Check out our New Publishers' Select for Free Articles

BBA - Biomembranes (v.1788, #12)

Editorial Board (pp. i).

Protein structure and ionic selectivity in calcium channels: Selectivity filter size, not shape, matters by Attila Malasics; Dirk Gillespie; Wolfgang Nonner; Douglas Henderson; Bob Eisenberg; Dezső Boda (pp. 2471-2480).

Calcium channels have highly charged selectivity filters (4 COO⁻ groups) that attract cations in to balance this charge and minimize free energy, forcing the cations (Na⁺ and Ca²⁺) to compete for space in the filter. A reduced model was developed to better understand the mechanism of ion selectivity in calcium channels. The charge/space competition (CSC) mechanism implies that Ca²⁺ is more efficient in balancing the charge of the filter because it provides twice the charge as Na⁺ while occupying the same space. The CSC mechanism further implies that the main determinant of Ca²⁺ versus Na⁺ selectivity is the density of charged particles in the selectivity filter, i.e., the volume of the filter (after fixing the number of charged groups in the filter). In this paper we test this hypothesis by changing filter length and/or radius (shape) of the cylindrical selectivity filter of our reduced model. We show that varying volume and shape together has substantially stronger effects than varying shape alone with volume fixed. Our simulations show the importance of depletion zones of ions in determining channel conductance calculated with the integrated Nernst–Planck equation. We show that confining the protein side chains with soft or hard walls does not influence selectivity.

Keywords: Calcium channel; Selectivity; Permeation; Grand canonical; Monte Carlo simulation

Structural and functional analysis of extracellular loop 2 of the Na⁺/H⁺ exchanger by Brian L. Lee; Xiuju Li; Yongsheng Liu; Brian D. Sykes; Larry Fliegel (pp. 2481-2488).

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pHi) by removing one intracellular H⁺ in exchange for one extracellular Na⁺. It has a large 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments and several membrane-associated segments. Extracellular regions of this domain are believed to contribute to cation coordination, transport and sensitivity to inhibitors. In this study we characterized the structure and function of extracellular loop 2. Mutation of residues Pro153, Pro154 and Phe155 demonstrated that these residues were critical for efficient NHE1 function. Mutations to Ala resulted in decreases in cation affinity and in decreases in activity of the protein, these were more marked in both Pro154 and Phe155. NMR spectroscopy was used to characterize the solution structure of a peptide NAc-Gly150-Phe155-NH₂. The peptide showed at least three different conformers in solution due to cis–trans isomerization of the Thr¹⁵²–Pro¹⁵³ and Pro¹⁵³–Pro¹⁵⁴ peptide bonds. The trans–trans conformation appeared to be in an extended conformation, whereas the cis–trans conformation showed a propensity to form a beta turn. Our results show that the EL2 region is critical to NHE1 function and that a peptide of the EL2 region can adopt different structures in solution potentially forming a beta turn that is important in function of the full protein Mutation of Pro¹⁵⁴ could disrupt the beta turn, affecting helix packing and the protein structure and function.

Keywords: Cation transport; Membrane; Na; ⁺; /H; ⁺; exchanger; pH regulation

Functional characterization of a Na⁺-coupled dicarboxylate transporter from Bacillus licheniformis by Melodie A. Strickler; Jason A. Hall; Olga Gaiko; Ana M. Pajor (pp. 2489-2496).

The Na⁺-coupled dicarboxylate transporter, SdcL, from Bacillus licheniformis is a member of the divalent anion/Na⁺ symporter (DASS) family that includes the bacterial Na⁺/dicarboxylate cotransporter SdcS (from Staphyloccocus aureus) and the mammalian Na⁺/dicarboxylate cotransporters, NaDC1 and NaDC3. The transport properties of SdcL produced in Escherichia coli are similar to those of its prokaryotic and eukaryotic counterparts, involving the Na⁺-dependent transport of dicarboxylates such as succinate or malate across the cytoplasmic membrane with a K_m of ∼6 μM. SdcL may also transport aspartate, α-ketoglutarate and oxaloacetate with low affinity. The cotransport of Na⁺ and dicarboxylate by SdcL has an apparent stoichiometry of 2:1, and a K_0.5 for Na⁺ of 0.9 mM. Our findings represent the characterization of another prokaryotic protein of the DASS family with transport properties similar to its eukaryotic counterparts, but with a broader substrate specificity than other prokaryotic DASS family members. The broader range of substrates carried by SdcL may provide insight into domains of the protein that allow a more flexible or larger substrate binding pocket.

