Ateam led by Professor XIONGXiaoli, ProfessorHEJunandProfessor CHENLing from the Guangzhou Institutesof Biomedicine and Healthof theChinese Academy of Sciencesintroduceddesigned disulfide bondsinto the SARS-CoV-1 spike (S) protein to allow imaging of previously unobserved rare conformationsbycryo-electron microscopy (cryo-EM). They found thatrare SARS-CoV-1 S conformations share parallel features with some conformations of SARS-CoV-2 and other Sarbecovirus S proteinsand analyzedpossible biological functions of these conserved features.
The study, entitled "Disulfide stabilization reveals conserved dynamic features between SARS-CoV-1 and SARS-CoV-2 spikes", was published inLife Science Alliance.
CoronavirusS proteinbinds virus receptor and mediates virus cell entry by promoting membrane fusion between virus and cell.Itis the primary target of coronavirus vaccine development. Due to the SARS-CoV-2 pandemic, structure and function of theS proteinhave been a subject of intense research in recent years.
SARS-CoV-2S proteinexhibitscomplicated structural dynamics.Three distinct prefusion conformations for the SARS-CoV-2S proteinare identified: locked, closed and open. The locked conformation has a tightly packed structure with features incompatible with the RBD “up” open conformation which is capable of binding the ACE2 receptor.It istransient under neutral pH, whilemore stable in acidic pH. The instability of locked conformations may be the reason why theywerenot observed in previous structural studies of SARS-CoV-1 S protein.
The team previously developed three pairs of disulfide bonds (x1, x2, x3) for SARS-CoV-2 S protein with abilities to stabilize RBD "down" conformations including rare locked conformations. In this study, the researchers introduced these three pairs of disulfides individually into SARS-CoV-1 S protein and obtained purified SARS-CoV-1 S-x1, S-x2, and S-x3 proteins. The S-x3 protein was tested to immunize mice and found to induce strong neutralizing sera with comparable titers compared with unmodified SARS-CoV-1S protein.
Theyimaged the engineeredSARS-CoV-1S proteins by cryo-EM and discovered that theengineered SARS-CoV-1S proteins can adopt two different locked conformations: locked-1 (where three S protomers are in the locked-1 conformation) and locked-2 (where three S protomers are in the locked-2 conformation). Similar conformations have been reported forSARS-CoV-2S protein.
The team also noticed asymmetric mixed locked conformations, namely, locked-112 (where two S protomers are in the locked-1 conformation and one is in the locked-2 conformation) and locked-122 (where two S protomers are in the locked-2 conformation and one is in the locked-1 conformation).
Furthermore, extrusion of Fusion Peptide Proximal Region (FPPR) was observedinS-x1 proteinclosed conformation structure,but not forthe x1 disulfide stabilizedSARS-CoV-2S protein. This feature demonstrates conformation flexibility of FPPR. Moreover, differentfrom SARS-CoV-2S protein, low pH condition was unable to convertSARS-CoV-1S protein into locked conformation. These results reveal similarities in structural conformations and differences in structural dynamics betweenSARS-CoV-1 and SARS-CoV-2 S proteins.
In summary, this study identifiedthat the S protein ofSARS-CoV-1 is similar to that of SARS-CoV-2,whichcan adopt two distinct locked conformations (locked-1 and locked-2). Together with the other observedS proteinconformations, the study showcases the complex structural dynamics of theS proteins of SARS-CoV-1 and SARS-CoV-2.
Interestingly, to date, locked-2 conformation has beenmostlyobserved for S proteins of other Sarbecoviruses. Structural analysis suggests that although differences exist between locked-1 and locked-2 conformations, both locked conformations utilize interactions located within domains C and D to rigidify the locked spike structures and to inhibit RBD motion. Conservation of mechanisms that inhibits RBD opening among Sarbecovirus S proteins in locked conformations suggests that locked conformations may play a conserved role in the lifecycle of Sarbecoviruses. The team proposed that the locked conformation may play a functional role in the virus assembly process, but this proposal requires further investigation to be confirmed.
Fig.Cryo-EM structures of engineered SARS-CoV-1 spikes in different conformations(Image by GIBH)
Contacts:
XiongXiaoli, PhD and HeJun, Ph.D
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences
Guangzhou, China, 510530
Email: xiong_xiaoli@gibh.ac.cn; he_jun@gibh.ac.cn
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