Phospholipid binding of the dengue virus envelope E protein segment containing the conserved His residue.

Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.

Interaction of Lassa virus fusion and membrane proximal peptides with late endosomal membranes.

Mammarenaviruses include many significant worldwide-widespread human pathogens, among them Lassa virus (LASV), having a dramatic morbidity and mortality rate. They are a potential high-risk menace to the worldwide public health since there are no treatments and there is a high possibility of animal-to-human and human-to-human viral transmission. These viruses enter into the cells by endocytosis fusing its membrane envelope with the late endosomal membrane thanks to the glycoprotein GP2, a membrane fusion protein of class I. This protein contains different domains, among them the N-terminal fusion peptide (NFP), the internal fusion loop (IFL), the membrane proximal external region (MPER) and the transmembrane domain (TMD). All these domains are implicated in the membrane fusion process. In this work, we have used an all-atom molecular dynamics study to know the binding of these protein domains with a complex membrane mimicking the late endosome one. We show that the NFP/IFL domain is capable of spontaneously inserting into the membrane without a significant change of secondary structure, the MPER domain locates at the bilayer interface with an orientation parallel to the membrane surface and tends to interact with other MPER domains, and the TMD domain tilts inside the bilayer. Moreover, they predominantly interact with negatively charged phospholipids. Overall, these membrane-interacting domains would characterise a target that would make possible to find effective antiviral molecules against LASV in particular and Mammarenaviruses in general.

Envelope E protein of dengue virus and phospholipid binding to the late endosomal membrane.

Flaviviruses include many significant human pathogens, comprising dengue, West Nile, Yellow fever, Japanese encephalitis, Zika and tick-borne encephalitis viruses and many others, affecting millions of people in the world. These viruses have produced important epidemics in the past, they continue to do it and they will undoubtedly continue to do so in the future. Flaviviruses enter into the cells via receptor-mediated endocytosis by fusing its membrane with the endosomal membrane in a pH-dependent manner with the help of the envelope E protein, a prototypical class II membrane fusion protein. The envelope E protein has a conserved fusion peptide at its distal end, which is responsible in the first instance of inserting the protein into the host membrane. Since the participation of other segments of the E protein in the fusion process should not be ruled out, we have used atomistic molecular dynamics to study the binding of the distal end of domain II of the envelope E protein from Dengue virus (DENV) with a complex membrane similar to the late-endosome one. Our work shows that not only the fusion peptide participates directly in the fusion, but also two other sequences of the protein, next to the fusion peptide it in the three-dimensional structure, are jointly wrapped in the fusion process. Overall, these three sequences represent a new target that would make it possible to obtain effective antivirals against DENV in particular and Flaviviruses in general.

Location, Orientation and Aggregation of Bardoxolone-ME, CDDO-ME, in a Complex Phospholipid Bilayer Membrane.

Bardoxolone methyl (CDDO-Me), a synthetic derivative of the naturally occurring triterpenoid oleanolic acid, displays strong antioxidant, anticancer and anti-inflammatory activities, according to different bibliographical sources. However, the understanding of its molecular mechanism is missing. Furthermore, CDDO-Me has displayed a significant cytotoxicity against various types of cancer cells. CDDO-Me has a noticeable hydrophobic character and several of its effects could be attributed to its ability to be incorporated inside the biological membrane and therefore modify its structure and specifically interact with its components. In this study, we have used full-atom molecular dynamics to determine the location, orientation and interactions of CDDO-Me in phospholipid model membranes. Our results support the location of CDDO-Me in the middle of the membrane, it specifically orients so that the cyano group lean towards the phospholipid interface and it specifically interacts with particular phospholipids. Significantly, in the membrane the CDDO-Me molecules specifically interact with POPE and POPS. Moreover, CDDO-Me does not aggregates in the membrane but it forms a complex conglomerate in solution. The formation of a complex aggregate in solution might hamper its biological activity and therefore it should be taken into account when intended to be used in clinical assays. This work should aid in the development of these molecules opening new avenues for future therapeutic developments.

Epigallocatechin-3-gallate location and interaction with late endosomal and plasma membrane model membranes by molecular dynamics.

