Cryo-EM structure of a single-chain β1-adrenoceptor – AmpC β-lactamase fusion protein

Abstract The insertion of fusion proteins has enabled the crystallization of a wide range of G-protein-coupled receptors (GPCRs). Here, we explored the possibility of using a larger fusion protein, inserted into the third intracellular loop (ICL3) of β1-adrenoceptor (β1AR) via rigid chimeric helix fusions. The aim was to engineer a single-chain fusion protein that comprises sufficient mass and rigidity to allow single-particle cryo-EM data collection, without depending on binding proteins, such as G-proteins or nanobodies. Through parsing of the protein data bank (PDB), we identified the protein AmpC β-lactamase as a suitable candidate. Both termini of this protein are α-helical and the helices are antiparallel to each other. The distance between their centroids measures ∼11 Å. Such a geometry is ideal to design extended chimeric helices with transmembrane (TM) helices 5 and 6 of β1AR, and the insertion of the protein adds ∼39 kDa of mass to the receptor. We expressed the β1AR - AmpC β-lactamase fusion protein in mammalian cells. The binding of the antagonists propranolol and cyanopindolol to the purified fusion protein was confirmed by CPM-based thermofluor assays. The cryo-EM structure was solved to a nominal overall resolution of 3.6 Å and the seven helix architecture and helix eight were clearly resolved. Superimposition of the structure with known X-ray crystal structures of β1AR suggests that the protein is in its inactive conformation. The fusion protein described here provides a basis for high-throughput structure elucidation of class A GPCRs by cryo-EM for drug discovery research as well as for the elucidation of inactive state or wild-type GPCR structures. The fusion protein geometry theoretically fits a wide range of class A GPCRs and therefore can be applied to a multitude of receptors.