Computational epitope map of SARS-CoV-2 spike protein

The primary immunological target of COVID-19 vaccines is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface and mediates viral entry into the host cell. To identify possible antibody binding sites, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation, and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for structure-based vaccine design. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics. The protective glycan shield and the high flexibility of its hinges give the stalk overall low epitope scores. Our computational epitope-mapping procedure is general and should thus prove useful for other viral envelope proteins whose structures have been characterized.

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Map of SARS-CoV-2 spike epitopes not shielded by glycans

10.1101/2020.07.03.186825 ◽

2020 ◽

Cited By ~ 6

Author(s):

Mateusz Sikora ◽

Sören von Bülow ◽

Florian E. C. Blanc ◽

Michael Gecht ◽

Roberto Covino ◽

...

Keyword(s):

Viral Entry ◽

Vaccine Development ◽

Protein S ◽

Antibody Binding ◽

Structural Rigidity ◽

Steric Accessibility ◽

Dynamics Simulations ◽

Viral Surface ◽

Atom System ◽

Immunological Target

The severity of the COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, calls for the urgent development of a vaccine. The primary immunological target is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface to mediate viral entry into the host cell. To identify possible antibody binding sites not shielded by glycans, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for vaccine development. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics in epitope mapping.

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Influenza hemagglutinin drives viral entry via two sequential intramembrane mechanisms

10.1101/2020.01.30.926816 ◽

2020 ◽

Author(s):

Anna Pabis ◽

Robert J. Rawle ◽

Peter M. Kasson

Keyword(s):

Viral Entry ◽

Transmembrane Domain ◽

Critical Role ◽

Cellular Membrane ◽

Membrane Curvature ◽

Viral Envelope ◽

Enveloped Viruses ◽

Influenza Hemagglutinin ◽

Hemagglutinin Protein ◽

Dynamics Simulations

AbstractEnveloped viruses enter cells via a process of membrane fusion between the viral envelope and a cellular membrane. For influenza virus, mutational data have shown that the membrane-inserted portions of the hemagglutinin protein play a critical role in achieving fusion. In contrast to the relatively well-understood ectodomain, a predictive mechanistic understanding of the intramembrane mechanisms by which influenza hemagglutinin drives fusion has been elusive. We have used molecular dynamics simulations of fusion between a full-length hemagglutinin proteoliposome and a lipid bilayer to analyze these mechanisms. In our simulations, hemagglutinin first acts within the membrane to increase lipid tail protrusion and promote stalk formation and then acts to engage the distal leaflets of each membrane and promote stalk widening, curvature, and eventual fusion. These two sequential mechanisms, one occurring prior to stalk formation and one after, are consistent with experimental measurements we report of single-virus fusion kinetics to liposomes of different sizes. The resulting model also helps explain and integrate prior mutational and biophysical data, particularly the mutational sensitivity of the fusion peptide N-terminus and the length sensitivity of the transmembrane domain. We hypothesize that entry by other enveloped viruses may also utilize sequential processes of acyl tail exposure followed by membrane curvature and distal leaflet engagement.

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Dynamic Interactions of Fully Glycosylated SARS-CoV-2 Spike Protein with Various Antibodies

10.1101/2021.05.10.443519 ◽

2021 ◽

Author(s):

Yiwei Cao ◽

Yeol Kyo Choi ◽

Martin Frank ◽

Hyeonuk Woo ◽

Sang-Jun Park ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Antibody Binding ◽

Viral Envelope ◽

Binding Modes ◽

Dynamic Interactions ◽

Health Crisis ◽

S Protein ◽

Protein Receptor ◽

Dynamics Simulations ◽

Antibody Interactions

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a public health crisis, and the vaccines that can induce highly potent neutralizing antibodies are essential for ending the pandemic. The spike (S) protein on the viral envelope mediates human angiotensin-converting enzyme 2 (ACE2) binding and thus is the target of a variety of neutralizing antibodies. In this work, we built various S trimer-antibody complex structures on the basis of the fully glycosylated S protein models described in our previous work, and performed all-atom molecular dynamics simulations to get insight into the structural dynamics and interactions between S protein and antibodies. Investigation of the residues critical for S-antibody binding allows us to predict the potential influence of mutations in SARS-CoV-2 variants. Comparison of the glycan conformations between S-only and S-antibody systems reveals the roles of glycans in S-antibody binding. In addition, we explored the antibody binding modes, and the influences of antibody on the motion of S protein receptor binding domains. Overall, our analyses provide a better understanding of S-antibody interactions, and the simulation-based S-antibody interaction maps could be used to predict the influences of S mutation on S-antibody interactions, which will be useful for the development of vaccine and antibody-based therapy.

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Simulation of Molecular Dynamics of SARS-CoV-2 S-Protein in the Presence of Multiple Arbidol Molecules: Interactions and Binding Mode Insights

Viruses ◽

10.3390/v14010119 ◽

2022 ◽

Vol 14 (1) ◽

pp. 119

Author(s):

Sophia S. Borisevich ◽

Edward M. Khamitov ◽

Maxim A. Gureev ◽

Olga I. Yarovaya ◽

Nadezhda B. Rudometova ◽

...

