Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation

Abstract

23Pandemic coronavirus disease 2019 is caused by the emerging severe 24 acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there are no 25 efficacious vaccines or therapeutics that are urgently needed. We expressed three 26 versions of spike (S) proteins-receptor binding domain (RBD), S1 subunit and S 27 ectodomain-in insect cells. RBD appears monomer in solutions, whereas S1 and S 28 associate into homotrimer with substantial glycosylation. The three proteins confer 29 excellent antigenicity with six convalescent COVID-19 patient sera. Cryo-electron 30 microscopy (cryo-EM) analyses indicate that the SARS-CoV-2 S trimer dominate in a 31 unique conformation distinguished from the classic prefusion conformation of 32 coronaviruses by the upper S1 region at lower position ~15 Å proximal to viral 33 membrane. Such conformation is proposed as an early prefusion state for the SARS-34 CoV-2 spike that may broaden the knowledge of coronavirus and facilitate vaccine 35 development.36

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

“…2G). This finding is consistent with the thermal denaturation profile of wild type S protein purified from High Five TM insect cells, despite the overall structural differences [31].…”

“…2G). This finding is consistent with the thermal denaturation profile of wild type S protein purified from High Five TM insect cells, despite the overall structural differences [46]. Additional work will be required to assign these transitions to specific domains or modules within the full length COVID-19 spike protein.…”

“…Interestingly, the structures determined from protein expressed in mammalian cells differ from a recently reported cryo-EM structure of wild type S protein produced from High Five TM insect cells. This structure appeared more compact overall and the model could not accommodate the presence of the SD1 domain [31]. Whether these disparities are the consequence of differences in expression hosts (e.g., glycosylation or other processing functions) or the lack of stabilizing mutations, remains an open question, although RBD recognition and antigenicity was preserved.…”

Unlocking COVID therapeutic targets: A structure-based rationale against SARS-CoV-2, SARS-CoV and MERS-CoV Spike

“… [36] , [37] , [38] . Several studies have shown that the ectodomain and the RBD expression in eukaryotic systems are stable and appear in the monomer conformation in high yield; and that soluble ACE2 binds to both monomer and trimer conformations [37] , [39] , [40] , [41] , [42] . In this context, to fully characterize the S protein, it is important to explore both the monomer and trimer in structural studies.…”

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

“…This structure appeared to be more compact overall, and the model could not accommodate the presence of the SD1 domain. 34 Whether these disparities are the consequence of differences in expression hosts (e.g., glycosylation or other processing functions) or the lack of stabilizing mutations remains an open question, although RBD recognition and antigenicity were preserved.…”