A benzene-mapping approach for uncovering cryptic pockets in membrane-bound proteins

ABSTRACTMolecular dynamics (MD) simulations in combination with small organic probes present in the solvent have previously been used as a method to reveal cryptic pockets that may not have been identified in experimental structures. We report such a method implemented within the CHARMM forcefield to effectively explore cryptic pockets on the surfaces of membrane-embedded proteins using benzene as a probe molecule. This relies on modified non-bonded parameters in addition to repulsive potentials between membrane lipids and benzene molecules. The method was tested on part of the outer shell of the dengue virus (DENV), for which research into a safe and effective neutralizing antibody or drug molecule is still ongoing. In particular, the envelope (E) protein, associated with the membrane (M) protein, is a lipid membrane-embedded complex which forms a dimer in the mature viral envelope. Solvent mapping was performed for the full, membrane-embedded EM protein complex and compared with similar calculations performed for the isolated, soluble E protein ectodomain dimer in solvent. Ectodomain-only simulations with benzene exhibited unfolding effects not observed in the more physiologically relevant membrane-associated systems. A cryptic pocket which has been experimentally shown to bind n-octyl-β-D-glucoside detergent was consistently revealed in all benzene-containing simulations. The addition of benzene also enhanced the flexibility and hydrophobic exposure of cryptic pockets at a key, functional interface in the E protein, and revealed a novel, potentially druggable pocket that may be targeted to prevent conformational changes associated with viral entry into the cell.GRAPHICAL ABSTRACT

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Chapter 2b: The molecular antigenic structure of the TBEV

Tick-borne encephalitis - The Book ◽

10.33442/26613980_2b-3 ◽

2020 ◽

Keyword(s):

Conformational Changes ◽

Neutralizing Antibodies ◽

Lipid Membrane ◽

Cell Entry ◽

Antigenic Structure ◽

E Proteins ◽

Amino Acid Sequence Level ◽

Endosomal Membrane ◽

E Protein ◽

Golgi Network

TBEV-particles are assembled in an immature, noninfectious form in the endoplasmic reticulum by the envelopment of the viral core (containing the viral RNA) by a lipid membrane associated with two viral proteins, prM and E. Immature particles are transported through the cellular exocytic pathway and conformational changes induced by acidic pH in the trans-Golgi network allow the proteolytic cleavage of prM by furin, a cellular protease, resulting in the release of mature and infectious TBE-virions. The E protein controls cell entry by mediating attachment to as yet ill-defined receptors as well as by low-pH-triggered fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. Because of its key functions in cell entry, the E protein is the primary target of virus neutralizing antibodies, which inhibit these functions by different mechanisms. Although all flavivirus E proteins have a similar overall structure, divergence at the amino acid sequence level is up to 60 percent (e.g. between TBE and dengue viruses), and therefore cross-neutralization as well as (some degree of) cross-protection are limited to relatively closely related flaviviruses, such as those constituting the tick-borne encephalitis serocomplex.

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Multiple Virtual Screening Strategies for the Discovery of Novel Compounds Active Against Dengue Virus: A Hit Identification Study

Scientia Pharmaceutica ◽

10.3390/scipharm88010002 ◽

2019 ◽

Vol 88 (1) ◽

pp. 2 ◽

Cited By ~ 4

Author(s):

Kowit Hengphasatporn ◽

Arthur Garon ◽

Peter Wolschann ◽

Thierry Langer ◽

Shigeta Yasuteru ◽

...

Keyword(s):

Virtual Screening ◽

Dengue Infection ◽

Md Simulations ◽

Viral Envelope ◽

Screening Process ◽

Active Inhibitor ◽

Effective Prevention ◽

E Protein ◽

Drug Databases ◽

Promising Target

Dengue infection is caused by a mosquito-borne virus, particularly in children, which may even cause death. No effective prevention or therapeutic agents to cure this disease are available up to now. The dengue viral envelope (E) protein was discovered to be a promising target for inhibition in several steps of viral infection. Structure-based virtual screening has become an important technique to identify first hits in a drug screening process, as it is possible to reduce the number of compounds to be assayed, allowing to save resources. In the present study, pharmacophore models were generated using the common hits approach (CHA), starting from trajectories obtained from molecular dynamics (MD) simulations of the E protein complexed with the active inhibitor, flavanone (FN5Y). Subsequently, compounds presented in various drug databases were screened using the LigandScout 4.2 program. The obtained hits were analyzed in more detail by molecular docking, followed by extensive MD simulations of the complexes. The highest-ranked compound from this procedure was then synthesized and tested on its inhibitory efficiency by experimental assays.

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Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609780113 ◽

2016 ◽

Vol 113 (45) ◽

pp. 12768-12773 ◽

Cited By ~ 44

Author(s):

Leopold Kong ◽

David E. Lee ◽

Rameshwar U. Kadam ◽

Tong Liu ◽

Erick Giang ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Vaccine Design ◽

Neutralizing Antibody ◽

Md Simulations ◽

Receptor Binding Site ◽

Structural Investigations ◽

Thermophilic Organisms

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world’s population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen–deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.