Keywords: Sodium; Succinate; Malate; Transport; DASS family

Conformational and dynamics simulation study of antimicrobial peptide hedistin—heterogeneity of its helix–turn–helix motif by Guohua Xu; Min Wu; Lin Wang; Xu Zhang; Shufen Cao; Maili Liu; Yanfang Cui (pp. 2497-2508).

Hedistin is an antimicrobial peptide isolated from the coelomocytes of Nereis diversicolor, possessing activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus. The three-dimensional structure of hedistin in both aqueous solution and deuterated dodecylphosphocholine (DPC) micelles was examined using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques. And, the early events of the antibacterial process of hedistin were simulated using palmitoyl-oleoyl-phophatidylcholine (POPC) lipid bilayers and molecular dynamics (MD) simulation methods. Hedistin lacks secondary structure in aqueous solution, however, in DPC micelles, it features with a heterogeneous helix–turn–helix moiety and exhibits obvious amphipathic nature. The turn region (residues Val9–Thr12) in the moiety is a four-residue hinge, lying in between the first N-terminal α-helix (residues Leu5–Lys8) and the second α-helix (residues Val13–Ala17) regions and causing an ∼120° angle between the axes of the two helices. The segmental and nonlinear nature of hedistin structure is referred to as the heterogeneity of its helix–turn–helix motif which was found to be corresponding to a kind of discrete dynamics behavior, herein coined as its dynamical heterogeneity, at the early stage (0–50 ns) of the MD simulations. That is, the first helix segment, prior to (at 310 K) or following (at 363 K) the second helix, binds to the lipid head-group region and subsequently permeates into the hydrophobic lipid tail region, and the hinge is the last portion entering the lipid environment. This result implies that hedistin may adopt a “carpet” model action when disrupting bacterial membrane.

Keywords: Abbreviations; DPC; dodecylphosphocholine; CD; circular dichroism; NMR; nuclear magnetic resonance; MD; molecular dynamics; AMPs; antimicrobial peptides; MRSA; methicillin resistant; Staphylococcus aureus; VRE; vancomycin resistant enterococci; POPC; palmitoyl-oleoyl-phophatidylcholine; SDS; sodium dodecyl sulphate; DQF-COSY; double-quantum filtered correlation spectroscopy; TOCSY; total correlation spectroscopy; NOESY; nuclear Overhauser effect spectroscopy; DSS; sodium 2, 2-dimethyl-2-silapentane-5-dulfonate salt; SPC; simple point charge; PME; Particle mesh Ewald; CSI; chemical shift index; DSSP; dictionary of secondary structure of proteinsAntimicrobial peptide; Hedistin; Nuclear magnetic resonance; Helix–turn–helix motif; Molecular dynamics simulation; Heterogeneity

Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate by Peter Guterstam; Fatemeh Madani; Hisaaki Hirose; Toshihide Takeuchi; Shiroh Futaki; Samir EL Andaloussi; Astrid Gräslund; Ülo Langel (pp. 2509-2517).

Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with varying hydrophobicity and arginine content is investigated. The cellular CPP uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell-based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2′- O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.

Keywords: Abbreviations; CPP; cell-penetrating peptide; pyrenebutyrate; 4-(1-pyrenyl)-butyric acid; FACS; fluorescence assisted cell sorting; POPC; palmitoyl-2-oleoyl-phosphatidylcholine; POPG; palmitoyl-2-oleoyl-phosphatidylglycerol; ON; oligonucleotide; LNA; locked nucleic acid; 2OMe; 2′-; O; -methyl RNA; LUV; large unilamellar vesicle; R9; oligoarginine nonamer; Pen; penetratin; TP10; transportan 10; pVEC; vascular endothelial cadherin peptideCell-penetrating peptide; Oligonucleotide delivery; Pyrenebutyrate; Cellular translocation; Locked nucleic acid; Splice switching

Structural study of liposomes loaded with a GM3 lactone analogue for the targeting of tumor epitopes by Sandra Ristori; Emanuela Di Cola; Carlotta Lunghi; Barbara Richichi; Cristina Nativi (pp. 2518-2525).

Therapeutic vaccination with tumor antigens is a new approach in cancer treatment, which aims at inducing immune response while avoiding the side effects generally associated to many conventional therapies. To improve the efficacy of vaccines, suitable carriers may be used. Herein the insertion of a thioether analogue of GM3 lactone (SNeuAC-C14) into liposomes is reported. SNeuAC-C14 is a potential vaccine for the targeting of saccharide-based tumor epitopes. Different liposome formulations were designed to act as carriers and to generate recognition by tumor epitopes. The structural study of pure and loaded liposomes was carried out by synchrotron Small Angle X-ray Scattering and was complemented by Dynamic Light Scattering and Zeta potential measurements. This provided detailed information on relevant properties of the investigated host-guest structures and showed that the active unit of SNeuAC-C14, i.e. its spiro tricyclic moiety, was located in the polar head region of the liposome bilayer, which is an important requirement for recognition phenomena. Moreover, it was found that most of the SNeuAC-C14/liposome complexes were positively charged. The obtained results allow these systems to be considered as candidates to promote immunoresponse in tumor cells.

