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 (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.