Prediction of amphipathic in-plane membrane anchors in monotopic proteins using a SVM classifier

BACKGROUND: Membrane proteins are estimated to represent about 25% of open reading frames in fully sequenced genomes. However, the experimental study of proteins remains difficult. Considerable efforts have thus been made to develop prediction methods. Most of these were conceived to detect transmembrane helices in polytopic proteins. Alternatively, a membrane protein can be monotopic and anchored via an amphipathic helix inserted in a parallel way to the membrane interface, so-called in-plane membrane (IPM) anchors. This type of membrane anchor is still poorly understood and no suitable prediction method is currently available. RESULTS: We report here the "AmphipaSeeK" method developed to predict IPM anchors. It uses a set of 21 reported examples of IPM anchored proteins. The method is based on a pattern recognition Support Vector Machine with a dedicated kernel. CONCLUSION: AmphipaSeeK was shown to be highly specific, in contrast with classically used methods (e.g. hydrophobic moment). Additionally, it has been able to retrieve IPM anchors in naively tested sets of transmembrane proteins (e.g. PagP). AmphipaSeek and the list of the 21 IPM anchored proteins is available on NPS@, our protein sequence analysis server

NMR structure and ion channel activity of the p7 protein from hepatitis C virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1H and 13C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.Instituto de Física La Plat

NMR structure and ion channel activity of the p7 protein from hepatitis C virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1H and 13C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.Instituto de Física La Plat

Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein

Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important

Structure-Based High-Throughput Epitope Analysis of Hexon Proteins in B and C Species Human Adenoviruses (HAdVs)

Human adenoviruses (HAdVs) are the etiologic agent of many human infectious diseases. The existence of at least 54 different serotypes of HAdVs has resulted in difficulties in clinical diagnosis. Acute respiratory tract disease (ARD) caused by some serotypes from B and C species is particularly serious. Hexon, the main coat protein of HAdV, contains the major serotype-specific B cell epitopes; however, few studies have addressed epitope mapping in most HAdV serotypes. In this study, we utilized a novel and rapid method for the modeling of homologous proteins based on the phylogenetic tree of protein families and built three-dimensional (3D) models of hexon proteins in B and C species HAdVs. Based on refined hexon structures, we used reverse evolutionary trace (RET) bioinformatics analysis combined with a specially designed hexon epitope screening algorithm to achieve high-throughput epitope mapping of all 13 hexon proteins in B and C species HAdVs. This study has demonstrated that all of the epitopes from the 13 hexon proteins are located in the proteins' tower regions; however, the exact number, location, and size of the epitopes differ among the HAdV serotypes

Mechanism of Inhibition of Enveloped Virus Membrane Fusion by the Antiviral Drug Arbidol

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection

Linearized esculentin-2EM shows pH dependent antibacterial activity with an alkaline optimum

Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5 to 26.9 %), while similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3 to 5.1 mN m-1) and lyse (↑ 15.1 to 32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1-23) in the N → C direction, with - increasing overall from circa - 0.8 to - 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms