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Cholesterol-Mediated Clustering of the HIV Fusion Protein gp41 in Lipid Bilayers.
Tran, N, Oh, Y, Sutherland, M, Cui, Q, Hong, M
Journal of molecular biology. 2022;(2):167345
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Abstract
The envelope glycoprotein (Env) of the human immunodeficient virus (HIV-1) is known to cluster on the viral membrane surface to attach to target cells and cause membrane fusion for HIV-1 infection. However, the molecular structural mechanisms that drive Env clustering remain opaque. Here, we use solid-state NMR spectroscopy and molecular dynamics (MD) simulations to investigate nanometer-scale clustering of the membrane-proximal external region (MPER) and transmembrane domain (TMD) of gp41, the fusion protein component of Env. Using 19F solid-state NMR experiments of mixed fluorinated peptides, we show that MPER-TMD trimers form clusters with interdigitated MPER helices in cholesterol-containing membranes. Inter-trimer 19F-19F cross peaks, which are indicative of spatial contacts within ∼2 nm, are observed in cholesterol-rich virus-mimetic membranes but are suppressed in cholesterol-free model membranes. Water-peptide and lipid-peptide cross peaks in 2D 1H-19F correlation spectra indicate that the MPER is well embedded in model phosphocholine membranes but is more exposed to the surface of the virus-mimetic membrane. These experimental results are reproduced in coarse-grained and atomistic molecular dynamics simulations, which suggest that the effects of cholesterol on gp41 clustering is likely via indirect modulation of the MPER orientation. Cholesterol binding to the helix-turn-helix region of the MPER-TMD causes a parallel orientation of the MPER with the membrane surface, thus allowing MPERs of neighboring trimers to interact with each other to cause clustering. These solid-state NMR data and molecular dynamics simulations suggest that MPER and cholesterol cooperatively govern the clustering of gp41 trimers during virus-cell membrane fusion.
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Single-molecule fluorescence vistas of how lipids regulate membrane proteins.
Ward, AE, Ye, Y, Schuster, JA, Wei, S, Barrera, FN
Biochemical Society transactions. 2021;(4):1685-1694
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Abstract
The study of membrane proteins is undergoing a golden era, and we are gaining unprecedented knowledge on how this key group of proteins works. However, we still have only a basic understanding of how the chemical composition and the physical properties of lipid bilayers control the activity of membrane proteins. Single-molecule (SM) fluorescence methods can resolve sample heterogeneity, allowing to discriminate between the different molecular populations that biological systems often adopt. This short review highlights relevant examples of how SM fluorescence methodologies can illuminate the different ways in which lipids regulate the activity of membrane proteins. These studies are not limited to lipid molecules acting as ligands, but also consider how the physical properties of the bilayer can be determining factors on how membrane proteins function.
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Functional Group Distributions, Partition Coefficients, and Resistance Factors in Lipid Bilayers Using Site Identification by Ligand Competitive Saturation.
Lind, C, Pandey, P, Pastor, RW, MacKerell, AD
Journal of chemical theory and computation. 2021;(5):3188-3202
Abstract
Small molecules such as metabolites and drugs must pass through the membrane of the cell, a barrier primarily comprising phospholipid bilayers and embedded proteins. To better understand the process of passive diffusion, knowledge of the ability of various functional groups to partition across bilayers and the associated energetics would be of utility. In the present study, the site identification by ligand competitive saturation (SILCS) methodology has been applied to sample the distributions of a diverse set of chemical solutes representing the functional groups of small molecules across phospholipid bilayers composed of 0.9:0.1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol and a mixture of 0.52:0.18:0.3 1,2-dioleoyl-sn-glycero-3-phospho-l-serine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol used in parallel artificial membrane permeability assay experiments. A combination of oscillating chemical potential grand canonical Monte Carlo and molecular dynamics in the SILCS simulations was applied to achieve solute sampling through the bilayers and surrounding aqueous environment from which the distribution of solutes and the functional groups they represent were obtained. Results show differential distribution of aliphatic versus aromatic groups with the former having increased sampling in the center of the bilayers versus in the region of the glycerol linker for the latter. Variations in the distribution of different polar groups are evident, with large differences between negative acetate and positive methylammonium with accumulation of the polar-neutral and acetate solutes above the bilayer head groups. Conversion of the distributions to absolute free energies allows for a detailed understanding of energetics of functional groups in different regions of the bilayers and for calculation of absolute free-energy profiles of multifunctional drug-like molecules across the bilayers from which partition coefficients and resistance factors suitable for insertion into the homogenous solubility-diffusion equation for calculation of permeability were obtained. Comparisons of the calculated bilayer/solution partition coefficients with 1-octanol/water experimental data for both drug-like molecules and the solutes show overall good agreement, validating the calculated distributions and associated absolute free-energy profiles.