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea and it has been reported to have many beneficial properties against many different types of illnesses and infections. However, the exact mechanism/s underlying its biological effects are unknown. It has been previously shown that EGCG is capable of binding to and disrupting the membrane, so that some of its effects on biological systems could be ascribed to its capacity to incorporate into the biological membrane and modulate its structure. In this work, we have used atomistic molecular dynamics (MD) to discern the location and orientation of EGCG in model membranes and the possible existence of specific interactions with membrane lipids. For that goal, we have used in our simulation two complex model membranes, one resembling the plasma membrane (PM) and the other one the late endosome (LE) membrane. Our results support that EGCG tends to associate with the membrane and exists inside it in a relatively stable and steady location with a low propensity to be associated with other EGCG molecules. Interestingly, EGCG forms hydrogen bonds with POPC and POPE in the PM system but POPC and BMP and no POPE in the LE. These data suggest that the broad beneficial effects of EGCG could be mediated, at least in part, through its membranotropic effects and therefore membrane functioning. Communicated by Ramaswamy H. Sarma.

Spontaneous membrane insertion of a dengue virus NS2A peptide.

Non-structural NS2A protein of Dengue virus is essential for viral replication but poorly characterized because of its high hydrophobicity. We have previously shown experimentally that NS2A possess a segment, peptide dens25, known to insert into membranes and interact specifically with negatively-charged phospholipids. To characterize its membrane interaction we have used two types of molecular dynamics membrane model systems, a highly mobile membrane mimetic (HMMM) and an endoplasmic reticulum (ER) membrane-like model. Using the HMMM system, we have been able of demonstrating the spontaneous binding of dens25 to the negatively-charged phospholipid 1,2-divaleryl-sn-glycero-3-phosphate containing membrane whereas no binding was observed for the membrane containing the zwitterionic one 1,2-divaleryl-sn-glycero-3-phosphocholine. Using the ER-like membrane model system, we demonstrate the spontaneous insertion of dens25 into the middle of the membrane, it maintained its three-dimensional structure and presented a nearly parallel orientation with respect to the membrane surface. Both charged and hydrophobic amino acids, presenting an interfacial/hydrophobic pattern characteristic of a membrane-proximal segment, are responsible for membrane binding and insertion. Dens25 might control protein/membrane interaction and be involved in membrane rearrangements critical for the viral cycle. These data should help us in the development of inhibitor molecules that target NS2A segments involved in membrane reorganisation.

Molecular dynamics study of the membrane interaction of a membranotropic dengue virus C protein-derived peptide.

Dengue virus C protein, essential in the dengue virus life cycle, possesses a segment, peptide PepC, known to bind membranes composed of negatively charged phospholipids. To characterize its interaction with the membrane, we have used the molecular dynamics HMMM membrane model system. This approach is capable of achieving a stable system and sampling the peptide/lipid interactions which determine the orientation and insertion of the peptide upon membrane binding. We have been able to demonstrate spontaneous binding of PepC to the 1,2-divaleryl-sn-glycero-3-phosphate/1,2-divaleryl-sn-glycero-3-phosphocholine membrane model system, whereas no binding was observed at all for the 1,2-divaleryl-sn-glycero-3-phosphocholine one. PepC, adopting an α-helix profile, did not insert into the membrane but did bind to its surface through a charge anchor formed by its three positively charged residues. PepC, maintaining its three-dimensional structure along the whole simulation, presented a nearly parallel orientation with respect to the membrane when bound to it. The positively charged amino acid residues Arg-2, Lys-6, and Arg-16 are mainly responsible for the peptide binding to the membrane stabilizing the structure of the bound peptide. The segment of dengue virus C protein where PepC resides is a fundamental protein-membrane interface which might control protein/membrane interaction, and its positive amino acids are responsible for membrane binding defining its specific location in the bound state. These data should help in our understanding of the molecular mechanism of DENV life cycle as well as making possible the future development of potent inhibitor molecules, which target dengue virus C protein structures involved in membrane binding.

Oleuropein aglycone in lipid bilayer membranes. A molecular dynamics study.

Olive oil has been recognized to possess many therapeutic applications. Its beneficial effects arise from many causes, but one of them lies on the presence of oleuropein aglycone (OA). OA presents a plethora of pharmacological beneficial properties. Although there is a great research going on the effect of polyphenols and their derivatives on different aspects of health, much less knowledge is available of the molecular basis of their beneficial effects. Due to the prominent hydrophobic character of OA and its high phospholipid/water partition coefficient, some of its possible effects on biological systems might be related to its capacity to interact with and locate into the membrane. In this work we have aimed to locate the molecule of OA in two membrane model systems, i.e., POPC/Chol and POPC/POPG/Chol. OA locates in between the hydrocarbon acyl chains of the phospholipids but its specific location and molecular interactions differ depending on the lipid system. OA is nearer to the membrane surface in the POPC/Chol system but it is located at a deeper position in the POPC/POPG/Chol system. Furthermore, OA seems to interact stronger with POPG than with POPC, implying the existence of specific interactions with negatively-charged phospholipids. Some of the biological effects of OA could be due to its preferential location in the membrane depending on the membrane lipid composition as well as the existence of specific interactions with specific phospholipids.

Possible mechanism of structural transformations induced by StAsp-PSI in lipid membranes.

In the present work we have analyzed the effect of StAsp-PSI (plant-specific insert of potato aspartic protease) on the structural and thermotropic properties of the major phospholipid types of bacterial and animal cells. Results obtained suggest that StAsp-PSI induces a destabilization of the membrane bilayers, depending on the time of interaction between the protein and the bilayers, rather than on its concentration. This temporal delay would be consistent with a lateral diffusion of StAsp-PSI monomers to assemble into aggregates to form pores. Like with the results previously reported for the StAsp-PSI circular dichroism, data obtained here from IR spectroscopy show that there are slight changes in the StAsp-PSI secondary structure in the presence of lipid membranes; suggesting that these changes could be related with the StAsp-PSI self-association. Results obtained from steady-state fluorescence anisotropy and differential scanning calorimetry assays suggest that StAsp-PSI interacts with both uncharged and negatively charged phospholipids, modulates the phase polymorphic behavior of model membranes and partitions and buries differentially in the membrane depending on the presence of negatively charged phospholipids.

Cholesterol and membrane phospholipid compositions modulate the leakage capacity of the swaposin domain from a potato aspartic protease (StAsp-PSI).

Potato aspartic proteases (StAPs) and their swaposin domain (StAsp-PSI) are proteins with cytotoxic activity which involves plasma membrane destabilization. The ability of these proteins to produce cell death varies with the cellular type. Therefore, StAPs and StAsp-PSI selective cytotoxicity could be attributed to the different membrane lipid compositions of target cells. In this work we investigate the possible mechanism by which StAPs and StAsp-PSI produce selective membrane destabilization. Results obtained from leakage assays show that StAsp-PSI is a potent inducer of the leakage of LUVs containing anionic phospholipids, especially those containing phosphatidylglycerol. Based in these results, we suggest that the cytotoxic activity of StAsp-PSI on pathogenic microorganisms could be mediated by the attraction between the exposed positive domains of StAsp-PSI and the negatively charged microorganism membrane. On the other hand, our circular dichroism spectroscopic measurements and analysis by size exclusion chromatography and followed by electrophoresis, indicate that hydrophobic environment is necessary to StAsp-PSI oligomerization and both StAsp-PSI disulfide bounds and membrane with negative charged phospholipids are required by StAsp-PSI to produce membrane destabilization and then induce cell death in tumors and microorganism cell targets. Additionally, we demonstrate that the presence of cholesterol into the LUV membranes strongly diminishes the capacity of StAsp-PSI to produce leakage. This result suggests that the lack of hemolytic and cytotoxic activities on human lymphocytes of StAsp-PSI/StAPs may be partly due by the presence of cholesterol in these cell membrane types.

Membrane interaction of segment H1 (NS4B(H1)) from hepatitis C virus non-structural protein 4B.

NS4B protein from hepatitis C virus (HCV) is a highly hydrophobic protein inducing a rearrangement of endoplasmic reticulum membranes responsible of the HCV replication process. Different helical elements have been found in the N- and C- terminal domains of the protein, which seem to be responsible for many key aspects of the viral replication process. In this work we have carried out a study of the binding and interaction with model biomembranes of peptide NS4B(H1), patterned after segment H1, one of these C-terminal previously identified segments. We show that NS4B(H1) partitions into phospholipid membranes; its membrane activity is modulated by lipid composition, interacting preferentially with negatively charged phospholipids as well as with sphingomyelin. Furthermore, the change in its sequence prevents the resulting peptide from interacting with the membrane. These data would support its role in the interaction of NS4B with the membrane and suggest that the region where this peptide resides could be involved in the membrane alteration which must occur in the HCV replication and/or assembly process.

