Summary: HELIQUEST calculates the physicochemical properties and amino acid composition of an α-helix and screens databank to identify protein segments possessing similar features. This server is also dedicated to mutating helices manually or automatically by genetic algorithm to design analogues of defined features. Availability: http://heliquest.ipmc.cnrs.fr

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HELIQUEST: a web server to screen sequences with specific α-helical properties

Viruses are intracellular parasites that use the host cell they infect to produce new infectious progeny. Distinct steps of the virus life cycle occur in association with the cytoskeleton or cytoplasmic membranes, which are often modified during infection. Plus-stranded RNA viruses induce membrane proliferations that support the replication of their genomes. Similarly, cytoplasmic replication of some DNA viruses occurs in association with modified cellular membranes. We describe how viruses modify intracellular membranes, highlight similarities between the structures that are induced by viruses of different families and discuss how these structures could be formed.

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Modification of intracellular membrane structures for virus replication

The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic α-helix: its polar face is weakly charged, containing mainly serine and threonine residues. We show that this feature explains the specificity of ArfGAP1 for curved versus flat lipid membranes. We built an algorithm to identify other potential amphipathic α-helices rich in serine and threonine residues in protein databases. Among the identified sequences, we show that three act as membrane curvature sensors. In the golgin GMAP-210, the sensor may serve to trap small vesicles at the end of a long coiled coil. In Osh4p/Kes1p, which transports sterol between membranes, the sensor controls access to the sterol-binding pocket. In the nucleoporin Nup133, the sensor corresponds to an exposed loop of a β-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.

A general amphipathic |[alpha]|-helical motif for sensing membrane curvature

An all-atomistic force field (FF) has been developed for fully saturated phospholipids. The parametrization has been largely based on high-level ab initio calculations in order to keep the empirica ...

Derivation and systematic validation of a refined all-atom force field for phosphatidylcholine lipids.

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.

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

https://www.cell.com/biophysj/pdf/S0006-3495(13)05745-7.pdf

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

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. 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. 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.

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

We have investigated the thermodynamics of phospholipid flip-flop and desorption. Using a series of PC (phosphatidylcholine) lipids with different lengths of acyl tails, and number of unsaturated tails, we calculated potentials of mean force (PMFs) in atomistic molecular dynamics simulations. The PMFs describe the free energy for moving a single lipid molecule from water to the center of the respective lipid bilayer. The free energy to move the lipid from equilibrium to the bilayer center is assumed to be the free energy barrier for lipid flip-flop. We find that the free energy barrier for flip-flop is strongly dependent on the structure of the bilayer; ranging from 16 kJ mol−1 in the thin DLPC bilayer, to 90 kJ mol−1 in the DOPC bilayer. There are large deformations in the bilayers' structure, to accommodate the charged PC head group in the bilayer interior. We observe pore formation in all the bilayers, except for POPC and DOPC. The free energy for desorption is equal to the excess chemical potential of the lipid in the bilayer compared to bulk water. The increased chemical potential for PC lipids with longer acyl tails is in qualitative agreement with the critical micelle concentrations. We also determined PMFs for transferring water into the center of the series of lipid bilayers. Water has the same free energy of transfer to the center of all the bilayers, indicating the lipid PMFs differ due to bilayer deformations. Lipid bilayers are soft and deformable, allowing large structural changes, which are dependent on the composition of the bilayer. Our results show that similar PC lipids with only slightly different acyl tails, can have dramatically different thermodynamic behavior.

Thermodynamics of flip-flop and desorption for a systematic series of phosphatidylcholine lipids

Computer simulations offer a valuable way to study membrane systems, from simple lipid bilayers to large transmembrane protein complexes and lipid-nucleic acid complexes for drug delivery. Their accuracy depends on the quality of the force field parameters used to describe the components of a particular system. We have implemented the widely used CHARMM22 and CHARMM27 force fields in the GROMACS simulation package to (i) combine the CHARMM22 protein force field with two sets of united-atom lipids parameters; (ii) allow comparisons of the lipid CHARMM27 force field with other lipid force fields or lipid-protein force field combinations. Our tests do not show any particular issue with the combination of the all-atom CHARMM22 force field with united-atoms lipid parameters, although pertinent experimental data are lacking to assess the quality of the lipid-protein interactions. The conversion utilities allow automatic generation of GROMACS simulation files with CHARMM nucleic acids and protein parameters and topologies, starting from pdb files using the standard GROMACS pdb2gmx method. CMAP is currently not implemented. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010

Combination of the CHARMM27 force field with united-atom lipid force fields

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a monotopic membrane protein anchored to the membrane by an N-terminal in-plane amphipathic alpha-helix. This membrane anchor is essential for the assembly of a functional viral replication complex. Although amino acid sequences differ considerably, putative membrane anchors with amphipathic features were predicted in NS5A from related Flaviviridae family members, in particular bovine viral diarrhea virus (BVDV), the prototype representative of the genus Pestivirus. We report here the NMR structure of the membrane anchor 1-28 of NS5A from BVDV in the presence of different membrane mimetic media. This anchor includes a long amphipathic alpha-helix of 21 residues interacting in-plane with the membrane interface and including a putative flexible region. Molecular dynamic simulation at a water-dodecane interface used to mimic the surface separating a lipid bilayer and an aqueous medium demonstrated the stability of the helix orientation and the location at the hydrophobic-hydrophilic interface. The flexible region of the helix appears to be required to allow the most favorable interaction of hydrophobic and hydrophilic side chain residues with their respective environment at the membrane interface. Despite the lack of amino acid sequence similarity, this amphipathic helix shares common structural features with that of the HCV counterpart, including a stable, hydrophobic N-terminal segment separated from the more hydrophilic C-terminal segment by a local, flexible region. These structural conservations point toward conserved roles of the N-terminal in-plane membrane anchors of NS5A in replication complex formation of HCV, BVDV, and other related viruses.

Nicolas Sapay

Papers

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

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

Thermodynamics of flip-flop and desorption for a systematic series of phosphatidylcholine lipids

Combination of the CHARMM27 force field with united-atom lipid force fields

NMR structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5A from bovine viral diarrhea virus.