Keywords: Liposome; Vaccine adjuvant; Structural investigation; SAXS

Differential endocytic trafficking of neuropathy-associated antibodies to GM1 ganglioside and cholera toxin in epithelial and neural cells by Ramiro Iglesias-Bartolomé; Alejandra Trenchi; Romina Comín; Ana L. Moyano; Gustavo A. Nores; Jose L. Daniotti (pp. 2526-2540).

Gangliosides are glycolipids mainly present at the plasma membrane (PM). Antibodies to gangliosides have been associated with a wide range of neuropathy syndromes. Particularly, antibodies to GM1 ganglioside are present in patients with Guillain–Barré syndrome (GBS). We investigated the binding and intracellular fate of antibody to GM1 obtained from rabbits with experimental GBS in comparison with the transport of cholera toxin (CTx), which binds with high affinity to GM1. We demonstrated that antibody to GM1 is rapidly and specifically endocytosed in CHO-K1 cells. After internalization, the antibody transited sorting endosomes to accumulate at the recycling endosome. Endocytosed antibody to GM1 is recycled back to the PM and released into the culture medium. In CHO-K1 cells, antibody to GM1 colocalized with co-endocytosed CTx at early and recycling endosomes, but not in Golgi complex and endoplasmic reticulum, where CTx was also located. Antibody to GM1, in contraposition to CTx, showed a reduced internalization to recycling endosomes in COS-7 cells and neural cell lines SH-SY5Y and Neuro2A. Results from photobleaching studies revealed differences in the lateral mobility of antibody to GM1 in the PM of analyzed cell lines, suggesting a relationship between the efficiency of endocytosis and lateral mobility of GM1 at the PM. Taken together, results indicate that two different ligands of GM1 ganglioside (antibody and CTx) are differentially endocytosed and trafficked, providing the basis to gain further insight into the mechanisms that operate in the intracellular trafficking of glycosphingolipid-binding toxins and pathological effects of neuropathy-associated antibodies.

Keywords: Abbreviations; CHO; Chinese hamster ovary; CTx; cholera toxin; CTxB; cholera toxin B subunit; ER; endoplasmic reticulum; DMEM; Dulbecco's modiﬁed Eagle's medium; FBS; fetal bovine serum; FRAP; fluorescence recovery after photobleaching; GalNAc-T; UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase; GBS; Guillain–Barré syndrome; GFP; green fluorescent protein; PBS; phosphate-buffered saline; PM; plasma membrane; RT; room temperature; TGN; trans-Golgi network; Tf; transferrin; YFP; yellow fluorescent proteinGlycolipid antibodie; Ganglioside; Intracellular trafficking; Cholera toxin; Endocytic recycling; Guillain–Barré syndrome

Quantitative characterization of coexisting phases in DOPC/DPPC/cholesterol mixtures: Comparing confocal fluorescence microscopy and deuterium nuclear magnetic resonance by Janos Juhasz; Frances J. Sharom; James H. Davis (pp. 2541-2552).

The differential miscibility of membrane lipids is thought to be the basis for the formation of dynamic microdomain assemblies in cell membranes known as membrane rafts. Because of their relevance to the existence of rafts, there has been much interest in recent years in model membrane systems that display coexisting liquid ordered ( l_o) and liquid disordered phases ( l_d), such as the ternary mixture composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. Carefully equilibrating the samples at well controlled temperatures allows us to use a quantitative confocal fluorescence microscopy approach to measure the area fractions of coexisting fluid phases in DOPC/DPPC/cholesterol mixtures. We can then compare the behaviour of a large population of unilamellar vesicles with the domain fractions deduced from²H NMR experiments. The fluorescence results are established for the first time to be in quantitative agreement with those obtained using²H NMR spectroscopy within the two phase region of the phase diagram. We are also able to describe fine details of the phase separation and the approach to equilibrium not previously reported, in particular the existence of small spots of l_o phase at temperatures higher than that at which the samples display domain fluctuations. A better understanding of coexisting fluid phases in model systems will assist in interpreting the behaviour of rafts in more complex biological membranes.

