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, S816-G838) and a fusion loop (FL, D839-F855). 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.

Identifying Distinct Structural Features of the SARS-CoV-2 Spike Protein Fusion Domain Essential for Membrane Interaction.

The SARS‐CoV‐2 virus is responsible for the COVID‐19 pandemic the world experience since 2019. The protein responsible for the first steps of cell invasion, the spike protein, has probably received the most attention in light of its central role during infection. Computational approaches are among the tools employed by the scientific community in the enormous effort to study this new affliction. One of these methods, namely molecular dynamics (MD), has been used to characterize the function of the spike protein at the atomic level and unveil its structural features from a dynamic perspective. In this review, we focus on these main findings, including spike protein flexibility, rare S protein conformational changes, cryptic epitopes, the role of glycans, drug repurposing, and the effect of spike protein variants.

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Molecular dynamics studies reveal structural and functional features of the SARS‐CoV‐2 spike protein

Gasdermin-D (GSDMD) is the ultimate effector of pyroptosis, a form of programmed cell death associated with pathogen invasion and inflammation. After proteolytic cleavage by caspases, the GSDMD N-terminal domain (GSDMDNT) assembles on the inner leaflet of the plasma membrane and induces the formation of membrane pores. We use atomistic molecular dynamics simulations to study GSDMDNT monomers, oligomers, and rings in an asymmetric plasma membrane mimetic. We identify distinct interaction motifs of GSDMDNT with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylserine (PS) headgroups and describe their conformational dependence. Oligomers are stabilized by shared lipid binding sites between neighboring monomers acting akin to double-sided tape. We show that already small GSDMDNT oligomers support stable, water-filled, and ion-conducting membrane pores bounded by curled beta-sheets. In large-scale simulations, we resolve the process of pore formation from GSDMDNT arcs and lipid efflux from partial rings. We find that high-order GSDMDNT oligomers can crack under the line tension of 86 pN created by an open membrane edge to form the slit pores or closed GSDMDNT rings seen in atomic force microscopy experiments. Our simulations provide a detailed view of key steps in GSDMDNT-induced plasma membrane pore formation, including sublytic pores that explain nonselective ion flux during early pyroptosis. 

Sublytic gasdermin-D pores captured in atomistic molecular simulations

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The constellation of cholesterol-dependent processes associated with SARS-CoV-2 infection

Although the amino acid sequences of SARS-CoV-1 and SARS-CoV-2 fusion peptides (FPs) are highly conserved, the cryo-electron microscopy structures of the SARS-CoV-1 and SARS-CoV-2 spike proteins show that the helix length of SARS-CoV-1 FP is longer than that of SARS-CoV-2 FP. In this work, we simulated the membrane-binding models of SARS-CoV-1 and SARS-CoV-2 FPs and compared the binding modes of the FPs with the POPC/POPE/cholesterol bilayer membrane. Our simulation results show that the SARS-CoV-2 FP binds to the bilayer membrane more effectively than the SARS-CoV-1 FP. It is seen that the short N-terminal helix of SARS-CoV-2 FP is more favorable to insert into the target membrane than the long N-terminal helix of SARS-CoV-1 FP. Meanwhile, the potential of mean force calculations showed that the SARS-CoV-2 FP would prefer only one binding mode (N-terminal binding), whereas the SARS-CoV-1 FP has two favorable membrane-binding modes (C-terminal and N-terminal binding modes). Moreover, in the case of SARS-CoV-1 FP binding to the target membrane, the transition between the two binding modes is relatively fast. Finally, we discovered that the membrane-binding mode would influence the helix length of SARS-CoV-1 FP, while the helix length of SARS-CoV-2 FP could be stably maintained in the membrane-bound configurations. This work suggests that the short helix might endow the FP with high membrane-anchoring strength. In particular, the membrane-penetrating residues (Phe, Ile, and Leu) of short α-helix interact with the cell membrane more strongly than those of long α-helix.

Different Binding Modes of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides to Cell Membranes: The Influence of Peptide Helix Length

During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. The viral spike (S) protein mediates both the initial contact with the host cell and the subsequent membrane fusion. Proteolytic cleavage of S at the S2' site exposes its fusion peptide (FP) as the new N-terminus. By binding to the host membrane, the FP anchors the virus to the host cell. The reorganization of S2 between virus and host then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 μs long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP, we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than 10-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.

Binding of SARS-CoV-2 Fusion Peptide to Host Endosome and Plasma Membrane.

During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. Initial contact with the host cell and membrane fusion are both mediated by the viral spike (S) protein. Proteolytic cleavage of S at the S2' site exposes its 40 amino acid long fusion peptide (FP). Binding of the FP to the host membrane anchors the S2 domain of S in both the viral and the host membrane. The reorganization of S2 then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 s-long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than ten-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.

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Stefan L. Schaefer

Papers

Binding of SARS-CoV-2 Fusion Peptide to Host Endosome and Plasma Membrane.

Binding of SARS-CoV-2 fusion peptide to host membranes