The F1148 hydrophobic lock: A critical determinant of SARS-CoV-2 spike protein-mediated membrane fusion via the 3H/CH cavity

The S2 subunit of the coronavirus Spike protein undergoes extensive conformational refolding to drive membrane fusion during viral entry. Although the HR1/HR2 six-helix bundle (6-HB) is recognized as the core mediator of fusion, the molecular driving force governing its formation remains poorly elucidated. Here, through systematic mutagenesis of the AlphaFold-predicted stem helix (SH) region in S2, followed by analysis of the resulting SC2-VLP entry phenotypes, we identified key amino acid residues within conserved helices that are present in both prefusion and postfusion Spike conformations. These elements, which we term postfusion-preserved helices (PFPHs), were found to be critical for SC2-VLP entry. Structural analysis revealed a "hydrolock" interaction between F1148 in PFPH-1 and a conserved cavity formed by 3H (I742, C749)/CH (I993, L996, I997). Deep mutational scanning demonstrated that only hydrophobic residues at F1148 were functionally viable and essential for membrane fusion, underscoring the critical role of a hydrophobic lock ("hydrolock") interaction between F1148 and the 3H/CH cavity in membrane fusion. Furthermore, HA-replacement mutagenesis and anti-HA neutralization assays showed that significant neutralization activity was restricted to HA insertions proximal to PFPH-1, selectively inhibiting membrane fusion without affecting receptor binding. Notably, the 3H/CH cavity remains structurally stable across Spike conformations, being sequentially occupied by prefusion-L977, intermediate-F782, and postfusion-F1148. We propose a model wherein hydrolock interactions drive S2 refolding and fusion by displacing intermediate interactions. This study provides mechanistic insights into Spike dynamics and highlights hydrolock interactions as a promising target for broad-spectrum antiviral strategies.