A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike

Xia, Shuai; Yan, Lei; Xu, Wei; Agrawal, Anurodh Shankar; Algaissi, Abdullah; Tseng, Chien-Te K.; Wang, Qian; Du, Lanying; Tan, Wenjie; Wilson, Ian A.; Jiang, Shibo; Yang, Bei; Lu, Lu

The coding sequence of EK1 peptide was individually fused to the 3′ end of the HR1 domain from SARS-CoV, MERS-CoV, and HCoV-229E (residues 892 to 970, 984 to 1062, and 785 to 873, respectively) through a six-amino acid linker (L6: SGGRGG). The resulting sequences encoding different HR1-L6-EK1 fusion proteins were then subcloned into a modified pET-28a vector, which contains a His 6 -SUMO tag upstream of the multiple cloning site. The resulting constructs, pET-28a-His 6 -SUMO-HR1-L6-EK1, were then expressed in Escherichia coli BL21 (DE3) at 16°C overnight in LB medium. We initially purified these fusion proteins using His-Talon resin (Clontech). Eluted fractions from the His-Talon column were then mixed with Ulp1 enzyme [1:100 (w/w)] and dialyzed against buffer A [20 mM tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM dithiothreitol] at 4°C overnight. After SUMO tag cleavage, the samples were reloaded onto the His-Talon column, and flow-through fractions containing untagged HR1-L6-EK1 were pooled, concentrated, and gel-filtered in buffer B [20 mM tris (pH 8.0) and 150 mM NaCl] on a HiLoad 16/60 Superdex 75 (GE Healthcare) column. Peak fractions that contain HR1-L6-EK1 trimer were pooled and concentrated to 10 mg/ml through centrifugation (EMD Millipore). and 30% PEG550MME, respectively. Diffraction data were collected at beamline BL19U1 of Shanghai Synchrotron Radiation Facility (SSRF), China, and processed with the HKL3000 program (57) . A summary of the data collection statistics is provided in table S3. The structure of HR1(SARS)-L6-EK1 was solved by molecular replacement, as implemented in the PHASER program of PHENIX (58) . The programs used a SARS-CoV fusion core structure [Protein Data Bank (PDB): 2bez] as the search model. For structure determination of HR1 (MERS)-L6-EK1 and HR1(229E)-L6-EK1, the MERS-CoV fusion core [PDB: 4mod] and SARS-CoV fusion core [PDB: 2bez] were used as search models. The structural models were further improved by cycles of manual building and refinement using the COOT (59) and PHENIX (58) programs. The quality of these models were analyzed with MolProbity (60) . A summary of the structure refinement statistics is also given in table S3. The figures were all prepared using the PyMOL program (The PyMOL Molecular Graphics System, Version 2.1, Schrödinger LLC). The electrostatic calculations were performed with PDB2PQR (61).