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The Transporter-Mediated Cellular Uptake and Efflux of Pharmaceutical Drugs and Biotechnology Products: How and Why Phospholipid Bilayer Transport Is Negligible in Real Biomembranes.
Kell, DB
Molecules (Basel, Switzerland). 2021;(18)
Abstract
Over the years, my colleagues and I have come to realise that the likelihood of pharmaceutical drugs being able to diffuse through whatever unhindered phospholipid bilayer may exist in intact biological membranes in vivo is vanishingly low. This is because (i) most real biomembranes are mostly protein, not lipid, (ii) unlike purely lipid bilayers that can form transient aqueous channels, the high concentrations of proteins serve to stop such activity, (iii) natural evolution long ago selected against transport methods that just let any undesirable products enter a cell, (iv) transporters have now been identified for all kinds of molecules (even water) that were once thought not to require them, (v) many experiments show a massive variation in the uptake of drugs between different cells, tissues, and organisms, that cannot be explained if lipid bilayer transport is significant or if efflux were the only differentiator, and (vi) many experiments that manipulate the expression level of individual transporters as an independent variable demonstrate their role in drug and nutrient uptake (including in cytotoxicity or adverse drug reactions). This makes such transporters valuable both as a means of targeting drugs (not least anti-infectives) to selected cells or tissues and also as drug targets. The same considerations apply to the exploitation of substrate uptake and product efflux transporters in biotechnology. We are also beginning to recognise that transporters are more promiscuous, and antiporter activity is much more widespread, than had been realised, and that such processes are adaptive (i.e., were selected by natural evolution). The purpose of the present review is to summarise the above, and to rehearse and update readers on recent developments. These developments lead us to retain and indeed to strengthen our contention that for transmembrane pharmaceutical drug transport "phospholipid bilayer transport is negligible".
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Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers.
Shcherbakov, AA, Hisao, G, Mandala, VS, Thomas, NE, Soltani, M, Salter, EA, Davis, JH, Henzler-Wildman, KA, Hong, M
Nature communications. 2021;(1):172
Abstract
The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.
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A certain proportion of docosahexaenoic acid tends to revert structural and dynamical effects of cholesterol on lipid membranes.
Pedroni, VI, Sierra, MB, Alarcón, LM, Verde, AR, Appignanesi, GA, Morini, MA
Biochimica et biophysica acta. Biomembranes. 2021;(6):183584
Abstract
This work investigates how docosahexaenoic acid (DHA) modifies the effect of Cholesterol (Chol) on the structural and dynamical properties of dipalmitoylphosphatidylcholine (DPPC) membrane. We employ low-cost and non-invasive methods: zeta potential (ZP), conductivity, density, and ultrasound velocity, complemented by molecular dynamics simulations. Our studies reveal that 30% of DHA added to the DPPC-Chol system tends to revert Chol action on a model lipid bilayer. Results obtained in this work shed light on the effect of polyunsaturated fatty acids - particularly DHA - on lipid membranes, with potential preventive applications in many diseases, e.g. neuronal as, Alzheimer's disease, and viral, as Covid-19.
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Membrane Activity and Channel Formation of the Adenylate Cyclase Toxin (CyaA) of Bordetella pertussis in Lipid Bilayer Membranes.
Knapp, O, Benz, R
Toxins. 2020;(3)
Abstract
The Gram-negative bacterium Bordetella pertussis is the cause of whooping cough. One of its pathogenicity factors is the adenylate cyclase toxin (CyaA) secreted by a Type I export system. The 1706 amino acid long CyaA (177 kDa) belongs to the continuously increasing family of repeat in toxin (RTX) toxins because it contains in its C-terminal half a high number of nine-residue tandem repeats. The protein exhibits cytotoxic and hemolytic activities that target primarily myeloid phagocytic cells expressing the αMβ2 integrin receptor (CD11b/CD18). CyaA represents an exception among RTX cytolysins because the first 400 amino acids from its N-terminal end possess a calmodulin-activated adenylate cyclase (AC) activity. The entry of the AC into target cells is not dependent on the receptor-mediated endocytosis pathway and penetrates directly across the cytoplasmic membrane of a variety of epithelial and immune effector cells. The hemolytic activity of CyaA is rather low, which may have to do with its rather low induced permeability change of target cells and its low conductance in lipid bilayer membranes. CyaA forms highly cation-selective channels in lipid bilayers that show a strong dependence on aqueous pH. The pore-forming activity of CyaA but not its single channel conductance is highly dependent on Ca2+ concentration with a half saturation constant of about 2 to 4 mM.