Interaction of the N-terminal segment of HCV protein NS5A with model membranes.

We have identified a membrane-active region in the HCV NS5A protein by performing an exhaustive study of membrane rupture induced by a NS5A-derived peptide library on model membranes having different phospholipid compositions. We report the identification in NS5A of a highly membranotropic region located at the suggested membrane association domain of the protein. We report the binding and interaction with model membranes of two peptides patterned after this segment, peptides 1A and 1B, derived from the strains 1a_H77 and 1b_HC-4J respectively. We show that they insert into phospholipid membranes, interact with them, and are located in a shallow position in the membrane. The NS5A region where this segment resides might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the replication complex, and consequently, directly implicated in the HCV life cycle.

A membranotropic region in the C-terminal domain of hepatitis C virus protein NS4B interaction with membranes.

We have identified a membrane-active region in the HCV NS4B protein by studying membrane rupture induced by a NS4B-derived peptide library on model membranes. This segment corresponds to one of two previously predicted amphipathic helix and define it as a new membrane association domain. We report the binding and interaction with model membranes of a peptide patterned after this segment, peptide NS4B(H2), and show that NS4B(H2) strongly partitions into phospholipid membranes, interacts with them, and is located in a shallow position in the membrane. Furthermore, changes in the primary sequence cause the disruption of the hydrophobicity along the structure and prevent the resulting peptide from interacting with the membrane. Our results suggest that the region where the NS4B(H2) is located might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the replication complex. Our findings therefore identify an important region in the HCV NS4B protein which might be implicated in the HCV life cycle and possibly in the formation of the membranous web.

Biophysical characterization of the fusogenic region of HCV envelope glycoprotein E1.

We have studied the binding and interaction of the peptide E1(FP) with various model membranes. E1(FP) is derived from the amino acid segment 274-291 of the hepatitis C virus envelope glycoprotein E1, which was previously proposed to host the peptide responsible for fusion to target membranes. In the present study we addressed the changes which take place upon E1(FP) binding in both the peptide and the phospholipid bilayer, respectively, through a series of complementary experiments. We show that peptide E1(FP) binds to and interacts with phospholipid model membranes, modulates the polymorphic phase behavior of membrane phospholipids, is localized in a shallow position in the membrane and interacts preferentially with cholesterol. The capability of modifying the biophysical properties of model membranes supports its role in HCV-mediated membrane fusion and suggests that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.

Interaction of a peptide corresponding to the loop domain of the S2 SARS-CoV virus protein with model membranes.

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein is responsible for the fusion between the membranes of the virus and the target cell. In the case of the S2 domain of protein S, it has been found a highly hydrophobic and interfacial domain flanked by the heptad repeat 1 and 2 regions; significantly, different peptides pertaining to this domain have shown a significant leakage effect and an important plaque formation inhibition, which, similarly to HIV-1 gp41, support the role of this region in the fusion process. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 1073-1095 of the SARS-CoV S glycoprotein, peptide SARS(L) in the presence of different membrane model systems, as well as the structural changes taking place in both the lipid and the peptide induced by the binding of the peptide to the membrane. Our results show that SARS(L) strongly partitions into phospholipid membranes and organizes differently in lipid environments, displaying membrane activity modulated by the lipid composition of the membrane. These data would support its role in SARS-CoV mediated membrane fusion and suggest that the region where this peptide resides could be involved in the merging of the viral and target cell membranes.

Screening a peptide library by DSC and SAXD: comparison with the biological function of the parent proteins.

