Cryo-EM structure of a cell-free synthesized full-length human β1-adrenergic receptor in complex with Gs

SUMMARY The third intracellular loop (ICL3) of the β1-adrenergic receptor (β1AR) plays a critical role in regulating G protein coupling, yet the structural basis has remained unclear due to truncations of ICL3 in all available structures of the β1AR in complex with Gs or a G protein mimetic nanobody. To address this, we used cell-free cotranslational insertion of full-length human β1AR into nanodiscs and determined its cryo-EM structure in complex with Gs. In this structure, ICL3 extends transmembrane helix 5, resulting in enhanced interactions with Gαs and in a slight rotation of the engaged G protein. This repositioning enables new polar interactions between Gαs, ICL2 and helix 8, while ICL1 and helix 8 form additional contacts with Gβ. These structural insights, supported by mutational analysis, demonstrate that ICL3 enhances G protein activation and downstream cAMP signaling by promoting more extensive interactions between the receptor and the heterotrimeric G protein. Competing Interest Statement FM and JK are shareholders of Cube Biotech, which sells products and services related to membrane protein characterization. All other authors declare no competing financial interests. The article is the authors' original work, has not received prior publication and is not under consideration for publication elsewhere. Footnotes ↵5 Lead contact