The effect of rhamnolipids on fungal membrane models as described by their interactions with phospholipids and sterols: An in silico study.

Introduction: Rhamnolipids (RLs) are secondary metabolites naturally produced by bacteria of the genera Pseudomonas and Burkholderia with biosurfactant properties. A specific interest raised from their potential as biocontrol agents for crop culture protection in regard to direct antifungal and elicitor activities. As for other amphiphilic compounds, a direct interaction with membrane lipids has been suggested as the key feature for the perception and subsequent activity of RLs. Methods: Molecular Dynamics (MD) simulations are used in this work to provide an atomistic description of their interactions with different membranous lipids and focusing on their antifungal properties. Results and discussion: Our results suggest the insertion of RLs into the modelled bilayers just below the plane drawn by lipid phosphate groups, a placement that is effective in promoting significant membrane fluidification of the hydrophobic core. This localization is promoted by the formation of ionic bonds between the carboxylate group of RLs and the amino group of the phosphatidylethanolamine (PE) or phosphatidylserine (PS) headgroups. Moreover, RL acyl chains adhere to the ergosterol structure, forming a significantly higher number of van der Waals contact with respect to what is observed for phospholipid acyl chains. All these interactions might be essential for the membranotropic-driven biological actions of RLs.

A toolkit for covalent docking with GOLD: from automated ligand preparation with KNIME to bound protein-ligand complexes.

Motivation: Current covalent docking tools have limitations that make them difficult to use for performing large-scale structure-based covalent virtual screening (VS). They require time-consuming tasks for the preparation of proteins and compounds (standardization, filtering according to the type of warheads), as well as for setting up covalent reactions. We have developed a toolkit to help accelerate drug discovery projects in the phases of hit identification by VS of ultra-large covalent libraries and hit expansion by exploration of the binding of known covalent compounds. With this application note, we offer the community a toolkit for performing automated covalent docking in a fast and efficient way. Results: The toolkit comprises a KNIME workflow for ligand preparation and a Python program to perform the covalent docking of ligands with the GOLD docking engine running in a parallelized fashion. Availability and implementation: The KNIME workflow entitled 'Evotec_Covalent_Processing_forGOLD.knwf' for the preparation of the ligands is available in the KNIME Hub https://hub.knime.com/emilie_pihan/spaces. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Antimicrobial Bombinin-like Peptide 3 Selectively Recognizes and Inserts into Bacterial Biomimetic Bilayers in Multiple Steps.

Bombinins are a wide family of antimicrobial peptides from Xenopus skin. By sequence clustering, we highlighted at least three families named A, B, and H, which might exert antibacterial activity by different modes of action. In this work, we study bombinin-like peptide 3 (BLP-3) as a nonhemolytic representative of the quite unexplored class A due to its appealing activity toward WHO-priority-list bacteria such as Neisseria, Pseudomonas aeruginosa, and Staphylococcus aureus. A marked preference for cardiolipin and phosphatidylglycerol head groups, typically found in bacteria, is proven with biomimetic membranes studied by liquid and solid NMR and MD simulations. BLP-3 gets structured upon interaction and penetrates deeply into the bilayer in two steps involving a superficial insertion of key side chains and subsequent internalization. All along the pathway, a fundamental role is played by lysine residues in the conserved region 11-19, which act in synergy with other key residues.

LIMONADA: A database dedicated to the simulation of biological membranes.

Cellular membranes are composed of a wide diversity of lipid species in varying proportions and these compositions are representative of the organism, cellular type and organelle to which they belong. Because models of these molecular systems simulated by MD steadily gain in size and complexity, they are increasingly representative of specific compositions and behaviors of biological membranes. Due to the number of lipid species involved, of force fields and topologies and because of the complexity of membrane objects that have been simulated, LIMONADA has been developed as an open database allowing to handle the various aspects of lipid membrane simulation. LIMONADA presents published membrane patches with their simulation files and the cellular membrane it models. Their compositions are then detailed based on the lipid identification from LIPID MAPS database plus the lipid topologies and the force field used. LIMONADA is freely accessible on the web at https://limonada.univ-reims.fr/.

Contributions and Limitations of Biophysical Approaches to Study of the Interactions between Amphiphilic Molecules and the Plant Plasma Membrane.

Some amphiphilic molecules are able to interact with the lipid matrix of plant plasma membranes and trigger the immune response in plants. This original mode of perception is not yet fully understood and biophysical approaches could help to obtain molecular insights. In this review, we focus on such membrane-interacting molecules, and present biophysically grounded methods that are used and are particularly interesting in the investigation of this mode of perception. Rather than going into overly technical details, the aim of this review was to provide to readers with a plant biochemistry background a good overview of how biophysics can help to study molecular interactions between bioactive amphiphilic molecules and plant lipid membranes. In particular, we present the biomimetic membrane models typically used, solid-state nuclear magnetic resonance, molecular modeling, and fluorescence approaches, because they are especially suitable for this field of research. For each technique, we provide a brief description, a few case studies, and the inherent limitations, so non-specialists can gain a good grasp on how they could extend their toolbox and/or could apply new techniques to study amphiphilic bioactive compound and lipid interactions.

