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.

Overall structure of MERS-CoV and SARS-CoV S trimers. We produced the MERS-CoV and SARS-CoV S trimers by fusing a T4 fibritin trimerization motif and 6X Histag at the C-terminal end of the ectodomain construct. For the MERS-CoV S protein, we also mutated the S2 cleavage site to enhance the stability. The resulting uncleaved MERS-CoV S ectodomain forms a trimer that can bind to the dimeric CD26 receptor protein, and then precipitate easily ( Supplementary Fig. 1 ). We then used thrombin enzyme to remove the C-terminal T4 fibritin trimerization motif and 6X Histag, and found that the MERS-CoV S ectodomain trimer protein can be separated into two peaks in the gel filtration profile ( Supplementary Fig. 2 ). One peak is the tag-removed MERS-CoV S ectodomain trimer, and the other is the mixed disassociated S1 and S2 subunits ( Supplementary Fig. 2 ). The cleaved form of the MERS-CoV S ectodomain was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and further N-terminal sequencing revealed that the S ectodomain protein was cleaved after residue R748 ( Supplementary Fig. 3) , three residues ahead of the S2 cleavage site. This indicated that once the MERS-CoV S ectodomain is cleaved into S1/S2 form, the S1 subunit tends to dissociate from S2. By contrast, the SARS-CoV S protein remains uncleaved after thrombin cleavage, and binds its receptor Angiotensin I Converting Enzyme 2(ACE2), confirmed by gel filtration survive assay ( Supplementary Fig. 4) .