Analysis of β ₂ AR-G _s and β ₂ AR-G _i complex formation by NMR spectroscopy

Abstract

The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the β2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the β2AR-Gs complex revealed the interaction interface of β2AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549–555 (2011)], yet, the dynamic process of the β2AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the β2AR in response to the full agonist BI-167107 and Gs and Gi1. These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the β2AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the β2AR-Gs and the β2AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs. These differences between the β2AR-Gs and β2AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.

Identification of ligand-specific G protein-coupled receptor states and prediction of downstream efficacy via data-driven modeling

“…Although downstream efficacy is an important metric for drug discovery purposes, alternative methods are required to characterize the molecular basis of receptor activation. Spectroscopy experiments have proven useful for this purpose ( Gregorio et al, 2017 ; Imai et al, 2020 ; Kofuku et al, 2012 ; Manglik et al, 2015 ; Ma et al, 2020 ; Nygaard et al, 2013 ; Ueda et al, 2019 ; Weis and Kobilka, 2018 ), yet they are often difficult to compare quantitatively to atomistic simulations due to chemical modifications introduced and/or complex interpretation of measured signals. 19 F-fluorine NMR and double electron-electron resonance (DEER) spectroscopy experiments have shown that the conformational ensembles of carazolol and the apo receptor have similar TM6 distance distributions ( Manglik et al, 2015 ), in agreement with the similarity in their microswitch expectation values and free energy landscapes ( Figure 2b,f and Figure 2—figure supplement 8e ).…”

Identification of ligand-specific G-protein coupled receptor states and prediction of downstream efficacy via data-driven modeling

“…Although downstream efficacy is an important metric for drug discovery purposes, alternative methods are required to characterize the molecular basis of receptor activation. Spectroscopy experiments have proven useful for this purpose (Gregorio et al, 2017;Imai et al, 2020;Kofuku et al, 2012;Manglik et al, 2015;Ma et al, 2020;Nygaard et al, 2013;Ueda et al, 2019;Weis and Kobilka, 2018), yet they are often difficult to compare quantitatively to atomistic simulations due to chemical modifications introduced and/or complex interpretation of measured signals. 19F-fluorine NMR and double-electron resonance (DEER) spectroscopy experiments have shown that the conformational ensembles of carazolol and the apo receptor have similar TM6 distance distributions (Manglik et al, 2015), in agreement with the similarity in their microswitch expectation values and free energy landscapes (Fig.…”

Allosteric communication regulates ligand‐specific GPCR activity

“…GPCRs are dynamic proteins and exist in an equilibrium among multiple conformational states even in the absence of agonist binding. The well characterized conformational states through crystallography [1][2][3]19], and electron microscopy [20][21][22] There are NMR [17,18,[24][25][26][27][28][29], DEER [30][31][32][33], FRET [34][35][36][37] and other experimental studies [38][39][40] that clearly illustrate that ligand selectively bind and stabilize specific receptor conformations [40][41][42][43] that shifts the equilibrium and the relative population of the various conformational states.…”