ADAPTABLE: a comprehensive web platform of antimicrobial peptides tailored to the user's research.

Antimicrobial peptides (AMPs) are part of the innate immune response to pathogens in all of the kingdoms of life. They have received significant attention because of their extraordinary variety of activities, in particular, as candidate drugs against the threat of super-bacteria. A systematic study of the relation between the sequence and the mechanism of action is urgently needed, given the thousands of sequences already in multiple web resources. ADAPTABLE web platform (http://gec.u-picardie.fr/adaptable) introduces the concept of "property alignment" to create families of property and sequence-related peptides (SR families). This feature provides the researcher with a tool to select those AMPs meaningful to their research from among more than 40,000 nonredundant sequences. Selectable properties include the target organism and experimental activity concentration, allowing selection of peptides with multiple simultaneous actions. This is made possible by ADAPTABLE because it not only merges sequences of AMP databases but also merges their data, thereby standardizing values and handling non-proteinogenic amino acids. In this unified platform, SR families allow the creation of peptide scaffolds based on common traits in peptides with similar activity, independently of their source.

Exploring the Dual Interaction of Natural Rhamnolipids with Plant and Fungal Biomimetic Plasma Membranes through Biophysical Studies.

Rhamnolipids (RLs) are potential biocontrol agents for crop culture protection. Their mode of action has been proposed as dual, combining plant protection activation and antifungal activities. The present work focuses on the interaction of natural RLs with plant and fungi membrane models at the molecular scale. Representative models were constructed and the interaction with RLs was studied by Fourier transform infrared (FTIR) and deuterium nuclear magnetic resonance (²H NMR) spectroscopic measurements. Molecular dynamic (MD) simulations were performed to investigate RL insertion in lipid bilayers. Our results showed that the RLs fit into the membrane models and were located near the lipid phosphate group of the phospholipid bilayers, nearby phospholipid glycerol backbones. The results obtained with plant plasma membrane models suggest that the insertion of RLs inside the lipid bilayer did not significantly affect lipid dynamics. Oppositely, a clear fluidity increase of fungi membrane models was observed. This effect was related to the presence and the specific structure of ergosterol. The nature of the phytosterols could also influence the RL effect on plant plasma membrane destabilization. Subtle changes in lipid dynamics could then be linked with plant defense induction and the more drastic effects associated with fungal membrane destabilization.

FATSLiM: a fast and robust software to analyze MD simulations of membranes.

When studying biological membranes, Molecular Dynamics (MD) simulations reveal to be quite complementary to experimental techniques. Because the simulated systems keep increasing both in size and complexity, the analysis of MD trajectories need to be computationally efficient while being robust enough to perform analysis on membranes that may be curved or deformed due to their size and/or protein-lipid interactions. This work presents a new software named FATSLiM ('Fast Analysis Toolbox for Simulations of Lipid Membranes') that can extract physical properties from MD simulations of membranes (with or without interacting proteins). Because it relies on the calculation of local normals, FATSLiM does not depend of the bilayer morphology and thus can handle with the same accuracy vesicles for instance. Thanks to an efficiency-driven development, it is also fast and consumes a rather low amount of memory. AVAILABILITY AND IMPLEMENTATION: FATSLiM (http://fatslim.github.io) is a stand-alone software written in Python. Source code is released under the GNU GPLv3 and is freely available at https://github.com/FATSLiM/fatslim A complete online documentation including instructions for platform-independent installation is available at http://pythonhosted.org/fatslim CONTACT: sebastien.buchoux@u-picardie.frSupplementary information: Supplementary data are available at Bioinformatics online.

Membrane lipids protected from oxidation by red wine tannins: a proton NMR study.

Dietary polyphenols widespread in vegetables and beverages like red wine and tea have been reported to possess antioxidant properties that could have positive effects on human health. In this study, we propose a new in situ and non-invasive method based on proton liquid-state nuclear magnetic resonance (NMR) to determine the antioxidant efficiency of red wine tannins on a twice-unsaturated phospholipid, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLiPC), embedded in a membrane model. Four tannins were studied: (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG). The lipid degradation kinetics was determined by measuring the loss of the bis-allylic protons during oxidation induced by a radical initiator, 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH). The antioxidant efficiency, i.e. the ability of tannins to slow down the lipid oxidation rate, was shown to be higher for galloylated tannins, ECG and EGCG. Furthermore, the mixture of four tannins was more efficient than the most effective tannin, EGCG, demonstrating a synergistic effect. To better understand the antioxidant action mechanism of polyphenols on lipid membranes, the tannin location was investigated by NMR and molecular dynamics. A correlation between antioxidant action of tannins and their location at the membrane interface (inserted at the glycerol backbone level) could thus be established.

