A Mechanistic Understanding of the Modes of Ca2+ Ion Binding to the SARS-CoV‑1 Fusion Peptide and
Their Role in the Dynamics of Host Membrane Penetration
Version 2 2024-01-25, 17:05Version 2 2024-01-25, 17:05
Version 1 2024-01-25, 13:05Version 1 2024-01-25, 13:05
journal contribution
posted on 2024-01-25, 17:05authored byJuliana
Debrito Carten, George Khelashvili, Miya K. Bidon, Marco R. Straus, Tiffany Tang, Javier A. Jaimes, Gary R. Whittaker, Harel Weinstein, Susan Daniel
The
SARS-CoV-1 spike glycoprotein contains a fusion peptide (FP)
segment that mediates the 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-Ca2+ binding that likely promote membrane insertion. We first individually
mutated the negatively charged residues in the SARS CoV-1 FP to assay
their roles in cell entry and syncytia formation, finding that charge
loss in the D802A or D830A mutants greatly reduced syncytia formation
and pseudoparticle transduction of VeroE6 cells. Interestingly, one
mutation (D812A) led to a modest increase in cell transduction, further
indicating that FP function likely depends on calcium binding at
specific residues and in specific combinations. To interpret these
results mechanistically and identify specific modes of FP-Ca2+ binding that modulate membrane insertion, we performed molecular
dynamics simulations of the SARS-CoV-1 FP and Ca2+ions.
The preferred residue pairs for Ca2+ binding we identified
(E801/D802, E801/D830, and D812/E821) include the two residues found
to be essential for S function in our biological studies (D802 and
D830). The three preferred Ca2+ binding pairs were also
predicted to promote FP membrane insertion. We also identified a Ca2+ binding pair (E821/D825) predicted to inhibit FP membrane
insertion. We then carried out simulations in the presence of membranes
and found that binding of Ca2+ to SARS-CoV-1 FP residue
pairs E801/D802 and D812/E821 facilitates membrane insertion by enabling
the peptide to adopt conformations that shield the negative charges
of the FP to reduce repulsion by the membrane phospholipid headgroups.
This calcium binding mode also optimally positions the hydrophobic
LLF region of the FP for membrane penetration. Conversely, Ca2+ binding to the FP E801/D802 and D821/D825 pairs eliminates
the negative charge screening and instead creates a repulsive negative
charge that hinders membrane penetration of the LLF motif. These
computational results, taken together with our biological studies,
provide an improved and nuanced mechanistic understanding of the dymanics
of SARS-CoV-1 calcium binding and their potential effects on host
cell entry.