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Transferosomes as nanocarriers for drugs across the skin: Quality by design from lab to industrial scale.
Fernández-García, R, Lalatsa, A, Statts, L, Bolás-Fernández, F, Ballesteros, MP, Serrano, DR
International journal of pharmaceutics. 2020;:118817
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Abstract
Transferosomes, also known as transfersomes, are ultradeformable vesicles for transdermal applications consisting of a lipid bilayer with phospholipids and an edge activator and an ethanol/aqueous core. Depending on the lipophilicity of the active substance, it can be encapsulated within the core or amongst the lipid bilayer. Compared to liposomes, transferosomes are able to reach intact deeper regions of the skin after topical administration delivering higher concentrations of active substances making them a successful drug delivery carrier for transdermal applications. Most transferosomes contain phosphatidylcholine (C18) as it is the most abundant lipid component of the cell membrane, and hence, it is highly tolerated for the skin, decreasing the risk of undesirable effects, such as hypersensitive reactions. The most common edge activators are surfactants such as sodium deoxycholate, Tween® 80 and Span® 80. Their chain length is optimal for intercalation within the C18 phospholipid bilayer. A wide variety of drugs has been successfully encapsulated within transferosomes such as phytocompounds like sinomenine or apigenin for rheumatoid arthritis and leukaemia respectively, small hydrophobic drugs but also macromolecules like insulin. The main factors to develop optimal transferosomal formulations (with high drug loading and nanometric size) are the optimal ratio between the main components as well as the critical process parameters for their manufacture. Application of quality by design (QbD), specifically design of experiments (DoE), is crucial to understand the interplay among all these factors not only during the preparation at lab scale but also in the scale-up process. Clinical trials of a licensed topical ketoprofen transferosomal gel have shown promising results in the alleviation of symptons in orthreothritis with non-severe skin and subcutaneous tissue disorders. However, the product was withdrawn from the market which probably was related to the higher cost of the medicine linked to the expensive manufacturing process required in the production of transferosomes compared to other conventional gel formulations. This example brings out the need for a careful formulation design to exploit the best properties of this drug delivery system as well as the development of manufacturing processes easily scalable at industrial level.
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Transmembrane potential of physiologically relevant model membranes: Effects of membrane asymmetry.
Lin, X, Gorfe, AA
The Journal of chemical physics. 2020;(10):105103
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Abstract
Transmembrane potential difference (Vm) plays important roles in regulating various biological processes. At the macro level, Vm can be experimentally measured or calculated using the Nernst or Goldman-Hodgkin-Katz equation. However, the atomic details responsible for its generation and impact on protein and lipid dynamics still need to be further elucidated. In this work, we performed a series of all-atom molecular dynamics (MD) simulations of symmetric model membranes of various lipid compositions and cation contents to evaluate the relationship between membrane asymmetry and Vm. Specifically, we studied the impact of the asymmetric distribution of POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine), PIP2 (phosphatidylinositol 4,5-bisphosphate), as well as Na+ and K+ on Vm using atomically detailed MD simulations of symmetric model membranes. The results suggest that, for an asymmetric POPC-POPC/POPS bilayer in the presence of NaCl, the presence of the monovalent anionic lipid POPS in the inner leaflet polarizes the membrane (ΔVm < 0). Intriguingly, replacing a third of the POPS lipids by the polyvalent anionic signaling lipid PIP2 counteracts this effect, resulting in a smaller negative membrane potential. We also found that replacing Na+ ions in the inner region by K+ depolarizes the membrane (ΔVm > 0). These divergent effects arise from variations in the strength of cation-lipid interactions and are correlated with changes in lipid chain order and head-group orientation.
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Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria.
Horne, JE, Brockwell, DJ, Radford, SE
The Journal of biological chemistry. 2020;(30):10340-10367
Abstract
β-Barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonization and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP-folding landscape and discuss the factors that guide folding in vitro and in vivo We particularly focus on the composition, architecture, and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance it is an attractive target. To bring down this vital OMP-supported barrier to antibiotics, we must first understand how bacterial cells build it.