Biophysical and Structural Characterization of HR1-HR2 Interaction of the SARS-CoV-2 Spike Protein Using Mimetic Recombinant Constructs

The COVID-19 pandemic has been recognized as the most significant global outbreak in recent decades. While several vaccines have been developed and administered worldwide, they face persistent challenges such as declining immunity and emergence of new viral strains that can evade them. To combat these obstacles, it is crucial to develop new antiviral treatments and vaccines that target highly conserved regions of the virus. One promising therapeutic approach against SARS- CoV-2 is to disrupt the membrane fusion mechanism that enables the virus to enter the cell. This process is promoted by the Spike (S) protein, which is a trimer of heterodimers (with S1 and S2 subunits), and involves the interaction between two highly conserved heptad-repeat regions (HR1 and HR2) of S2 to form a 6-helix bundle (6HB) structure. Our previous research with HIV-1, a virus that shares a similar fusion mechanism with SARS-CoV-2, has demonstrated that single-chain chimeric proteins that mimic the HR1 region of the gp41 subunit, functionally equivalent to SARS-CoV-2 S2 subunit, can bind strongly HR2 and constitute potent fusion inhibitors [1]. In this work, we describe the design, engineering, and biophysical characterization of a series of single-chain proteins (named CoVS-HR1) that imitate the HR1 region in its trimeric coiled-coil conformation. While a first generation of proteins folded in-helical structures as expected, they tended to oligomerize and some of them showed low stability [2], a second generation of re-engineered proteins showed much better biophysical characteristics in terms of structure, stability and oligomerization state and allowed a detailed study of their binding to their complementary HR2 peptides [3]. Finally, we present the high-resolution crystallographic structure of the complex between a CoVS-HR1 protein and an HR2 peptide, which allowed a deep analysis of the interaction and highlighted the most implicated residues. It also allowed us to identify several int