Keyword(s):

Molecular Dynamics ◽

Antiviral Activity ◽

Viral Entry ◽

Binding Sites ◽

Surface Protein ◽

Protein S ◽

Binding Mode ◽

Structural Flexibility ◽

Binding Interface ◽

Dynamics Simulations

In this work, we evaluated the antiviral activity of Arbidol (Umifenovir) against SARS-CoV-2 using a pseudoviral system with the glycoprotein S of the SARS-CoV-2 virus on its surface. In order to search for binding sites to protein S of the virus, we described alternative binding sites of Arbidol in RBD and in the ACE-2-RBD complex. As a result of our molecular dynamics simulations combined with molecular docking data, we note the following fact: wherever the molecules of Arbidol bind, the interaction of the latter affects the structural flexibility of the protein. This interaction may result both in a change in the shape of the domain–enzyme binding interface and simply in a change in the structural flexibility of the domain, which can subsequently affect its affinity to the enzyme. In addition, we examined the possibility of Arbidol binding in the stem part of the surface protein. The possibility of Arbidol binding in different parts of the protein is not excluded. This may explain the antiviral activity of Arbidol. Our results could be useful for researchers searching for effective SARS-CoV-2 virus inhibitors targeting the viral entry stage.

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Altered O-glycosylation Level of SARS-CoV-2 Spike Protein by Host O-glycosyltransferase Strengthens Its Trimeric Structure

10.1101/2021.04.06.438614 ◽

2021 ◽

Author(s):

Zhijue Xu ◽

Xin Ku ◽

Jiaqi Tian ◽

Han Zhang ◽

Jingli Hou ◽

...

Keyword(s):

Membrane Fusion ◽

Protein S ◽

Viral Envelope ◽

Spike Protein ◽

Viral Envelope Protein ◽

S Protein ◽

Mass Spectrometric Approach ◽

Host Cell Entry ◽

Dynamics Simulations ◽

Glycan Heterogeneity

SummaryThe trimeric spike protein (S) mediates host-cell entry and membrane fusion of SARS-CoV-2. S protein is highly glycosylated, whereas its O-glycosylation is still poorly understood. Herein, we site-specifically examine the O-glycosylation of S protein through a mass spectrometric approach with HCD-triggered-ETD model. We identify 15 high-confidence O-glycosites and at least 10 distinct O-glycan structures on S protein. Peptide microarray assays prove that human ppGalNAc-T6 actively participates in O-glycosylation of S protein. Importantly, the upregulation of ppGalNAc-T6 expression can profoundly enhance the O-glycosylation level by generating new O-glycosites and increasing both O-glycan heterogeneity and intensities. Further molecular dynamics simulations reveal that the O-glycosylation on the protomer-interface regions, which are mainly modified by ppGalNAc-T6, can potentially stabilize the trimeric S protein structure. Our work provides deep molecular insights of how viral infection harnesses the host O-glycosyltransferases to dynamically regulate the O-glycosylation level of the viral envelope protein responsible for membrane fusion.

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Influenza hemagglutinin drives viral entry via two sequential intramembrane mechanisms

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914188117 ◽

2020 ◽

Vol 117 (13) ◽

pp. 7200-7207 ◽

Cited By ~ 5

Author(s):

Anna Pabis ◽

Robert J. Rawle ◽

Peter M. Kasson

Keyword(s):

Viral Entry ◽

Transmembrane Domain ◽

Critical Role ◽

Cellular Membrane ◽

Membrane Curvature ◽

Viral Envelope ◽

Enveloped Viruses ◽

Influenza Hemagglutinin ◽

Hemagglutinin Protein ◽

Dynamics Simulations

Enveloped viruses enter cells via a process of membrane fusion between the viral envelope and a cellular membrane. For influenza virus, mutational data have shown that the membrane-inserted portions of the hemagglutinin protein play a critical role in achieving fusion. In contrast to the relatively well-understood ectodomain, a predictive mechanistic understanding of the intramembrane mechanisms by which influenza hemagglutinin drives fusion has been elusive. We used molecular dynamics simulations of fusion between a full-length hemagglutinin proteoliposome and a lipid bilayer to analyze these mechanisms. In our simulations, hemagglutinin first acts within the membrane to increase lipid tail protrusion and promote stalk formation and then acts to engage the distal leaflets of each membrane and promote stalk widening, curvature, and eventual fusion. These two sequential mechanisms, one occurring before stalk formation and one after, are consistent with our experimental measurements of single-virus fusion kinetics to liposomes of different sizes. The resulting model also helps explain and integrate previous mutational and biophysical data, particularly the mutational sensitivity of the fusion peptide N terminus and the length sensitivity of the transmembrane domain. We hypothesize that entry by other enveloped viruses may also use sequential processes of acyl tail exposure, followed by membrane curvature and distal leaflet engagement.