Membrane interacting peptides: from killers to helpers.

Membrane interacting peptides are reviewed in terms of structure and mode of action on lipid membranes. Helical, ß-stranded, peptides containing both helices and strands, cyclic, lipopeptides and short linear peptides are seen to considerably modulate membrane function. Among peptides that lead to membrane alteration or permeation, antimicrobial peptides play an important role and some of them may be foreseen as potential new antibiotics. Alternatively, peptides that do not destroy the membrane are also very important in modulating the structure and dynamics of the lipid bilayer and play important roles in membrane protein functions. Peptide lipid complexes are shown to be very variable in structure and dynamics: "carpet", "barrel stave", toroid and disordered pores, electrostatic wedge and molecular electroporation models are discussed. Their assembly is reviewed in terms of electric, amphipathic and dynamic properties of both lipids and peptides.

Mild pretreatment and enzymatic saccharification of cellulose with recycled ionic liquids towards one-batch process.

The development of second-generation bioethanol involves minimizing the energy input throughout the processing steps. We report here that efficient ionic liquid pretreatments of cellulose can be achieved with short duration times (20 min) at mild temperature (45°C) with [Emim](+)[MeO(H)PO(2)](-) and at room temperature (25 °C) with [Emim](+)[CH(3)COO](-). In these conditions, yields of glucose were increased by a factor of 3. In addition, the recycling of these two imidazolium-based ILs can be performed in maintaining their efficiency to pretreat cellulose. The short time and mild temperature of cellulose solubilization allowed a one-batch processing of [Emim](+)[MeO(H)PO(2)](-) IL-pretreatment and saccharification. In the range from 0 to 100% IL in an aqueous enzymatic medium, the glucose yields were improved at IL proportions between 10 and 40%. The maximum yield at 10% IL is very promising to consider one batch process as efficient as two-step process.

Key role of polyphosphoinositides in dynamics of fusogenic nuclear membrane vesicles.

The role of phosphoinositides has been thoroughly described in many signalling and membrane trafficking events but their function as modulators of membrane structure and dynamics in membrane fusion has not been investigated. We have reconstructed models that mimic the composition of nuclear envelope precursor membranes with naturally elevated amounts of phosphoinositides. These fusogenic membranes (membrane vesicle 1(MV1) and nuclear envelope remnants (NER) are critical for the assembly of the nuclear envelope. Phospholipids, cholesterol, and polyphosphoinositides, with polyunsaturated fatty acid chains that were identified in the natural nuclear membranes by lipid mass spectrometry, have been used to reconstruct complex model membranes mimicking nuclear envelope precursor membranes. Structural and dynamic events occurring in the membrane core and at the membrane surface were monitored by solid-state deuterium and phosphorus NMR. "MV1-like" (PC∶PI∶PIP∶PIP(2), 30∶20∶18∶12, mol%) membranes that exhibited high levels of PtdIns, PtdInsP and PtdInsP(2) had an unusually fluid membrane core (up to 20% increase, compared to membranes with low amounts of phosphoinositides to mimic the endoplasmic reticulum). "NER-like" (PC∶CH∶PI∶PIP∶PIP(2), 28∶42∶16∶7∶7, mol%) membranes containing high amounts of both cholesterol and phosphoinositides exhibited liquid-ordered phase properties, but with markedly lower rigidity (10-15% decrease). Phosphoinositides are the first lipids reported to counterbalance the ordering effect of cholesterol. At the membrane surface, phosphoinositides control the orientation dynamics of other lipids in the model membranes, while remaining unchanged themselves. This is an important finding as it provides unprecedented mechanistic insight into the role of phosphoinositides in membrane dynamics. Biological implications of our findings and a model describing the roles of fusogenic membrane vesicles are proposed.

Enzymatic hydrolysis of ionic liquid-pretreated celluloses: contribution of CP-MAS 13C NMR and SEM.