Keywords: Confocal fluorescence microscopy; Lipid rafts; Ternary phase equilibria; Deuterium nuclear magnetic resonance; Liquid ordered phase; Domain area fraction

Interaction of nicotinamide and picolinamide with phosphatidylcholine and phosphatidylethanolamine membranes: A combined approach using dipole potential measurements and quantum chemical calculations by Ana Borba; Fabiana Lairion; Anibal Disalvo; Rui Fausto (pp. 2553-2562).

Interaction between the bioactive compounds nicotinamide and picolinamide and phospholipids (phosphatidylcholines and phosphatidylethanolamines) was investigated by a combined approach using dipole potential measurements and quantum chemical calculations. It is shown that nicotinamide and picolinamide interactions with phosphatidylcholines are of two main types: (i) specific interactions with the phosphate group of the lipid, for which H-bonding between NH₂ group of the substrate and the phosphate plays a dominant role, (ii) conjugated less specific weaker interactions involving both the phosphate and carbonyl groups of the head group, which propagate to the lipid alkyl chains and increase their conformational disorder. For phosphatidylethanolamines, picolinamide was found to decrease the dipole potential of the membrane in a similar way as for phosphatidylcholines, while nicotinamide is ineffective. These findings are correlated with the specific properties of phosphatidylethanolamines (reduced exposure of phosphate groups) and structural differences in the two substrates, in particular: different separation of the nitrogen atoms in the molecules, existence of a strong intramolecular hydrogen bond in picolinamide (NH...N_(ring)), which is absent in nicotinamide, and non-planarity of nicotinamide molecules, in contrast to picolinamide ones. Additional information on the lipid/substrate interactions was extracted from the analysis of the changes produced in the relevant vibrational frequencies of the lipid and substrate upon binding. The present study gives molecular support to the argument that changes of dipole potentials are due to effects on the constitutive dipolar PO and CO groups. In addition, it is also shown that according to the specific binding of the substrate to one or both of those, the conformational state of the acyl chains may be affected. These entropy effects may be in the origin of the well-known interdependence of the properties of one monolayer with respect to the other in bilayer membranes.

Keywords: Nicotinamide; Picolinamide; Phosphatidylcholine; Phosphatidylethanolamine; Interaction; Dipole potential; Quantum chemical calculation; Infrared spectra; Monolayer properties’ interdependence

Solid-state NMR study of proteorhodopsin in the lipid environment: Secondary structure and dynamics by Lichi Shi; Evelyn M.R. Lake; Mumdooh A.M. Ahmed; Leonid S. Brown; Vladimir Ladizhansky (pp. 2563-2574).

Proteorhodopsins are typical retinal-binding light-driven proton pumps of heptahelical architecture widely distributed in marine and freshwater bacteria. Recently, we have shown that green proteorhodopsin (GPR) can be prepared in a lipid-bound state that gives well-resolved magic angle spinning (MAS) NMR spectra in samples with different patterns of reverse labelling. Here, we present 3D and 4D sequential chemical shift assignments identified through experiments conducted on a uniformly¹³C,¹⁵N-labelled sample. These experiments provided the assignments for 153 residues, with a particularly high density in the transmembrane regions (∼74% of residues). The extent of assignments permitted a detailed examination of the secondary structure and dynamics in GPR. In particular, we present experimental evidence of mobility of the protein's termini and of the A–B, C–D, and F–G loops, the latter being possibly coupled to the GPR ion-transporting function.

Keywords: Solid-state NMR; Membrane protein; Proteorhodopsin; Secondary structure; Protein dynamics; Carboxyl ionization

Influence of membrane curvature on the structure of the membrane-associated pleckstrin homology domain of phospholipase C-δ1 by Naoko Uekama; Takahiro Aoki; Toshihiro Maruoka; Seiji Kurisu; Akiko Hatakeyama; Satoru Yamaguchi; Masashi Okada; Hitoshi Yagisawa; Katsuyuki Nishimura; Satoru Tuzi (pp. 2575-2583).