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The two redox states of the human NEET proteins’ [2Fe–2S] clusters

JBIC Journal of Biological Inorganic Chemistry ◽

10.1007/s00775-021-01890-8 ◽

2021 ◽

Vol 26 (7) ◽

pp. 763-774

Author(s):

Ke Zuo ◽

Henri-Baptiste Marjault ◽

Kara L. Bren ◽

Giulia Rossetti ◽

Rachel Nechushtai ◽

...

Keyword(s):

Protein S ◽

Spectroscopic Studies ◽

Reduced Form ◽

Rieske Protein ◽

Kinetic Measurements ◽

Redox States ◽

Extra Electron ◽

Dynamics Simulations ◽

Reduced State

AbstractThe NEET proteins constitute a unique class of [2Fe–2S] proteins. The metal ions bind to three cysteines and one histidine. The proteins’ clusters exist in two redox states; the oxidized protein (containing two FeIII ions) can transfer the cluster to apo-acceptor protein(s), while the reduced form (containing one ferrous ion) remains bound to the protein frame. Here, we perform in silico and in vitro studies on human NEET proteins in both reduced and oxidized forms. Quantum chemical calculations on all available human NEET proteins structures suggest that reducing the cluster weakens the Fe–NHis and Fe–SCys bonds, similar to what is seen in other Fe–S proteins (e.g., ferredoxin and Rieske protein). We further show that the extra electron in the [2Fe–2S]+ clusters of one of the NEET proteins (mNT) is localized on the His-bound iron ion, consistently with our previous spectroscopic studies. Kinetic measurements demonstrate that the mNT [2Fe–2S]+ is released only by an increase in temperature. Thus, the reduced state of human NEET proteins [2Fe–2S] cluster is kinetically inert. This previously unrecognized kinetic inertness of the reduced state, along with the reactivity of the oxidized state, is unique across all [2Fe–2S] proteins. Finally, using a coevolutionary analysis, along with molecular dynamics simulations, we provide insight on the observed allostery between the loop L2 and the cluster region. Specifically, we show that W75, R76, K78, K79, F82 and G85 in the latter region share similar allosteric characteristics in both redox states. Graphic abstract

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Conformational Ensembles of an Intrinsically Disordered Protein pKID with and without a KIX Domain in Explicit Solvent Investigated by All-Atom Multicanonical Molecular Dynamics

Biomolecules ◽

10.3390/biom2010104 ◽

2012 ◽

Vol 2 (1) ◽

pp. 104-121 ◽

Cited By ~ 16

Author(s):

Koji Umezawa ◽

Jinzen Ikebe ◽

Mitsunori Takano ◽

Haruki Nakamura ◽

Junichi Higo

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Binding Site ◽

Complex Structure ◽

Intrinsically Disordered Protein ◽

Bound State ◽

Conformational Ensembles ◽

Intrinsically Disordered ◽

Dynamics Simulations ◽

High Flexibility

The phosphorylated kinase-inducible activation domain (pKID) adopts a helix–loop–helix structure upon binding to its partner KIX, although it is unstructured in the unbound state. The N-terminal and C-terminal regions of pKID, which adopt helices in the complex, are called, respectively, αA and αB. We performed all-atom multicanonical molecular dynamics simulations of pKID with and without KIX in explicit solvents to generate conformational ensembles. Although the unbound pKID was disordered overall, αA and αB exhibited a nascent helix propensity; the propensity of αA was stronger than that of αB, which agrees with experimental results. In the bound state, the free-energy landscape of αB involved two low free-energy fractions: native-like and non-native fractions. This result suggests that αB folds according to the induced-fit mechanism. The αB-helix direction was well aligned as in the NMR complex structure, although the αA helix exhibited high flexibility. These results also agree quantitatively with experimental observations. We have detected that the αB helix can bind to another site of KIX, to which another protein MLL also binds with the adopting helix. Consequently, MLL can facilitate pKID binding to the pKID-binding site by blocking the MLL-binding site. This also supports experimentally obtained results.

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Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e17-06-0400 ◽

2017 ◽

Vol 28 (26) ◽

pp. 3801-3814 ◽

Cited By ~ 2

Author(s):

Sunandini Chandra ◽

Raju Kalaivani ◽

Manoj Kumar ◽

Narayanaswamy Srinivasan ◽

Debi P. Sarkar

Keyword(s):

Membrane Fusion ◽

Viral Entry ◽

Sendai Virus ◽

Viral Envelope ◽

Bioactive Molecules ◽

Host Cells ◽

Actin Dynamics ◽

Envelope Glycoproteins ◽

Animal Cells ◽

Viral Envelopes

Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion–mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206—an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.

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The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

Journal of Virology ◽

10.1128/jvi.73.2.1293-1301.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1293-1301 ◽

Cited By ~ 21

Author(s):

Kazunori Inabe ◽

Masako Nishizawa ◽

Shigeru Tajima ◽

Kazuyoshi Ikuta ◽

Yoko Aida

Keyword(s):

Viral Entry ◽

Envelope Protein ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Transient Transfection ◽

Viral Envelope ◽

Tyrosine Residue ◽

Wild Type ◽

Viral Envelope Protein

ABSTRACT The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.

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