In a study published in Life Science Alliance, a team led by Prof. XIONG Xiaoli, Prof. HE Jun and Prof. CHEN Ling from the Guangzhou Institutes of Biomedicine and Health of the Chinese Academy of Sciences introduced designed disulfide bonds into the SARS-CoV-1 spike (S) protein to allow imaging of previously unobserved rare conformations by cryo-electron microscopy (cryo-EM). They found that rare SARS-CoV-1 S conformations share parallel features with some conformations of SARS-CoV-2 and other Sarbecovirus S proteins, and analyzed possible biological functions of these conserved features. 

Coronavirus S protein binds virus receptor and mediates virus cell entry by promoting membrane fusion between virus and cell. SARS-CoV-2 S protein exhibits complicated structural dynamics. Three distinct prefusion conformations have been 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 is transient under neutral pH, while more stable in acidic pH. The instability of locked conformations may be the reason why they were not observed in previous structural studies of SARS-CoV-1 S protein. 

The team has 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 team 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 was found to induce strong neutralizing sera with comparable titers compared with the unmodified SARS-CoV-1 S protein. 

The researchers imaged the engineered SARS-CoV-1 S proteins by cryo-EM, and discovered that they 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 for SARS-CoV-2 S protein. Also, they 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, the researchers observed the extrusion of fusion peptide proximal region (FPPR) in S-x1 protein closed conformation structure, but not in the x1 disulfide stabilized SARS-CoV-2 S protein. This feature demonstrated conformation flexibility of FPPR. Different from SARS-CoV-2 S protein, low pH condition was unable to convert SARS-CoV-1 S protein into locked conformation. These results revealed the similarities in structural conformations and differences in structural dynamics between SARS-CoV-1 and SARS-CoV-2 S proteins. 

This study identified that the S protein of SARS-CoV-1 is similar to that of SARS-CoV-2, which can adopt two distinct locked conformations, locked-1 and locked-2. It showcased the complex structural dynamics of the S proteins of SARS-CoV-1 and SARS-CoV-2. 

To date, locked-2 conformation has been mostly observed 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.