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Chapter 2b: The molecular antigenic structure of the TBEV

Tick-borne encephalitis - The Book ◽

10.33442/26613980_2b-4 ◽

2021 ◽

Author(s):

Franz-Xaver Heinz ◽

Karin Stiasny

Keyword(s):

Conformational Changes ◽

Neutralizing Antibodies ◽

Lipid Membrane ◽

Cell Entry ◽

Antigenic Structure ◽

E Proteins ◽

Amino Acid Sequence Level ◽

Endosomal Membrane ◽

E Protein ◽

Golgi Network

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Antiviral activity of silymarin and baicalein against dengue virus

Scientific Reports ◽

10.1038/s41598-021-98949-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zhao Xuan Low ◽

Brian Ming OuYong ◽

Pouya Hassandarvish ◽

Chit Laa Poh ◽

Babu Ramanathan

Keyword(s):

Antiviral Activity ◽

Dengue Virus ◽

Viral Entry ◽

Dengue Infection ◽

Antiviral Agents ◽

Antiviral Drug ◽

Vero Cells ◽

Viral Envelope ◽

E Protein

AbstractDengue is an arthropod-borne viral disease that has become endemic and a global threat in many countries with no effective antiviral drug available currently. This study showed that flavonoids: silymarin and baicalein could inhibit the dengue virus in vitro and were well tolerated in Vero cells with a half-maximum cytotoxic concentration (CC50) of 749.70 µg/mL and 271.03 µg/mL, respectively. Silymarin and baicalein exerted virucidal effects against DENV-3, with a selective index (SI) of 10.87 and 21.34, respectively. Baicalein showed a better inhibition of intracellular DENV-3 progeny with a SI of 7.82 compared to silymarin. Baicalein effectively blocked DENV-3 attachment (95.59%) to the Vero cells, while silymarin prevented the viral entry (72.46%) into the cells, thus reducing viral infectivity. Both flavonoids showed promising antiviral activity against all four dengue serotypes. The in silico molecular docking showed that silymarin could bind to the viral envelope (E) protein with a binding affinity of − 8.5 kcal/mol and form hydrogen bonds with the amino acids GLN120, TRP229, ASN89, and THR223 of the E protein. Overall, this study showed that silymarin and baicalein exhibited potential anti-DENV activity and could serve as promising antiviral agents for further development against dengue infection.

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Structure of Acidic pH Dengue Virus Showing the Fusogenic Glycoprotein Trimers

Journal of Virology ◽

10.1128/jvi.02411-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 743-750 ◽

Cited By ~ 37

Author(s):

Xinzheng Zhang ◽

Ju Sheng ◽

S. Kyle Austin ◽

Tabitha E. Hoornweg ◽

Jolanda M. Smit ◽

...

Keyword(s):

Dengue Virus ◽

Conformational Changes ◽

Lipid Membrane ◽

Neutralizing Antibody ◽

Intermediate Stage ◽

Host Cells ◽

Acidic Ph ◽

E Proteins ◽

Fab Fragments ◽

Domain Iii

ABSTRACTFlaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However, the dynamic nature of the fusogenic trimer has made the determination of its structure a challenge. Here we have used Fab fragments of the neutralizing antibody DV2-E104 to stop the conformational change of dengue virus at an intermediate stage of the fusion process. Using cryo-electron microscopy, we show that in this intermediate stage, the E glycoproteins form 60 trimers that are similar to the predicted “open” fusogenic trimer.IMPORTANCEThe structure of a dengue virus has been captured during the formation of fusogenic trimers. This was accomplished by binding Fab fragments of the neutralizing antibody DV2-E104 to the virus at neutral pH and then decreasing the pH to 5.5. These trimers had an “open” conformation, which is distinct from the “closed” conformation of postfusion trimers. Only two of the three E proteins within each spike are bound by a Fab molecule at domain III. Steric hindrance around the icosahedral 3-fold axes prevents binding of a Fab to the third domain III of each E protein spike. Binding of the DV2-E104 Fab fragments prevents domain III from rotating by about 130° to the postfusion orientation and thus precludes the stem region from “zipping” together the three E proteins along the domain II boundaries into the “closed” postfusion conformation, thus inhibiting fusion.

