This work was funded by German Research Foundation (DFG) through CRC1423, project number 421152132, subproject C01 (to P.W.H.) and subprojects A01, A05 and Z03 (to P.S.), Stiftung Charité and the Einstein Center Digital for Future to P.W.H. P.S. is further supported through CRC 1078–Project ID 221545957–SFB 1078, subproject B06; through the cluster of excellence ‘UniSysCat‘ (under Germany’s Excellence Strategy-EXC2008/1-390540038 and through the European Union’s Horizon 2020 MSCA Program under grant agreement 956314 (ALLODD). This work was also funded by National Institutes of Health grant R01NS028471 (to B.K.K.), by National Natural Science Foundation of China (Grant 32122041 to X.L.) and by Tsinghua University Initiative Scientific Research Program (to X.L.). We are grateful to A. Inoue (Tohoku University, Japan) for providing the CRISPR–Cas9-edited triple knockout barr1/barr2/β2AR HEK293A cells and to H. Schihada (Philipps-Universität Marburg, Germany) for providing the Gs-CASE sensor DNA material and advice for the BRET dissociation assay. We thank B. Bauer (Charité–Universitätsmedizin Berlin, Germany) for assistance in molecular biology and purifying reagents. B.K.K. and P.W.H. acknowledge the Einstein Foundation and the Berlin Institute of Health for their support. We are grateful to M. Heck (Charité–Universitätsmedizin Berlin, Germany) for advice on the statistical analysis of the BRET 2 assay and M. Heck and K. P. Hofmann (Charité–Universitätsmedizin Berlin, Germany) for helpful discussions. P.F.S. also holds external affiliations with the Institute of Theoretical Chemistry, University of Vienna, Austria, the Universidad Nacional de Colombia, Bogotá, Colombia, the Center for noncoding RNA in Technology and Health at the University of Copenhagen and the Santa Fe Institute, Santa Fe, New Mexico, USA. We gratefully acknowledge the scientific support and HPC resources provided by the Erlangen National High Performance Computing Center (NHR@FAU) of the Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) under the NHR project p101ae. NHR funding is provided by federal and Bavarian state authorities. NHR@FAU hardware is partially funded by DFG (440719683).

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting guanine nucleotide exchange. Here, we investigate the coupling of G proteins with GPCRs and describe the events that ultimately lead to the ejection of GDP from its binding pocket in the Gα subunit, the rate-limiting step during G-protein activation. Using molecular dynamics simulations, we investigate the temporal progression of structural rearrangements of GDP-bound Gs protein (Gs·GDP; hereafter GsGDP) upon coupling to the β2-adrenergic receptor (β2AR) in atomic detail. The binding of GsGDP to the β2AR is followed by long-range allosteric effects that significantly reduce the energy needed for GDP release: the opening of α1-αF helices, the displacement of the αG helix and the opening of the α-helical domain. Signal propagation to the Gs occurs through an extended receptor interface, including a lysine-rich motif at the intracellular end of a kinked transmembrane helix 6, which was confirmed by site-directed mutagenesis and functional assays. From this β2AR-GsGDP intermediate, Gs undergoes an in-plane rotation along the receptor axis to approach the β2AR-Gsempty state. The simulations shed light on how the structural elements at the receptor-G-protein interface may interact to transmit the signal over 30 Å to the nucleotide-binding site. Our analysis extends the current limited view of nucleotide-free snapshots to include additional states and structural features responsible for signaling and G-protein coupling specificity.

Peer reviewed