A long QT mutation substitutes cholesterol for phosphatidylinositol-4,5-bisphosphate in KCNQ1 channel regulation.

INTRODUCTION: Phosphatidylinositol-4,5-bisphosphate (PIP2) is a cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that R539W is paradoxically less sensitive to intracellular magnesium inhibition than the WT channel, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it. METHODS AND RESULTS: COS-7 cells were transfected with WT or mutated KCNE1-KCNQ1 channel, and patch-clamp recordings were performed in giant-patch, permeabilized-patch or ruptured-patch configuration. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when membrane PIP2 levels are decreasing. As opposed to R243H and R555C mutants, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. A molecular model of a fragment of the KCNQ1 C-terminus and the membrane bilayer suggested that a potential novel interaction of R539W with cholesterol stabilizes the channel opening and hence prevents rundown upon PIP2 depletion. We then carried out the same rundown experiments under cholesterol depletion and observed an accelerated R539W rundown that is consistent with this model. CONCLUSIONS: We show for the first time that a mutation may shift the channel interaction with PIP2 to a preference for cholesterol. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.

Domain formation and permeabilization induced by the saponin α-hederin and its aglycone hederagenin in a cholesterol-containing bilayer.

Saponins and triterpenic acids have been shown to be able to interact with lipid membranes and domains enriched with cholesterol (rafts). How saponins are able to modulate lipid phase separation in membranes and the role of the sugar chains for this activity is unknown. We demonstrate in a binary membrane model composed of DMPC/Chol (3:1 mol/mol) that the saponin α-hederin and its aglycone presenting no sugar chain, the triterpenic acid hederagenin, are able to induce the formation of lipid domains. We show on multilamellar vesicles (MLV), giant unilamellar vesicles (GUV), and supported planar bilayers (SPB) that the presence of sugar units on the sapogenin accelerates domain formation and increases the proportion of sterols within these domains. The domain shape is also influenced by the presence of sugars because α-hederin and hederagenin induce the formation of tubular and spherical domains, respectively. These highly curved structures should result from the induction of membrane curvature by both compounds. In addition to the formation of domains, α-hederin and hederagenin permeabilize GUV. The formation of membrane holes by α-hederin comes along with the accumulation of lipids into nonbilayer structures in SPB. This process might be responsible for the permeabilizing activity of both compounds. In LUV, permeabilization by α-hederin was sterol-dependent. The biological implications of our results and the mechanisms involved are discussed in relation to the activity of saponins and triterpenic acids on membrane rafts, cancer cells, and hemolysis.

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.

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.

Effects of surfactin on membrane models displaying lipid phase separation.

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.

Multi-scale simulation of the simian immunodeficiency virus fusion peptide.

Fusion peptides of type I fusion glycoproteins are structural elements of several enveloped viruses which enable the fusion between host and virus membranes. It is generally suggested that these peptides can promote the early fusion steps by inducing membrane curvature and that they adopt a tilted helical conformation in membranes. Although this property has been the subject of several experimental and in silico studies, an extensive sampling of the membrane peptide interaction has not yet been done. In this study, we performed coarse-grained molecular dynamic simulations in which the lipid bilayer self-assembles around the peptide. The simulations indicate that the SIV fusion peptide can adopt two different orientations in a DPPC bilayer, a major population which adopts a tilted interfacial orientation and a minor population which is perpendicular to the bilayer. The simulations also indicate that for the SIV mutant that does not induce fusion in vitro the tilt is abolished.

In silico predictions of 3D structures of linear and cyclic peptides with natural and non-proteinogenic residues.

We extended the use of Peplook, an in silico procedure for the prediction of three-dimensional (3D) models of linear peptides to the prediction of 3D models of cyclic peptides and thanks to the ab initio calculation procedure, to the calculation of peptides with non-proteinogenic amino acids. Indeed, such peptides cannot be predicted by homology or threading. We compare the calculated models with NMR and X-ray models and for the cyclic peptides, with models predicted by other in silico procedures (Pep-Fold and I-Tasser). For cyclic peptides, on a set of 38 peptides, average root mean square deviation of backbone atoms (BB-RMSD) was 3.8 and 4.1 Å for Peplook and Pep-Fold, respectively. The best results are obtained with I-Tasser (2.5 Å) although evaluations were biased by the fact that the resolved Protein Data Bank models could be used as template by the server. Peplook and Pep-Fold give similar results, better for short (up to 20 residues) than for longer peptides. For peptides with non-proteinogenic residues, performances of Peplook are sound with an average BB-RMSD of 3.6 Å for 'non-natural peptides' and 3.4 Å for peptides combining non-proteinogenic residues and cyclic structure. These results open interesting possibilities for the design of peptidic drugs.

Monolayer properties of uronic acid bicatenary derivatives at the air-water interface: effect of hydroxyl group stereochemistry evidenced by experimental and computational approaches.

