Surface Charge and Stability of SARS-CoV-2 Spike Protein Ectodomains in Omicron Variants

The SARS-CoV-2 spike (S) protein, particularly its ectodomain (ECD), plays a crucial role in viral attachment and fusion with host cells. The ECD is subject to significant alteration by mutations, particularly in variants of concern (VoCs) such as Omicron. Omicron variants, notably BA.1 and BA.2, exhibit significant increases in surface positive charge, which enhance binding to heparan sulfate proteoglycans and facilitate ACE2 receptor engagement. However, it is known that such alterations may also impact protein stability, a critical factor given the metastable prefusion state of the S protein. To characterize stability, we analyzed studies on the thermal behavior of the ECD using differential scanning fluorometry (DSF) and calorimetry (DSC). Two distinct thermal transitions of the ECD were identified: a lower temperature Tm1 linked to local ECD structural changes, likely involving the receptor-binding domain (RBD), and a higher Tm2 associated with full ECD unfolding. BA.1 and BA.2 exhibit a decreased Tm1 compared to the wild type, that may influence the flexibility of the RBD and its ability to undergo up/down transitions. Notably, Tm2 remains unchanged. In conclusion, even if mutations as those affecting charged amino acids might destabilize the ECD on their own, other mutations counteract this effect, preserving both the stability of the entire ECD and the advantage of the altered surface potential in BA.1 and BA.2 and later Omicron variants. These insights offer valuable contributions to our understanding of the complex relationship between viral evolution and structural integrity.