The SARS-CoV-2 spike protein is the
primary antigenic determinant
of the virus and has been studied extensively, yet the process of
membrane fusion remains poorly understood. The fusion domain (FD)
of viral glycoproteins is well established as facilitating the initiation
of membrane fusion. An improved understanding of the structural plasticity
associated with these highly conserved regions aids in our knowledge
of the molecular mechanisms that drive viral fusion. Within the spike
protein, the FD of SARS-CoV-2 exists immediately following S2′
cleavage at the N-terminus of the S2 domain. Here we have shown that
following the introduction of a membrane at pH 7.4, the FD undergoes
a transition from a random coil to a more structurally well-defined
postfusion state. Furthermore, we have classified the domain into
two distinct regions, a fusion peptide (FP, S
816
–G
838
) and a fusion loop (FL, D
839
–F
855
). The FP forms a helix–turn–helix motif upon association
with a membrane, and the favorable entropy gained during this transition
from a random coil is likely the driving force behind membrane insertion.
Membrane depth experiments then revealed the FP is found inserted
within the membrane below the lipid headgroups, while the interaction
of the FL with the membrane is shallower in nature. Thus, we propose
a structural model relevant to fusion at the plasma membrane in which
the FP inserts itself just below the phospholipid headgroups and the
FL lays upon the lipid membrane surface.
SARS-CoV-2 may enter target cells through the process of membrane fusion at either the plasma ($pH 7.4-7.0) or endosomal ($pH 6.5-5.0) membrane in order to deliver its genetic information. The fusion domain (FD) of the spike glycoprotein is responsible for initiating fusion and is thus integral to the viral life cycle. The FD of SARS-CoV-2 is unique in that it consists of two structurally distinctive regions referred to as the fusion peptide (FP) and the fusion loop (FL); yet the molecular mechanisms behind how this FD perturbs the membrane to initiate fusion remains unclear. In this study via solution NMR, we witnessed only a slight conformational change in the FD between pH 7.4 and pH 5.0, resulting in a minor elongation of helix 1. However, we found that the FD's ability to mediate membrane fusion has a large and significant pH dependence, with fusion events being more readily induced at low pH. Interestingly, a biphasic relationship between the environmental pH and fusogenicity was discovered, suggesting a preference for the FD to initiate fusion at the late endosomal membrane. Furthermore, the conserved disulfide bond and hydrophobic motif "LLF" were found to be critical for the function of the complete FD, with minimal activity witnessed when either was perturbed. In conclusion, these findings indicate that the SARS-CoV-2 FD preferably initiates fusion at a pH similar to the late endosome through a mechanism that heavily relies on the internal disulfide bond of the FL and hydrophobic LLF motif within the FP.
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