By screening uronic acid-based surfactant interfacial properties, the effect of the hydroxyl group stereochemistry (OH-4) on the conformation of bicatenary (disubstituted) derivatives at the air-water interface has been evidenced by experimental and computational approaches. Physical and optical properties of a monolayer characterized by Langmuir film balance, Brewster angle microscopy, and ellipsometry at 20 °C reveal that the derivative of glucuronate (C(14/14)-GlcA) forms a more expanded monolayer, and shows a transition state under compression, in the opposite to that of galacturonate (C(14/14)-GalA). Both films are very mechanically resistant (compression modulus > 300 mN m(-1)) and stable (collapse pressure exceeding 60 mN m(-1)), while that of C(14/14)-GalA exhibits a very high compression modulus up to 600 mN m(-1) like films in the solid state. Computational approaches provide single and assembly molecular models that corroborate the molecule expansion degree and interactions data from experimental results. Differences in the molecular conformation and film behaviours of uronic acid bicatenary derivatives at the air-water interface are attributed to the intra-H-bonding formation, which is more favourable with an OH-4 in the axial (C(14/14)-GalA) than in the equatorial position (C(14/14)-GlcA).

A new in-silico method for determination of helical transmembrane domains based on the PepLook scan: application to IL-2Rß and IL-2Rγc receptor chains.

BACKGROUND: Modeling of transmembrane domains (TMDs) requires correct prediction of interfacial residues for in-silico modeling and membrane insertion studies. This implies the defining of a target sequence long enough to contain interfacial residues. However, too long sequences induce artifactual polymorphism: within tested modeling methods, the longer the target sequence, the more variable the secondary structure, as though the procedure were stopped before the end of the calculation (which may in fact be unreachable). Moreover, delimitation of these TMDs can produce variable results with sequence based two-dimensional prediction methods, especially for sequences showing polymorphism. To solve this problem, we developed a new modeling procedure using the PepLook method. We scanned the sequences by modeling peptides from the target sequence with a window of 19 residues. RESULTS: Using sequences whose NMR-structures are already known (GpA, EphA1 and Erb2-HER2), we first determined that the hydrophobic to hydrophilic accessible surface area ratio (ASAr) was the best criterion for delimiting the TMD sequence. The length of the helical structure and the Impala method further supported the determination of the TMD limits. This method was applied to the IL-2Rß and IL-2Rγ TMD sequences of Homo sapiens, Rattus norvegicus, Mus musculus and Bos taurus. CONCLUSIONS: We succeeded in reducing the variation in the TMD limits to only 2 residues and in gaining structural information.

The Pseudomonas aeruginosa membranes: a target for a new amphiphilic aminoglycoside derivative?

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.

Happy birthday cell penetrating peptides: already 20 years.

The recent discovery of new potent therapeutic molecules that do not reach the clinic due to poor delivery and low bioavailability has made of delivery a keystone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs). CPPs were discovered 20 years ago based on the potency of several proteins to enter cells. So far numerous CPPs have been described which can be grouped into two major classes, the first requiring chemical linkage with the drug for cellular internalization, the second involving formation of stable, non-covalent complexes with cargos. Nowadays, CPPs constitute as a very promising tool for non-invasive cellular import of cargos and have been successfully applied for ex vivo and in vivo delivery of therapeutic molecules varying from small chemical molecules, nucleic acids, proteins, peptides, liposomes to particles. This short introduction will highlight the major breakthroughs in the CPP history, which have driven these delivery agents to the clinic.

The role of proline in the membrane re-entrant helix of caveolin-1.

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro(110) residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the N terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the N terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells, the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the N terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation, and lipid droplet formulation. In addition, we find that this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent-resistant membranes of these cells. Thus, the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.

Acylated and unacylated ghrelin binding to membranes and to ghrelin receptor: towards a better understanding of the underlying mechanisms.

The O-octanoylation of human ghrelin is a natural post-translational modification that enhances its binding to model membranes and could potentially play a central role in ghrelin biological activities. Here, we aimed to clarify the mechanisms that drive ghrelin to the membrane and hence to its receptor that mediates most of its endocrinological effects. As the acylation enhances ghrelin lipophilicity and that ghrelin contains many basic residues, we examined the electrostatic attraction and/or hydrophobic interactions with membranes. Using various liposomes and buffer conditions in binding, zeta potential and isothermal titration calorimetry studies, we found that whereas acylated and unacylated ghrelin were both electrostatically attracted towards the membrane, only acylated ghrelin penetrated into the headgroup and the lipid backbone regions of negatively charged membranes. The O-acylation induced a 120-fold increase in ghrelin local concentration in the membrane. However, acylated ghrelin did not deeply penetrate the membrane nor did it perturb its organisation. Conformational studies by circular dichroism and attenuated total reflection Fourier transformed infrared as well as in silico modelling revealed that both forms of ghrelin mainly adopted the same structure in aqueous, micellar and bilayer environments even though acylated ghrelin structure is slightly more α-helical in a lipid bilayer environment. Altogether our results suggest that membrane acts as a "catalyst" in acylated ghrelin binding to the ghrelin receptor and hence could explain why acylated and unacylated ghrelin are both full agonists of this receptor but in the nanomolar and micromolar range, respectively.

Cholesterol interaction with proteins that partition into membrane domains: an overview.

