Biophysical evolution of the receptor binding domains of SARS-CoVs

Abstract

ABSTRACTWith hundreds of coronaviruses (CoVs) identified in bats that are capable of infecting humans, it is important to understand how CoVs that affected the human population have evolved. Seven known coronaviruses have infected humans, of which three CoVs caused severe disease with high mortality rates: SARS-CoV emerged in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Both SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor binding domain (RBD) of spike protein to bind to the ACE2 receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor binding motif (RBM) that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry towards ACE2, compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse RBMs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrants further optimization of protein subunit vaccines that use RBD as an antigen either by inserting stabilizing mutations or formulation screening.Statement of SignificanceThis study holds significant relevance in the context of the COVID-19 pandemic and the broader understanding of coronaviruses. A comparison of the receptor binding domains (RBDs) of SARS-CoV and SARS-CoV-2 reveals significant differences in their structure, stability, aggregation, and function. Despite divergent sequences, the RBDs share a similar fold and ACE2 receptor binding capability, likely through convergent evolution. These findings are crucial for understanding coronavirus evolution, interactions with human receptors, and the spillover of coronaviruses from animals to humans. The study also has implications for vaccine design strategies for SARS-CoVs, where the RBD is used as an antigen in protein subunit vaccines. By anticipating future outbreaks and enhancing our understanding of zoonotic spillover, this research contributes to safeguarding human health.