Insights Into SARS-CoV-2 S1-RBD-ACE2 Interaction Using Computational Tools and Membrane Microarray Platform

The Coronavirus Disease of 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has led to a global health crisis. Emerging variants like Alpha, Beta, Gamma, and Omicron raise concerns due to increased infectivity and competitiveness. For instance, several early variants contained the N501Y mutation in the S1 spike protein receptor binding domain (S1-RBD), which caused an increase in its affinity for ACE2. Notably, the Omicron variant, with 15 mutations, including a unique Q493R, demonstrated enhanced transmission. However, the molecular mechanisms underlying these were not entirely clear. To uncover the impact of these mutations, we first performed molecular dynamics (MD) simulations with the wild type (WT) and the N501Y mutant ACE2-S1-RBD complex, revealing an interfacial ‘freezing’ in the N501Y mutant. Similarly, the Omicron mutant showed, among other differences, a decrease in the interfacial dynamics. Importantly, the reversal of the Q493R mutation showed further interaction enhancement.

Subsequently, we utilized the hybrid live cell-supported lipid bilayer platform to reconstitute the initial interaction between S1-RBD and ACE2 expressed on living cells to delineate the role of endocytosis and cytoskeleton in the process. Specifically, we functionalized bilayers with the fluorescently labelled S1-RBD and monitored the interaction of ACE2 expressing A549 cells. This revealed clustering of S1-RBD on membrane and more importantly, a depletion of the Omicron S1-RBD from membrane corrals, which was not seen with the WT S1-RBD. Further, treatment of cells with pitstop2, but not blebbistatin, resulted in a decrease in the S1-RBD depletion. Endocytosis likely plays a role in the initial interaction between S1-RBD and host cells, with the omicron variant showing enhanced interaction and depletion. Understanding these changes is crucial for developing effective treatments. Ongoing research is essential to address the evolving nature of SARS-CoV-2 and emerging variants.