We have recently identified the membranotropic regions of the hepatitis C virus proteins E1, E2, core and p7 proteins by observing the effect of protein-derived peptide libraries on model membrane integrity. We have studied in this work the ability of selected sequences of these proteins to modulate the L(beta)-L(alpha) and L(alpha)-H(II) phospholipid phase transitions as well as check the viability of using both DSC and SAXD to screen a protein-derived peptide library. We demonstrate that it is feasible to screen a library of peptides corresponding to one or several proteins by both SAXD and DSC. This methodological combination should allow the identification of essential regions of membrane-interacting proteins which might be implicated in the molecular mechanism of membrane fusion and/or budding.

Membrane insertion of the three main membranotropic sequences from SARS-CoV S2 glycoprotein.

In order to complete the fusion process of SARS-CoV virus, several regions of the S2 virus envelope glycoprotein are necessary. Recent studies have identified three membrane-active regions in the S2 domain of SARS-CoV glycoprotein, one situated downstream of the minimum furin cleavage, which is considered the fusion peptide (SARSFP), an internal fusion peptide located immediately upstream of the HR1 region (SARSIFP) and the pre-transmembrane domain (SARSPTM). We have explored the capacity of these selected membrane-interacting regions of the S2 SARS-CoV fusion protein, alone or in equimolar mixtures, to insert into the membrane as well as to perturb the dipole potential of the bilayer. We show that the three peptides interact with lipid membranes depending on lipid composition and experiments using equimolar mixtures of these peptides show that different segments of the protein may act in a synergistic way suggesting that several membrane-active regions could participate in the fusion process of the SARS-CoV.

A second SARS-CoV S2 glycoprotein internal membrane-active peptide. Biophysical characterization and membrane interaction.

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. The S2 domain of protein S has been suggested to have two fusion peptides, one located at its N-terminus, downstream of the furin cleavage, and another, more internal, located immediately upstream of the HR1. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 873-888 of the SARS-CoV S glycoprotein, peptide SARS IFP, as well as the structural changes taking place in both the phospholipid and the peptide induced by the binding of the peptide to the membrane. We demonstrate that SARS IFP peptide binds to and interacts with phospholipid model membranes and shows a higher affinity for negatively charged phospholipids than for zwitterionic ones. SARS IFP peptide specifically decreases the mobility of the phospholipid acyl chains of negatively charged phospholipids and adopts different conformations in the membrane depending upon their composition. These data support its role in SARS-mediated membrane fusion and suggest that the regions where this peptide resides might assist the fusion peptide and/or the pretransmembrane segment of the SARS-CoV spike glycoprotein in the fusion process.

Structural and dynamic characterization of the interaction of the putative fusion peptide of the S2 SARS-CoV virus protein with lipid membranes.

The SARS coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a Class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. In the present work, we report a study of the binding and interaction with model membranes of a peptide pertaining to the putative fusion domain of SARS-CoV, SARS FP, as well as the structural changes that take place in both the phospholipid and the peptide molecules upon this interaction. From fluorescence and infrared spectroscopies, the peptide ability to induce membrane leakage, aggregation and fusion, as well as its affinity toward specific phospholipids, was assessed. We demonstrate that SARS FP strongly partitions into phospholipid membranes, more specifically with those containing negatively charged phospholipids, increasing the water penetration depth and displaying membrane-activity modulated by the lipid composition of the membrane. Interestingly, peptide organization is different depending if SARS FP is in water or bound to the membrane. These data suggest that SARS FP could be involved in the merging of the viral and target cell membranes by perturbing the membrane outer leaflet phospholipids and specifically interacting with negatively charged phospholipids located in the inner leaflet.

The pre-transmembrane region of the HCV E1 envelope glycoprotein: interaction with model membranes.

The previously identified membranotropic regions of the HCV E1 envelope glycoprotein, a class II membrane fusion protein, permitted us to identify different sequences which might be implicated in viral membrane fusion, membrane interaction and/or protein-protein binding. HCV E1 glycoprotein presents a membrano-active region immediately adjacent to the transmembrane segment, which could be involved in membrane destabilization similarly to the pre-transmembrane domains of class I fusion proteins. Consequently, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 309-340, peptide E1PTM, as well as the structural changes which take place in both the peptide and the phospholipid molecules induced by the binding of the peptide to the membrane. Here we demonstrate that peptide E1(PTM) strongly partitions into phospholipid membranes, interacts with negatively-charged phospholipids and locates in a shallow position in the membrane. These data support its role in HCV-mediated membrane fusion and suggest that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.