A mechanistic understanding of the modes of Ca ion binding to the SARS-CoV-1 fusion peptide and their role in the dynamics of host membrane penetration

The SARS-CoV-1 spike glycoprotein contains a fusion peptide (FP) segment that mediates fusion of the viral and host cell membranes. Calcium ions are thought to position the FP optimally for membrane insertion by interacting with negatively charged residues in this segment (E801, D802, D812, E821, D825, and D830); however, which residues bind to calcium and in what combinations supportive of membrane insertion are unknown. Using biological assays and molecular dynamics studies, we have determined the functional configurations of FP-Ca ⁺² binding which promote membrane insertion. We first mutated the negatively charged residues in the SARS CoV-1 FP to assay their role in cell entry and syncytia formation, finding that charge loss in the D802A or D830A mutants reduced syncytia formation and pseudoparticle transduction. Interestingly, the D812A mutation led to increased pseudoparticle transduction, indicating the Ca ²⁺ effect depends on binding at specific FP sites. To interpret mechanistically these results and learn how specific modes of FP-Ca ²⁺ binding modulate membrane insertion, we performed molecular dynamics simulations. Preferred residue pairs for Ca ²⁺ binding were identified (E801/D802; E801/D830; D812/E821) which promote FP membrane insertion. In contrast, binding to residues E821/D825 inhibited FP membrane insertion, which is also supported by our biological assays. Our findings show that Ca ²⁺ binding to SARS-CoV-1 FP residue pairs E801/D802 and D812/E821 facilitates membrane insertion, whereas binding to the E801/D802 and D821/D825 pairs is detrimental. These conclusions provide an improved and nuanced mechanistic understanding of calcium binding modes to FP residues and their dynamic effects on host cell entry.