sampled in the process and nuclear magnetic resonance detects conformational equilibria, the mechanism by which agonist-activated GPCRs interact with various effectors remains

elusive. Here, we used paramagnetic nuclear magnetic resonance for leucine amide resonances to visualize the structure of β2-adrenoreceptor in the full agonist-bound state, without

thermostabilizing mutations abolishing its activity. The structure exhibited a unique orientation of the intracellular half of the transmembrane helix 6, forming a cluster of

G-protein-interacting residues. Furthermore, analyses of efficacy-dependent chemical shifts of the residues near the pivotal PIF microswitch identified an equilibrium among three

conformations, including one responsible for the varied signal level in each ligand-bound state. Together, these results provide a structural basis for the dynamic activation of GPCRs and

shed light on GPCR-mediated signal transduction.

Atomic coordinates for β2AR-Δ in the fully activated state have been deposited in the PDB under accession code 6KR8. The NMR data and restraints used in the structure calculations have been

deposited in the Biological Magnetic Resonance Data Bank under accession number 36284. The other data that support the findings of this study are available from the corresponding author upon

reasonable request.

All code used in this study is available from the corresponding author upon reasonable request.

This work is supported by The Ministry of Education, Culture, Sports, Science and Technology and the Japan Society for the Promotion of Science KAKENHI grant number JP17H06097 and by the

development of innovative drug discovery technologies for middle-sized molecules from the Japan Agency for Medical Research and Development (to I.S.).

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

Shunsuke Imai, Tomoki Yokomizo, Yutaka Kofuku, Yutaro Shiraishi, Takumi Ueda & Ichio Shimada

S.I. designed the study, constructed β2AR-Δ and its variants, purified proteins, conducted GTP turnover assays with T.Y., acquired NMR spectra, analyzed the PRE data and calculated the PRE

structure and wrote the manuscript. Y.K. established the purification protocol of β2AR at the early stage of the project and constructed the plasmid for the expression of

cystathionine-γ-synthase. Y.S. prepared the virus stock for the coexpression of the Gs heterotrimer and cultured the cells by using the virus stock. T.U. performed the exchange Monte Carlo

calculation. I.S. designed the study, analyzed the data and wrote the manuscript.

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