Fusion Peptide of SARS-CoV-2 Spike Rearranges into a Wedge Inserted in Bilayered Micelles

Koppisetti, Rama K.; Fulcher, Yan G.; Doren, Steven R. Van

doi:10.1021/jacs.1c05435

Cited by 63 publications

(115 citation statements)

References 56 publications

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“…This model is somehow not consistent with a recent NMR study of the structure of SARS-2 FP 23 , which suggests that D830 (corresponding to D812 in SARS-1 FP) does not bind to Ca 2+ , and D839 and D843 (corresponding to E821 and D825 in SARS-1 FP) most likely participate in binding Ca 2+ , while the other three are moderated likely to bind Ca 2+ . This research is on SARS-2 FP, so it may be a different from our SARS-1 FP case.…”

Section: Discussioncontrasting

confidence: 81%

“…The recent NMR paper on SARS-2 FP structure mentioned above indicates that the FP inserts in the membrane as a wedge 23 , where the FP1 and FP2 are closely positioned together in membranes. Although that result is about the SARS-2 FP, it is very reasonable to believe that the SARS-1 FP can adopt a similar structure based on the similarity between the SARS-1 and SARS-2 FPs.…”

Section: Discussionmentioning

confidence: 99%

“…However, the limitation of the NMR and the MD simulation studies is that there is only one FP in their system: one FP in a bicelles in the NMR study 23 ; or one FP in a membrane patch in the MD simulation 27 . These environments exclude the possibility of inter-molecular interaction between the FPs.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, the FP adopts its active conformation only in the membranes 12 , which is generally not determined in the crystallographic structure as they are hydrophobic and intrinsically disordered. The structure of the SARS-2 FP has been solved only recently, using NMR in bicelles 23 . ESR is a useful technique to study the effect of FPs on the membrane structure with implications for the mechanism leading to membrane fusion.…”

Section: Introductionmentioning

confidence: 99%

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Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides

Lai

Freed

2021

Preprint

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Coronaviruses are a major infectious disease threat, and include the human pathogens of zoonotic origin SARS-CoV ("SARS-1"), SARS-CoV-2 ("SARS-2") and MERS-CoV ("MERS"). Entry of coronaviruses into host cells is mediated by the viral spike (S) protein. Previously, we identified that the domain immediately downstream of the S2' cleavage site is the bona fide FP (amino acids 798-835) for SARS-1 using ESR spectroscopy technology. We also found that the SARS-1 FP induces membrane ordering in a Ca2+ dependent fashion. In this study, we want to know which residues are involved in this Ca2+ binding, to build a topological model and to understand the role of the Ca2+. We performed a systematic mutation study on the negatively charged residues on the SARS-1 FP. While all six negatively charged residues contributes to the membrane ordering activity of the FP to some extent, D812 is the most important residue. We provided a topological model of how the FP binds Ca2+ ions: both FP1 and FP2 bind one Ca2+ ion, and there are two binding sites in FP1 and three in FP2. We also found that the corresponding residue D830 in the SARS-2 FP plays a similar critical role. ITC experiments show that the binding energies between the FP and Ca2+ as well as between the FP and membranes also decreases for all mutants. The binding of Ca2+, the folding of FP and the ordering activity correlated very well across the mutants, suggesting that the function of the Ca2+ is to help to folding of FP in membranes to enhance its activity. Using a novel pseudotyped virus particle (PP)-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole S proteins in its trimer form in real time. We found that the SARS-1 and SARS-2 PPs also induce membrane ordering as the separate FPs do, and the mutations of the negatively charged residues also greatly reduce the membrane ordering activity. However, the difference in kinetic between the PP and FP indicates a possible role of FP trimerization. This finding could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel to combat the ongoing COVID-19 pandemic.

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Section: Discussioncontrasting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides

Lai

Freed

2021

Preprint

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“…Then, furin cleaves the S2 region (S2 ) to start conformational changes for fusion between the viral envelope and the plasma membrane of the host cell. Therefore, the "fusion peptide", from residues 816 to 857 of protein S [3], is exposed for insertion into the host cell membrane. There follows a gathering between the envelope of the virus and the cell membrane, both formed by a lipid bilayer which will therefore fuse [4].…”

Section: Introductionmentioning

confidence: 99%

PDZ-Containing Proteins Targeted by the ACE2 Receptor

Caillet‐Saguy

Wolff

2021

Viruses

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Angiotensin-converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike (S) protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residue segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein-interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high-throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants’ values ranging from 3 to 81 μM. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role in ACE2 internalization and recycling that could be of benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in the neurological symptoms of COVID-19.

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A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS‐CoV‐2 Spike‐Mediated Viral Invasion

Wu,

Li,

Lin

et al. 2023

Advanced Science

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The receptor‐binding domain (RBD) of spike recognizing the receptor angiotensin‐converting enzyme 2 (ACE2) initiates membrane fusion between severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and cell membrane. Although the structure of the RBD_ACE2 complex has been well studied, its functional mechanism in membrane fusion is still not fully understood. Here, using an in vitro cell–vesicle content‐mixing assay, it is found that the cleavage at the S2’ site by thrombin (Thr) protease strongly accelerates membrane fusion, compared to that of cleavage at the S1/S2 site by PreScission (3C) protease. Moreover, mutations at the RBD_ACE2 interface resulted in a positive correlation between binding affinity and fusion probability. In both the cell–vesicle and cell–cell fusion assays, by crosslinking two membranes via the neutravidin (NTV)_biotin interaction or complementary DNA strands, it is found that spike drives membrane fusion in the absence of ACE2, and a suitable distance between two membranes is critical for spike‐mediated membrane fusion. Finally, unsuitable membrane crosslinkers significantly inhibited the fusion probability in the presence of ACE2. Taken together, the results suggest that the RBD_ACE2 complex may act as a crosslinker to bridge the viral and cell membranes at a suitable distance, which is critical, but also substitutable for spike‐mediated SARS‐CoV‐2 entry.

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Fusion Peptide of SARS-CoV-2 Spike Rearranges into a Wedge Inserted in Bilayered Micelles

Cited by 63 publications

References 56 publications

Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides

Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides

PDZ-Containing Proteins Targeted by the ACE2 Receptor

A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS‐CoV‐2 Spike‐Mediated Viral Invasion

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