The supramolecular structure of four model celluloses was altered prior to their enzymatic saccharification using two ionic liquid pretreatments: one with the commonly used 1-ethyl-3-methylimidazolium acetate ([Emim](+)[CH(3)COO](-)) and the other with the newly developed 1-ethyl-3-methylimidazolium methylphosphonate ([Emim](+)[MeO(H)PO(2)](-)). The estimation of crystallinity index (CrI) by solid state (13)C nuclear magnetic resonance for each untreated/pretreated celluloses was compared with the performances of their enzymatic hydrolysis. For α-cellulose, both pretreatments led to a significant decrease in CrI from 25% to 5% but had no effect on glucose yields. In contrast, The [Emim](+)[MeO(H)PO(2)](-) pretreatment on the long fibers of cellulose had no significant effect on the CrI although a conversion yield in glucose of 88% is obtained versus 32% without pretreatment. However, scanning electron microscopy analysis suggested a loss of fiber organization induced by both ionic liquid pretreatments leading to a larger accessibility by cellulases to the cellulose surface.

Cholesterol and lipid phases influence the interactions between serotonin receptor agonists and lipid bilayers.

Solid state NMR techniques have been used to investigate the effect that two serotonin receptor 1a agonists (quipazine and LY-165,163) have on the phase behavior of, and interactions within, cholesterol/phosphocholine lipid bilayers. The presence of agonist, and particularly LY-165,163, appears to widen the phase transitions, an effect that is much more pronounced in the presence of cholesterol. It was found that both agonists locate close to the cholesterol, and their interactions with the lipids are modulated by the lipid phases. As the membrane condenses into mixed liquid-ordered/disordered phases, quipazine is pushed up toward the surface of the bilayer, whereas LY-165,163 moves deeper into the lipid chain region. In light of our results, we discuss the role of lipid/drug interactions on drug efficacy.

Effect of monolayer lipid charges on the structure and orientation of protein VAMP1 at the air-water interface.

SNARE proteins are implicated in membrane fusion during neurotransmission and peptide hormone secretion. Relatively little is known about the molecular interactions of their trans- and juxtamembrane domains with lipid membranes. Here, we report the structure and the assembling behavior of one of the SNARE proteins, VAMP1/synaptobrevin1 incorporated in a lipid monolayer at an air-water interface which mimics the membrane environment. Our results show that the protein is extremely sensitive to surface pressure as well as the lipid composition. Monolayers of proteins alone or in the presence of the neutral phospholipid DMPC underwent structural transition from alpha-helix to beta-sheet upon surface compression. In contrast, the anionic phospholipid DMPG inhibited this transition in a concentration-dependent manner. Moreover, the orientation of the proteins was highly sensitive to the charge density of the lipid layers. Thus, the structure of VAMP1 is clearly controlled by protein-lipid interactions.

Surfactin-triggered small vesicle formation of negatively charged membranes: a novel membrane-lysis mechanism.

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri = 200) and is complete for Ri = 50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.

Plant sterols in "rafts": a better way to regulate membrane thermal shocks.

Specialized lipid domains (rafts) that are generally enriched in sterols and sphingolipids, are most likely present in cell membranes of animals, plants and fungi. While cholesterol and ergosterol are predominant in vertebrates and fungi, plants possess complex sterol profiles, dominated by sitosterol and stigmasterol in Arabidopsis thaliana. Fully hydrated model membranes of composition approaching those found in rafts of mammals, fungi and plants were investigated by means of solid-state 2H-NMR, using deuterated dipalmitoylphosphatidylcholine (2H(62)-DPPC). The dynamics of such membranes was determined through measuring of membrane ordering or disordering properties. The presence of the liquid-ordered, lo, phase, which may be an indicator of rigid sterol-sphingolipid domains, was detected in all binary or ternary mixtures of all sterols investigated. Of great interest, the dynamics of ternary mixtures mimicking rafts in plants (phytosterol/glucosylcerebroside/DPPC), showed a lesser temperature sensitivity to thermal shocks, on comparing to systems mimicking rafts in mammals and fungi. This effect was particularly marked with sitosterol. The presence of an ethyl group branched on the alkyl chain of sitosterol and stigmasterol is proposed as reinforcing the membrane cohesion by additional attractive van der Waals interactions with the alkyl chains of sphingolipids and phospholipids. As a side result, the elevated resolution of NMR spectra in the presence of sitosterol also suggests domains of smaller size than with other sterols. Finally, the role of phytosterols in maintaining plant membranes in a state of dynamics less sensitive to temperature shocks is discussed.

Dimerization of Neu/Erb2 transmembrane domain is controlled by membrane curvature.

Secondary structures of the proto-oncogenic Neu/ErbB2 transmembrane segment and its mutant analogue have been determined in phospholipids. It is found that the mutated peptide possesses less helical character possibly due to the valine/glutamic acid point mutation. Embedding peptides in lipid systems whose topology can change from small (100-200 A) tumbling objects to bilayer discs of 450 A diameter leads to the finding that coiled-coil interactions are only observed in the presence of a bilayer membrane of low curvature, independent of mutation. This strongly suggests that any event that may change membrane topology can therefore perturb the dimerization/ologomerization and subsequent phosphorylation cascade leading to cell growth or cancer processes.