Cryo-EM structure of a single-chain β1-adrenoceptor - AmpC β-lactamase fusion protein
The insertion of fusion proteins has enabled the crystallization of a wide range of G-protein-coupled receptors. Here, we adapted this engineering strategy to cryo-electron microscopy (cryo-EM). We inserted the soluble protein AmpC {beta}-lactamase into the third intracellular loop (ICL3) of ultra-thermostable {beta}1-adrenoceptor ({beta}1AR) via chimeric helix fusions. Biochemical and biophysical characterization showed that the resulting fusion protein after expression, solubilization and purification was monodisperse and able to bind the known {beta}1AR weak partial agonist cyanopindolol, and the antagonist propranolol. The protein particles comprised sufficient mass and discernable structural features to elucidate its cryo-EM structure in complex with cyanopindolol without any natural (G-proteins, arrestins) or artificial (Nanobodies, DARPins) binding partners, to an overall resolution of 4.2 [A]. The seven-helix architecture and helix eight, as well as both GPCR - AmpC {beta}-lactamase connections are clearly resolved. {beta}1AR is in its inactive conformation. 3D variability analysis revealed significant flexibility between the two protein domains and within the GPCR helices, offering insights into conformational dynamics. The map contains clear density for the cyanopindolol. The fusion protein geometry theoretically fits a wide range of class A GPCRs, presenting a powerful platform for structure elucidation of a diverse array of class A GPCR - ligand complexes by cryo-EM in the inactive receptor state. The approach furthermore holds potential for structure elucidation of GPCRs in the absence of ligands.