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/.

Blending Ferulic Acid Derivatives and Polylactic Acid into Biobased and Transparent Elastomeric Materials with Shape Memory Properties.

Thanks to its remarkable properties such as sustainability, compostability, biocompatibility, and transparency, poly-l-lactic acid (PLA) would be a suitable replacement for oil-based polymers should it not suffer from low flexibility and poor toughness, restricting its use to rigid plastic by excluding elastomeric applications. Indeed, there are few fully biobased and biodegradable transparent elastomers-PLA-based or not-currently available. In the last decades, many strategies have been investigated to soften PLA and enhance its toughness and elongation at break by using plasticizers, oligomers, or polymers. This work shows how a ferulic acid-derived biobased additive (BDF) blends with a common rigid and brittle commercial grade of polylactic acid to provide a transparent non-covalently cross-linked elastomeric material with shape memory behavior exhibiting an elongation at break of 434% (vs 6% for pristine PLA). Through a structure-activity relationship analysis conducted with BDF analogues and a modeling study, we propose a mechanism based on π-π stacking to account for the elastomeric properties. Blending ferulic acid derivatives with polylactic acid generates a new family of fully sustainable transparent elastomeric materials with functional properties such as shape memory.

Multiple C2 domains and transmembrane region proteins (MCTPs) tether membranes at plasmodesmata.

In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved a unique type of MCS, inside intercellular pores, the plasmodesmata, where endoplasmic reticulum (ER)-plasma membrane (PM) contacts coincide with regulation of cell-to-cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remain unknown. Here, we show that the multiple C2 domains and transmembrane region protein (MCTP) family, key regulators of cell-to-cell signalling in plants, act as ER-PM tethers specifically at plasmodesmata. We report that MCTPs are plasmodesmata proteins that insert into the ER via their transmembrane region while their C2 domains dock to the PM through interaction with anionic phospholipids. A Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects, impaired plasmodesmata function and composition, while MCTP4 expression in a yeast Δtether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-to-cell signalling.

Structural Analysis of Nonapeptides Derived from Elastin.

Elastin-derived peptides are released from the extracellular matrix remodeling by numerous proteases and seem to regulate many biological processes, notably cancer progression. The canonical elastin peptide is VGVAPG, which harbors the XGXXPG consensus pattern, allowing interaction with the elastin receptor complex located at the surface of cells. Besides these elastokines, another class of peptides has been identified. This group of bioactive elastin peptides presents the XGXPGXGXG consensus sequence, but the reason for their bioactivity remains unexplained. To better understand their nature and structure-function relationships, herein we searched the current databases for this nonapeptide motif and observed that the XGXPGXGXG elastin peptides define a specific group of tandemly repeated patterns. Further, we focused on four tandemly repeated human elastin nonapeptides, i.e., AGIPGLGVG, VGVPGLGVG, AGVPGLGVG, and AGVPGFGAG. These peptides were analyzed by means of optical spectroscopies and molecular dynamics. Ultraviolet-circular dichroism and Raman spectra are consistent with a mixture of ß-turn, ß-strand, and random-chain secondary elements in aqueous media. Quantitative analysis of their conformations suggested that turns corresponded to half of the total population of structural elements, whereas the remaining half were equally distributed between ß-strand and unordered chains. These distributions were confirmed by molecular dynamics simulations. Altogether, our data suggest that these highly dynamic peptides harbor a type II ß-turn located in their central part. We hypothesize that this structural element could explain their specific bioactivity.

Homology modeling and in vivo functional characterization of the zinc permeation pathway in a heavy metal P-type ATPase.

The P1B ATPase heavy metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements, enabling metal transport across the membrane. The detailed mechanism of metal permeation by HMA4 through the membrane remains elusive. Here, homology modeling of the HMA4 transmembrane region was conducted based on the crystal structure of a ZntA bacterial homolog. The analysis highlighted amino acids forming a metal permeation pathway, whose importance was subsequently investigated functionally through mutagenesis and complementation experiments in plants. Although the zinc pathway displayed overall conservation among the two proteins, significant differences were observed, especially in the entrance area with altered electronegativity and the presence of a ionic interaction/hydrogen bond network. The analysis also newly identified amino acids whose mutation results in total or partial loss of the protein function. In addition, comparison of zinc and cadmium accumulation in shoots of A. thaliana complemented lines revealed a number of HMA4 mutants exhibiting different abilities in zinc and cadmium translocation. These observations could be instrumental to design low cadmium-accumulating crops, hence decreasing human cadmium exposure.

Mechanism of Trypanosoma brucei gambiense resistance to human serum.

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.

Insight into the Self-Assembling Properties of Peptergents: A Molecular Dynamics Simulation Study.

By manipulating the various physicochemical properties of amino acids, the design of peptides with specific self-assembling properties has been emerging for more than a decade. In this context, short peptides possessing detergent properties (so-called "peptergents") have been developed to self-assemble into well-ordered nanostructures that can stabilize membrane proteins for crystallization. In this study, the peptide with "peptergency" properties, called ADA8 and extensively described by Tao et al., is studied by molecular dynamic simulations for its self-assembling properties in different conditions. In water, it spontaneously forms beta sheets with a ß barrel-like structure. We next simulated the interaction of this peptide with a membrane protein, the bacteriorhodopsin, in the presence or absence of a micelle of dodecylphosphocholine. According to the literature, the peptergent ADA8 is thought to generate a belt of ß structures around the hydrophobic helical domain that could help stabilize purified membrane proteins. Molecular dynamic simulations are here used to image this mechanism and provide further molecular details for the replacement of detergent molecules around the protein. In addition, we generalized this behavior by designing an amphipathic peptide with beta propensity, which was called ABZ12. Both peptides are able to surround the membrane protein and displace surfactant molecules. To our best knowledge, this is the first molecular mechanism proposed for "peptergency".

Membrane Interactions of Natural Cyclic Lipodepsipeptides of the Viscosin Group.

Many Pseudomonas spp. produce cyclic lipodepsipeptides (CLPs), which, besides their role in biological functions such as motility, biofilm formation and interspecies interactions, are antimicrobial. It has been established that interaction with the cellular membrane is central to the mode of action of CLPs. In this work, we focus on the CLPs of the so-called viscosin group, aiming to assess the impact of the main structural variations observed within this group on both the antimicrobial activity and the interaction with model membranes. The antimicrobial activity of viscosin, viscosinamide A, WLIP and pseudodesmin A were all tested on a broad panel of mainly Gram-positive bacteria. Their capacity to permeabilize or fuse PG/PE/cardiolipin model membrane vesicles is assessed using fluorescent probes. We find that the Glu2/Gln2 structural variation within the viscosin group is the main factor that influences both the membrane permeabilization properties and the minimum inhibitory concentration of bacterial growth, while the configuration of the Leu5 residue has no apparent effect. The CLP-membrane interactions were further evaluated using CD and FT-IR spectroscopy on model membranes consisting of PG/PE/cardiolipin or POPC with or without cholesterol. In contrast to previous studies, we observe no conformational change upon membrane insertion. The CLPs interact both with the polar heads and aliphatic tails of model membrane systems, altering bilayer fluidity, while cholesterol reduces CLP insertion depth.

Differential Interaction of Synthetic Glycolipids with Biomimetic Plasma Membrane Lipids Correlates with the Plant Biological Response.

Natural and synthetic amphiphilic molecules including lipopeptides, lipopolysaccharides, and glycolipids are able to induce defense mechanisms in plants. In the present work, the perception of two synthetic C14 rhamnolipids, namely, Alk-RL and Ac-RL, differing only at the level of the lipid tail terminal group have been investigated using biological and biophysical approaches. We showed that Alk-RL induces a stronger early signaling response in tobacco cell suspensions than does Ac-RL. The interactions of both synthetic RLs with simplified biomimetic membranes were further analyzed using experimental and in silico approaches. Our results indicate that the interactions of Alk-RL and Ac-RL with lipids were different in terms of insertion and molecular responses and were dependent on the lipid composition of model membranes. A more favorable insertion of Alk-RL than Ac-RL into lipid membranes is observed. Alk-RL forms more stable molecular assemblies than Ac-RL with phospholipids and sterols. At the molecular level, the presence of sterols tends to increase the RLs' interaction with lipid bilayers, with a fluidizing effect on the alkyl chains. Taken together, our findings suggest that the perception of these synthetic RLs at the membrane level could be related to a lipid-driven process depending on the organization of the membrane and the orientation of the RLs within the membrane and is correlated with the induction of early signaling responses in tobacco cells.

Complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: A review.

Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance. Lipid specificity is a key factor for the detailed understanding of the penetration and/or activity of lipid-interacting molecules and of mechanisms of some diseases. Further investigation in that way should improve drug discovery and development of membrane-active molecules in many domains such as health, plant protection or microbiology. In this review, we will present complementary biophysical approaches that can give information about lipid specificity at a molecular point of view. Examples of application will be given for different molecule types, from biomolecules to pharmacological drugs. A special emphasis is given to cyclic lipopeptides since they are interesting molecules in the scope of this review by combining a peptidic moiety and a lipidic tail and by exerting their activity via specific interactions with the plasma membrane.

Modeling of non-covalent complexes of the cell-penetrating peptide CADY and its siRNA cargo.

CADY is a cell-penetrating peptide spontaneously making non-covalent complexes with Short interfering RNAs (siRNAs) in water. Neither the structure of CADY nor that of the complexes is resolved. We have calculated and analyzed 3D models of CADY and of the non-covalent CADY-siRNA complexes in order to understand their formation and stabilization. Data from the ab initio calculations and molecular dynamics support that, in agreement with the experimental data, CADY is a polymorphic peptide partly helical. Taking into consideration the polymorphism of CADY, we calculated and compared several complexes with peptide/siRNA ratios of up to 40. Four complexes were run by using molecular dynamics. The initial binding of CADYs is essentially due to the electrostatic interactions of the arginines with siRNA phosphates. Due to a repetitive arginine motif (XLWR(K)) in CADY and to the numerous phosphate moieties in the siRNA, CADYs can adopt multiple positions at the siRNA surface leading to numerous possibilities of complexes. Nevertheless, several complex properties are common: an average of 14±1 CADYs is required to saturate a siRNA as compared to the 12±2 CADYs experimentally described. The 40 CADYs/siRNA that is the optimal ratio for vector stability always corresponds to two layers of CADYs per siRNA. When siRNA is covered by the first layer of CADYs, the peptides still bind despite the electrostatic repulsion. The peptide cage is stabilized by hydrophobic CADY-CADY contacts thanks to CADY polymorphism. The analysis demonstrates that the hydrophobicity, the presence of several positive charges and the disorder of CADY are mandatory to make stable the CADY-siRNA complexes.

Plasma membrane localization of Solanum tuberosum remorin from group 1, homolog 3 is mediated by conformational changes in a novel C-terminal anchor and required for the restriction of potato virus X movement].

The formation of plasma membrane (PM) microdomains plays a crucial role in the regulation of membrane signaling and trafficking. Remorins are a plant-specific family of proteins organized in six phylogenetic groups, and Remorins of group 1 are among the few plant proteins known to specifically associate with membrane rafts. As such, they are valuable to understand the molecular bases for PM lateral organization in plants. However, little is known about the structural determinants underlying the specific association of group 1 Remorins with membrane rafts. We used a structure-function approach to identify a short C-terminal anchor (RemCA) indispensable and sufficient for tight direct binding of potato (Solanum tuberosum) REMORIN 1.3 (StREM1.3) to the PM. RemCA switches from unordered to α-helical structure in a nonpolar environment. Protein structure modeling indicates that RemCA folds into a tight hairpin of amphipathic helices. Consistently, mutations reducing RemCA amphipathy abolished StREM1.3 PM localization. Furthermore, RemCA directly binds to biological membranes in vitro, shows higher affinity for Detergent-Insoluble Membranes lipids, and targets yellow fluorescent protein to Detergent-Insoluble Membranes in vivo. Mutations in RemCA resulting in cytoplasmic StREM1.3 localization abolish StREM1.3 function in restricting potato virus X movement. The mechanisms described here provide new insights on the control and function of lateral segregation of plant PM.

SAHBNET, an accessible surface-based elastic network: an application to membrane protein.

Molecular Dynamics is a method of choice for membrane simulations and the rising of coarse-grained forcefields has opened the way to longer simulations with reduced calculations times. Here, we present an elastic network, SAHBNET (Surface Accessibility Hydrogen-Bonds elastic NETwork), that will maintain the structure of soluble or membrane proteins based on the hydrogen bonds present in the atomistic structure and the proximity between buried residues. This network is applied on the coarse-grained beads defined by the MARTINI model, and was designed to be more physics-based than a simple elastic network. The SAHBNET model is evaluated against atomistic simulations, and compared with ELNEDYN models. The SAHBNET is then used to simulate two membrane proteins inserted in complex lipid bilayers. These bilayers are formed by self-assembly and the use of a modified version of the GROMACS tool genbox (which is accessible through the gcgs.gembloux.ulg.ac.be website). The results show that SAHBNET keeps the structure close to the atomistic one and is successfully used for the simulation of membrane proteins.

Modelling and Simulations of Extracellular Glycoproteins.

While the knowledge of protein structure and function has seen vast advances in previous decades, the understanding of how their posttranslational modifications, such as glycosylations, influence their structure and function remains poor. However, advances in in silico methodologies to study glycosylations in recent past have enabled us to study this and understand the role of glycosylations in protein structure and function in ways that would not be possible by conventional experimental methods. In this chapter, we will demonstrate how to leverage these methodologies to study glycoproteins and their structural and dynamic properties using molecular modelling techniques.

Multiscale modelling of the extracellular matrix.

The extracellular matrix is a complex three-dimensional network of molecules that provides cells with a complex microenvironment. The major constituents of the extracellular matrix such as collagen, elastin and associated proteins form supramolecular assemblies contributing to its physicochemical properties and organization. The structure of proteins and their supramolecular assemblies such as fibrils have been studied at the atomic level (e.g., by X-ray crystallography, Nuclear Magnetic Resonance and cryo-Electron Microscopy) or at the microscopic scale. However, many protein complexes are too large to be studied at the atomic level and too small to be studied by microscopy. Most extracellular matrix components fall into this intermediate scale, so-called the mesoscopic scale, preventing their detailed characterization. Simulation and modelling are some of the few powerful and promising approaches that can deepen our understanding of mesoscale systems. We have developed a set of modelling tools to study the self-organization of the extracellular matrix and large motion of macromolecules at the mesoscale level by taking advantage of the dynamics of articulated rigid bodies as a mean to study a larger range of motions at the cost of atomic resolution.

The Trypanosoma Brucei KIFC1 Kinesin Ensures the Fast Antibody Clearance Required for Parasite Infectivity.

Human innate immunity to Trypanosoma brucei involves the trypanosome C-terminal kinesin TbKIFC1, which transports internalized trypanolytic factor apolipoprotein L1 (APOL1) within the parasite. We show that TbKIFC1 preferentially associates with cholesterol-containing membranes and is indispensable for mammalian infectivity. Knockdown of TbKIFC1 did not affect trypanosome growth in vitro but rendered the parasites unable to infect mice unless antibody synthesis was compromised. Surface clearance of Variant Surface Glycoprotein (VSG)-antibody complexes was far slower in these cells, which were more susceptible to capture by macrophages. This phenotype was not due to defects in VSG expression or trafficking but to decreased VSG mobility in a less fluid, stiffer surface membrane. This change can be attributed to increased cholesterol level in the surface membrane in TbKIFC1 knockdown cells. Clearance of surface-bound antibodies by T. brucei is therefore essential for infectivity and depends on high membrane fluidity maintained by the cholesterol-trafficking activity of TbKIFC1.

Impacts of the carbonyl group location of ester bond on interfacial properties of sugar-based surfactants: experimental and computational evidences.

Interfacial properties of surfactants are dependent on the conformation adopted by the hydrophilic headgroup or/and the hydrophobic tail at the boundary limit of two immiscible phases. Here, we demonstrate the impacts of the carbonyl group (-CO-) location of the ester bond of sugar-based surfactants by comparing some properties of two closely related esters, octyl glucuronate and glucose octanoate, at the air-water interface. The carbonyl group location influences the rate and extent of interfacial adsorption and the rheology properties of sugar esters at the air-water interface, which were evaluated by dynamic surface tension and complex surface viscoelastic measurements. Octyl glucuronate adsorbs the fastest at the air-water interface whereas glucose octanoate reduces the dynamic surface tension at the lowest value and exhibits the highest film viscoelasticity. Differences are attributed to molecular conformation constraints inducing relevant changes to the surface coverage kinetic capacity and the interaction strengths of the octyl sugar ester adsorbed films at the air-water interface. All of the results are supported by the minimum cross-sectional area values per molecule determined by both experimental and computational approaches.

Low-diluted Phenacetinum disrupted the melanoma cancer cell migration.

Dynamic and reciprocal interactions generated by the communication between tumor cells and their matrix microenvironment, play a major role in the progression of a tumor. Indeed, the adhesion of specific sites to matrix components, associated with the repeated and coordinated formation of membrane protrusions, allow tumor cells to move along a determined pathway. Our study analyzed the mechanism of action of low-diluted Phenacetinum on murine cutaneous melanoma process in a fibronectin matrix environment. We demonstrated a reduction of dispersed cell migration, early and for as long as 24 h, by altering the formation of cell protrusions. Moreover, low-diluted Phenacetinum decreased cell stiffness highly on peripheral areas, due to a disruption of actin filaments located just under the plasma membrane. Finally, it modified the structure of the plasma membrane by accumulating large ordered lipid domains and disrupted B16 cell migration by a likely shift in the balance between ordered and disordered lipid phases. Whereas the correlation between the excess of lipid raft and cytoskeleton disrupting is not as yet established, it is clear that low-diluted Phenacetinum acts on the actin cytoskeleton organization, as confirmed by a decrease of cell stiffness affecting ultimately the establishment of an effective migration process.

Molecular Model for the Self-Assembly of the Cyclic Lipodepsipeptide Pseudodesmin A.

Self-assembly of peptides into supramolecular structures represents an active field of research with potential applications ranging from material science to medicine. Their study typically involves the application of a large toolbox of spectroscopic and imaging techniques. However, quite often, the structural aspects remain underexposed. Besides, molecular modeling of the self-assembly process is usually difficult to handle, since a vast conformational space has to be sampled. Here, we have used an approach that combines short molecular dynamics simulations for peptide dimerization and NMR restraints to build a model of the supramolecular structure from the dimeric units. Experimental NMR data notably provide crucial information about the conformation of the monomeric units, the supramolecular assembly dimensions, and the orientation of the individual peptides within the assembly. This in silico/in vitro mixed approach enables us to define accurate atomistic models of supramolecular structures of the bacterial cyclic lipodepsipeptide pseudodesmin A.

Linoleic and linolenic acid hydroperoxides interact differentially with biomimetic plant membranes in a lipid specific manner.

Linoleic and linolenic acid hydroperoxides (HPOs) constitute key intermediate oxylipins playing an important role as signaling molecules during plant defense processes in response to biotic or abiotic stress. They have also been demonstrated in vitro as antimicrobial agents against plant fungi and bacteria. To reach the phytopathogens in vivo, the HPOs biosynthesized in the plant cells must cross the plant plasma membrane (PPM) where they can also interact with plasma membrane lipids and have an effect on their organization. In the present study, we have investigated the interaction properties of HPOs with PPM at a molecular level using biophysical tools combining in vitro and in silico approaches and using plant biomimetic lipid systems. Our results have shown that HPOs are able to interact with PPM lipids and perturb their lateral organization. Glucosylceramide (GluCer) is a privileged partner, sitosterol lessens their binding and the presence of both GluCer and sitosterol further reduces their interaction. Hydrophobic effect and polar interactions are involved in the binding. The chemical structure of HPOs influences their affinity for PPM lipids. The presence of three double bonds in the HPO molecule gives rise to a higher affinity comparatively to two double bonds, which can be explained by their differential interaction with the lipid polar headgroups.