Biological membranes are complex structures composed largely of proteins and lipids. These components have very different structural and physical properties and consequently they do not form a single homogeneous mixture. Rather components of the mixture are more enriched in some regions than in others. This can be demonstrated with simple lipid mixtures that spontaneously segregate components so as to form different lipid phases that are immiscible with one another. The segregation of molecular components of biological membranes also involves proteins. One driving force that would promote the segregation of membrane components is the preferential interaction between a protein and certain lipid components. Among the varied lipid components of mammalian membranes, the structure and physical properties of cholesterol is quite different from that of other major membrane lipids. It would therefore be expected that in many cases proteins would have very different energies of interaction with cholesterol vs. those of other membrane lipids. This would be sufficient to cause segregation of components in membranes. The factors that facilitate the interaction of proteins with cholesterol are varied and are not yet completely understood. However, there are certain groups that are present in some proteins that facilitate interaction of the protein with cholesterol. These groups include saturated acyl chains of lipidated proteins, as well as certain amino acid sequences. Although there is some understanding as to why these particular groups favour interaction with cholesterol, our knowledge of these molecular features is not sufficiently developed to allow for the design of agents that will modify such binding.

Realistic modeling approaches of structure-function properties of CPPs in non-covalent complexes.

Transfers of cargoes into cells by means of carrier peptides are multi-steps biological phenomenon the mechanisms of which are unclear. We here discuss bases of realistic in silico molecular modeling approaches of the formation of non-covalent complexes considering CPPs and cargo diversities.

Insight into the cellular uptake mechanism of a secondary amphipathic cell-penetrating peptide for siRNA delivery.

Delivery of siRNA remains a major limitation to their clinical application, and several technologies have been proposed to improve their cellular uptake. We recently described a peptide-based nanoparticle system for efficient delivery of siRNA into primary cell lines: CADY. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA and improves their cellular uptake independently of the endosomal pathway. In the present work, we have combined molecular modeling, spectroscopy, and membrane interaction approaches in order to gain further insight into CADY/siRNA particle mechanism of interaction with biological membrane. We demonstrate that CADY forms stable complexes with siRNA and binds phospholipids tightly, mainly through electrostatic interactions. Binding to siRNA or phospholipids triggers a conformational transition of CADY from an unfolded state to an alpha-helical structure, thereby stabilizing CADY/siRNA complexes and improving their interactions with cell membranes. Therefore, we propose that CADY cellular membrane interaction is driven by its structural polymorphism which enables stabilization of both electrostatic and hydrophobic contacts with surface membrane proteoglycan and phospholipids.

Standardized evaluation of protein stability.

We compare mean force potential values of a large series of PDB models of proteins and peptides and find that, either as monomers or polymers, proteins longer than 200-250 residues have equivalent MFP values that are averaged to -65+/-3 kcal/aa. This value is named the standard or stability value. The standard value is reached irrespective of sequences and 3D folds. Peptides are too short to follow the rule and frequently exist as populations of conformers; one exception is peptides in amyloid fibrils. Fibrils surpass the standard value in accordance with their uppermost stability. In parallel, we calculate median MFP values of amino acids in stably folded PDB models of proteins: median values vary from -25 for Gly to -115 kcal/aa for Trp. These median values are used to score primary sequences of proteins: all sequences converge to a mean value of -63.5+/-2.5 kcal/aa, i.e., only 1.5 kcal less than the folded model standard. Sequences from unfolded proteins have lower values. This supports the conclusion that sequences carry in an important message and more specifically that diversity of amino acids in sequences is mandatory for stability. We also use the median amino acid MFP to score residue stability in 3D folds. This demonstrates that 3D folds are compromises between fragments of high and fragments of low scores and that functional residues are often but not always in the extreme score values. The approach opens to possibilities of evaluating any 3D model and of detecting functional residues and should help in conducting mutation assays.

Edgetic perturbation models of human inherited disorders.

Cellular functions are mediated through complex systems of macromolecules and metabolites linked through biochemical and physical interactions, represented in interactome models as 'nodes' and 'edges', respectively. Better understanding of genotype-to-phenotype relationships in human disease will require modeling of how disease-causing mutations affect systems or interactome properties. Here we investigate how perturbations of interactome networks may differ between complete loss of gene products ('node removal') and interaction-specific or edge-specific ('edgetic') alterations. Global computational analyses of approximately 50,000 known causative mutations in human Mendelian disorders revealed clear separations of mutations probably corresponding to those of node removal versus edgetic perturbations. Experimental characterization of mutant alleles in various disorders identified diverse edgetic interaction profiles of mutant proteins, which correlated with distinct structural properties of disease proteins and disease mechanisms. Edgetic perturbations seem to confer distinct functional consequences from node removal because a large fraction of cases in which a single gene is linked to multiple disorders can be modeled by distinguishing edgetic network perturbations. Edgetic network perturbation models might improve both the understanding of dissemination of disease alleles in human populations and the development of molecular therapeutic strategies.

'Edgetic' perturbation of a C. elegans BCL2 ortholog.

Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or 'edgetic', alleles. We established a proof of concept with CED-9, a Caenorhabditis elegans BCL2 ortholog. Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.