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Chapter 2b: The molecular and antigenic structure of TBEV

Tick-borne encephalitis - The Book ◽

10.33442/978-981-14-0914-1_2b ◽

2019 ◽

Author(s):

Franz-Xaver Heinz ◽

Karin Stiasny

Keyword(s):

Conformational Changes ◽

Neutralizing Antibodies ◽

Lipid Membrane ◽

Cell Entry ◽

Antigenic Structure ◽

E Proteins ◽

Amino Acid Sequence Level ◽

Endosomal Membrane ◽

E Protein ◽

Golgi Network

• TBEV-particles are assembled in an immature, noninfectious form in the endoplasmic reticulum by the envelopment of the viral core (containing the viral RNA) by a lipid membrane associated with two viral proteins, prM and E. • Immature particles are transported through the cellular exocytic pathway and conformational changes induced by acidic pH in the trans-Golgi network allow the proteolytic cleavage of prM by furin, a cellular protease, resulting in the release of mature and infectious TBE-virions. • The E protein controls cell entry by mediating attachment to as yet ill-defined receptors as well as by low-pH-triggered fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. • Because of its key functions in cell entry, the E protein is the primary target of virus neutralizing antibodies, which inhibit these functions by different mechanisms. • Although all flavivirus E proteins have a similar overall structure, divergence at the amino acid sequence level is up to 60 percent (e.g. between TBE and dengue viruses), and therefore cross-neutralization as well as (some degree of) cross-protection are limited to relatively closely related flaviviruses, such as those constituting the tick-borne encephalitis serocomplex.

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Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins

Journal of Virology ◽

10.1128/jvi.01469-16 ◽

2016 ◽

Vol 90 (23) ◽

pp. 10762-10773 ◽

Cited By ~ 9

Author(s):

Jacquelyn A. Stone ◽

Bhadra M. Vemulapati ◽

Birgit Bradel-Tretheway ◽

Hector C. Aguilar

Keyword(s):

Membrane Protein ◽

Membrane Fusion ◽

Conformational Changes ◽

Protein Interactions ◽

Viral Entry ◽

Nipah Virus ◽

Viral Envelope ◽

Host Cells ◽

Protein Protein Interactions ◽

Flow Cytometric

ABSTRACTThe paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions.IMPORTANCENipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in virus-host cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on coimmunoprecipitation, a technique with some pitfalls. We developed a flow-cytometric assay to study membrane protein-protein interactions, and using this assay we report a bidentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family.

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GS-5806 Inhibits Pre- to Postfusion Conformational Changes of the Respiratory Syncytial Virus Fusion Protein

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00761-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 7109-7112 ◽

Cited By ~ 26

Author(s):

Dharmaraj Samuel ◽

Weimei Xing ◽

Anita Niedziela-Majka ◽

Jinny S. Wong ◽

Magdeleine Hung ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Fusion Protein ◽

Conformational Changes ◽

Viral Entry ◽

Viral Envelope ◽

Host Cell Membrane ◽

Virus Fusion ◽

Molecule Inhibitor ◽

Syncytial Virus

ABSTRACTGS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproducedin vitrousing a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity.

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Closing the gap: an atomistic structural and functional perspective of S. mansoni universal stress G4LZI3 protein in complex with phenolic compounds

Current Drug Discovery Technologies ◽

10.2174/1570163817666201001122436 ◽

2020 ◽

Vol 17 ◽

Author(s):

Priscilla Masamba ◽

Geraldene Munsamy ◽

Abidemi Paul Kappo

Keyword(s):

Phenolic Compounds ◽

Conformational Changes ◽

Stress Protein ◽

Md Simulations ◽

Binding Energies ◽

Developmental Cycle ◽

Structure Based Drug Design ◽

Bioinformatic Tools ◽

Drug Of Choice ◽

Functional Perspective

Background: For decades, Praziquantel has been the undisputed drug of choice for all schistosome infections, but rising concerns due to the unelucidated mechanism of action of the drug and unavoidable reports of emerging drug resistant strains has necessitated the need for alternative treatment drug. Moreover, current apprehension has been reinforced by total dependence on the drug for treatment hence, the search for novel and effective anti-schistosomal drugs. Uses: This study made use of bioinformatic tools to determine the structural binding of the Universal G4LZI3 stress protein (USP) in complex with ten polyphenol compounds, thereby highlighting the effectiveness of these recently identified ‘lead’ molecules in the design of novel therapeutics targeted against schistosomiasis. Upregulation of the G4LZI3 USP throughout the schistosome multifaceted developmental cycle sparks interest in its potential role as a druggable target. The integration of in silico tools provides an atomistic perspective into the binding of potential inhibitors to target proteins. Conclusion: This study therefore, implemented the use of molecular dynamic (MD) simulations to provide functional and structural insight into key conformational changes upon binding of G4ZLI3 to these key phenolic compounds. Post-MD analyses revealed unique structural and conformational changes in the G4LZI3 protein in complex with curcumin and catechin respectively. These systems exhibited the highest binding energies, while the major interacting residues conserved in all the complexes provides a route map for structure-based drug design of novel compounds with enhanced inhibitory potency against the G4LZI3 protein. This study suggests an alternative approach for the development of anti-schistosomal drugs using natural compounds.

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