Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains

Yuan, Yuan; Cao, Duanfang; Zhang, Yanfang; Ma, Jun; Qi, Jianxun; Wang, Qihui; Lu, Guangwen; Wu, Ying; Yan, Jinghua; Shi, Yi; Zhang, Xinzheng; Gao, George F.

Protein expression and purification. The gene encoding MERS-CoV spike protein (GenBank accession number JX869059, residues 18-1,294, with an Arg751Ser mutation to abolish the protease cleavage site) and the SARS-CoV spike gene (GenBank accession number AY2,78,488, residues 14-1,193) were both synthesized and subcloned into the baculovirus transfer vector pFastbac1 (Invitrogen) with a N-terminal gp67 signal peptide, a C-terminal thrombin cleavage site followed by a T4 fibritin trimerization domain and a 6X Histag. The two kinds of S protein were produced with Bac-to-Bac baculovirus expression system (Invitrogen) separately. Transfection and virus amplification were conducted with Sf9 cells, and Hi5 cells (Invitrogen) were used to produce the recombinant proteins. Soluble S protein was captured from cell supernatants by metal affinity chromatography using a HisTrap HP 5 ml column (GE Healthcare). The eluted product was pooled and further purified by gel filtration chromatography with a Superose 6 10/300 GL (GE Healthcare) column equilibrated with a buffer containing 20 mM Tris-HCl (pH7.5) and 150 mM NaCl. Then, the S proteins were both cleaved with thrombin (Sigma, 3 units per mg S protein) at 4°C overnight to remove the C-terminal trimerization domain and 6 Â His-tag. A final round of size exclusion chromatography was conducted to purify the cleaved product with a Superose 6 10/300 GL column. The resulting S proteins reached a purity of 95% as shown by SDS-PAGE ( Supplementary Figs 2 and 4) . The coding sequence for N terminal domain (NTD, spanning residues 18-353) of MERS-CoV S protein (MERS-CoV S-NTD) was cloned into the EcoRI and XhoI restriction sites of pFastBac1 vector for baculovirus expression (Bac-to-Bac baculovirus expression system, Invitrogen). An N-terminal gp67B signal peptide and a C-terminal 6X Histag were added to facilitate protein secretion and purification. The MERS-CoV S-NTD protein was purified by Ni-NTA affinity column and Superdex200 gel filtration column (GE Healthcare). The protein was concentrated to 15 mg ml À 1 in buffer containing 20 mM Tris, pH 8.0 and 150 mM NaCl for crystal screening. The SARS-CoV S-NTD (spanning residues 14-292) was constructed, expressed and purified with the same strategy.