Structure and function of SARS-CoV-2 membrane fusion peptides

The Spike glycoprotein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),
etiological agent of COVID-19, is a membrane-anchored protein responsible for viral entry. The S2
subunit features two functional segments called fusion peptides (FP) that bind to target membranes
and mediate the fusion between viral and cell membranes. One segment, located at the N-terminus of
the S2 subunit, is considered the bona fide FP and displays characteristics of class I viral FPs. The
second membranotropic segment is more internally positioned (IFP) and exhibits features of class II
viral FPs. The function of FP has been reported as calcium-dependent, but little is known about the
possible binding sites and the effect of this ion on the FP fusogenic activity. Here we used a combined
biophysical approach to evaluate the structure and function of both FP and IFP, as well as to
understand the effect of Ca2+ ions on the structure and function of the N-terminal FP and determine its
binding sites. To do so, we utilized IFP, wild-type FP, and analogs containing strategic amino acid
substitutions that possibly represent the Ca2+ binding sites (E4Q, D5N, N9D, and D15N) in
combination with various spectroscopic techniques such as circular dichroism, fluorescence, electron
spin resonance, and nuclear magnetic resonance. Our data reveal the following findings: i) both
peptides acquire an α-helical secondary structure in micelles; ii) the membrane-ordering effect and the
lipid mixing activity of IFP are higher than those of FP; and iii) Ca2+ modulates the structure,
fusogenic activity, and membrane-ordering effect of FP. Based on our data, we propose the following
hypotheses: i) E4 and N9 residues from FP are the possible Ca2+ binding sites; ii) IFP is the most
fusion-active SARS-CoV-2 fusion peptide; and iii) the peptide-induced lipid packing and head group
ordering may induce bending moments in the bilayer and membrane dehydration. Three-dimensional
models of both FP and IFP in dodecylphosphocholine micelles were determined by solution nuclear
magnetic resonance (NMR) spectroscopy. While IFP adopts a flexible helical structure, FP exhibits a
π-helix correlation involving D5-N9, resulting in the formation of an α-aneurism around N9, with all
hydrophobic residues appearing on the same side of the helix. Additionally, the membrane surface
topology of IFP in lipid bilayers was demonstrated using magnetically aligned phospholipid bicelles
through oriented sample solid-state NMR. We discuss the importance of the peptide structures and
their membranotropic effects to their putative roles in the membrane fusion mechanism.