The effects of geometric properties of membranes on the structure of the phospholipase C-δ1 (PLC-δ1) pleckstrin homology (PH) domain were investigated using solid state¹³C NMR spectroscopy. Conformations of the PLC-δ1 PH domain at the surfaces of multilamellar vesicles (MLV), small unilamellar vesicles (SUV), and micelles were examined to evaluate the effects of membrane curvature on the PH domain. An increase in curvature of the water–hydrophobic layer interface hinders membrane-penetration of the amphipathic α2-helix of the PH domain that assists the membrane-association of the PH domain dominated by the phosphatidylinositol 4,5-bisphosphate (PIP₂) specific lipid binding site. The solid state¹³C NMR signal of Ala88 located at the α2-helix indicates that the conformation of the α2-helix at the micelle surface is similar to the solution conformation and significantly different from those at the MLV and SUV surfaces which were characterized by membrane-penetration and re-orientation. The signal of Ala112 which flanks the C-terminus of the β5/β6 loop that includes the α2-helix, showed downfield displacement with decrease in the interface curvature of the micelles, SUV and MLV. This reveals that the conformation of the C-terminus of the β5/β6 loop connecting the β-sandwich core containing the PIP₂ binding site and the amphipathic α2-helix is sensitive to alterations of the curvature of lipid bilayer surface. It is likely that these alterations in the conformation of the PLC-δ1 PH domain contribute to the regulatory mechanisms of the intracellular localization of PLC-δ1 in a manner dependent upon the structure of the molecular complex containing PIP₂.

Keywords: Phospholipase C-δ1; PH domain; Solid state NMR; Membrane curvature; Cellular signal transduction

Identification of the channel-forming domain of Clostridium perfringens Epsilon-toxin (ETX) by Oliver Knapp; Elke Maier; Roland Benz; Blandine Geny; Michel R. Popoff (pp. 2584-2593).

Epsilon-toxin (ETX) is a potent toxin produced by Clostridium perfringens strains B and D. The bacteria are important pathogens in domestic animals and cause edema mediated by ETX. This toxin acts most likely by heptamer formation and rapid permeabilization of target cell membranes for monovalent anions and cations followed by a later entry of calcium. In this study, we compared the primary structure of ETX with that of the channel-forming stretches of a variety of binding components of A-B-types of toxins such as Anthrax protective antigen (PA), C2II of C2-toxin and Ib of Iota-toxin and found a remarkable homology to amino acids 151–180 of ETX. Site-directed mutagenesis of amino acids within the putative channel-forming domain resulted in changes of cytotoxicity and effects on channel characteristics in lipid bilayer experiments including changes of selectivity and partial channel block by methanethiosulfonate (MTS) reagents and antibodies against His₆-tags from the trans-side of the lipid bilayer membranes.

Keywords: Epsilon-toxin (ETX); Channel formation; Channel-forming domain; Beta-PFT; Clostridium perfringens; Lipid bilayer membrane

Substrate discrimination by ergothioneine transporter SLC22A4 and carnitine transporter SLC22A5: Gain-of-function by interchange of selected amino acids by Petra Bacher; Susanne Giersiefer; Markus Bach; Christian Fork; Edgar Schömig; Dirk Gründemann (pp. 2594-2602).

ETT (originally designated as OCTN1; human gene symbol SLC22A4) and CTT (OCTN2; SLC22A5) are highly specific transporters of ergothioneine and carnitine, respectively. Despite a high degree of sequence homology, both carriers discriminate precisely between substrates: ETT does not transport carnitine, and CTT does not transport ergothioneine. Our aim was to turn ETT into a transporter for carnitine and CTT into a transporter for ergothioneine by a limited number of point mutations. From a multiple alignment of several mammalian amino acid sequences, those positions were selected for conversion that were momentously different between ETT and CTT from human but conserved among all orthologues. Mutants were expressed in 293 cells and assayed for transport of ergothioneine and carnitine. Several ETT mutants clearly catalyzed transport of carnitine, up to 35% relative to wild-type CTT. Amazingly, complementary substitutions in CTT did not provoke transport activity for ergothioneine. In similar contrast, carnitine transport by CTT mutants was abolished by very few substitutions, whereas ergothioneine transport by ETT mutants was maintained even with the construct most active in carnitine transport. To explain these results, we propose that ETT and CTT use dissimilar pathways for conformational change, in addition to incongruent substrate binding sites. In other words, carnitine is excluded from ETT by binding, and ergothioneine is excluded from CTT by turnover movement. Our data indicate amino acids critical for substrate discrimination not only in transmembrane segments 5, 7, 8, and 10, but also in segments 9 and 12 which were hitherto considered as unimportant.

Keywords: Abbreviations; CT; carnitine; CTT; carnitine transporter; ET; ergothioneine; ETT; ergothioneine transporter; LC; liquid chromatography; MPP; ⁺; 1-methyl-4-phenylpyridinium; MS; mass spectrometry; TMS; transmembrane segmentCarnitine transporter; Ergothioneine transporter; Gain-of-function; Site-directed mutagenesis; Substrate discrimination

Sections

Personal tools

BBA - Biomembranes (v.1788, #12)

Sections

Personal tools

Local resources

BBA - Biomembranes (v.1788, #12)