Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

Human TPST1 transmembrane domain triggers enzyme dimerisation and localisation to the Golgi compartment

TPST1 is a human tyrosylprotein sulfotransferase that uses 3'phosphoadenosine-5'phosphosulfate (PAPS) to transfer the sulfate moiety to proteins predominantly designated for secretion. To achieve a general understanding of the cellular role of human tyrosine-directed sulfotransferases, we investigated targeting, structure and posttranslational modification of TPST1. Golgi localisation of the enzyme in COS-7 and HeLa cells was visualised by fluorescence imaging techniques. PNGase treatment and mutational studies determined that TPST1 bears N-linked glycosyl residues exclusively at position Asn60 and Asn262. By alanine mutation of these asparagine residues, we could determine that the N-linked oligosaccharides do not have an influence on Golgi retention of TPST1. In concert with N and C-terminal flanking residues, the transmembrane domain of TPST1 was determined to act in targeting and retention of the enzyme to the trans-Golgi compartment. This domain exhibits a pronounced secondary structure in a lipid environment. Further in vivo FRET studies using the transmembrane domain suggest that the human tyrosylprotein sulfotransferase may be functional as homodimer/oligomer in the trans-Golgi compartment.     

The transmembrane domain of the SNARE protein VAMP2 is highly sensitive to its lipid environment

Neurotransmitter and hormone exocytosis depends on SNARE protein transmembrane domains and membrane lipids but their interplay is poorly understood. We investigated the interaction of the structure of VAMP2, a vesicular transmembrane SNARE protein, and membrane lipid composition by infrared spectroscopy using either the wild-type transmembrane domain (TMD), VAMP2_TM22, or a peptide mutated at the central residues G₁₀₀/C₁₀₃ (VAMP2_TM22VV) previously identified by us as being critical for exocytosis. Our data show that the structure of VAMP2_TM22, in terms of α-helices and β-sheets is strongly influenced by peptide/lipid ratios, by lipid species including cholesterol and by membrane surface charges. Differences observed in acyl chain alignments further underscore the role of the two central small amino acid residues G₁₀₀/C₁₀₃ within the transmembrane domain during lipid rearrangements in membrane fusion.     

Structural characterization of the transmembrane proximal region of the hepatitis C virus E1 glycoprotein

A detailed knowledge of the mechanism of virus entry represents one of the most promising approaches to develop new therapeutic strategies. However, viral fusion is a very complex process involving fusion glycoproteins present on the viral envelope. In the two hepatitis C virus envelope proteins, E1 and E2, several membranotropic regions with a potential role in the fusion process have been identified. Among these, we have selected the 314-342 E1 region. Circular Dichroism data indicate that the peptide exhibits a clear propensity to adopt a helical folding in different membrane mimicking media, such as mixtures of water with fluorinated alcohols and phospholipids, with a slight preference for negative charged bilayers. The 3D structure of E1_314-342 peptide, calculated by 2D-NMR in a low-polarity environment, consists of two helical stretches encompassing residues 319-323 and 329-338 respectively. The peptide, presenting a largely apolar character, interacts with liposomes, as indicated by fluorescence and electron spin resonance spectra. The strength of the interaction and the deepness of peptide insertion in the phospholipid membrane are modulated by the bilayer composition, the interaction with anionic phospholipids being among the strongest ever observed. The presence of cholesterol also affects the peptide-bilayer interaction, favoring the peptide positioning close to the bilayer surface. Overall, the experimental data support the idea that this region of E1 might be involved in membrane destabilization and viral fusion; therefore it may represent a good target to develop anti-viral molecules.     

Direct Visualization of Large and Protein-Free Hemifusion Diaphragms

Fusion of cellular membranes is a ubiquitous biological process requiring remodeling of two phospholipid bilayers. We believe it is very likely that merging of membranes proceeds via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets that expands radially into an hemifusion diaphragm (HD) and subsequently open to a fusion pore. Although considered to be a key intermediate in fusion, direct experimental verification of this structure is difficult due to its transient nature. Using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (GUVs) containing phosphatidylserine and fluorescent virus derived transmembrane peptides or membrane proteins in the presence of divalent cations. Time-resolved imaging revealed that fusion was preceded by displacement of peptides and fluorescent lipid analogs from the GUV-GUV adhesion region. A detailed analysis of this area being several μm in size revealed that peptides were completely sequestered as expected for an HD. Lateral distribution of lipid analogs was consistent with formation of an HD but not with the presence of two adherent bilayers. Formation and size of the HD were dependent on lipid composition and peptide concentration.     

A solid-state NMR study of changes in lipid phase induced by membrane-fusogenic LV-peptides

Membrane fusion requires restructuring of lipid bilayers mediated by fusogenic membrane proteins. Peptides that correspond to natural transmembrane sequences or that have been designed to mimic them, such as low-complexity “Leu-Val” (LV) peptide sequences, can drive membrane fusion, presumably by disturbing the lipid bilayer structure. Here, we assess how peptides of different fusogenicity affect membrane structure using solid state NMR techniques. We find that the more fusogenic variants induce an unaligned lipid phase component and a large degree of phase separation as observed in ³¹P 2D spectra. The data support the idea that fusogenic peptides accumulate PE in a non-bilayer phase which may be critical for the induction of fusion.     

Structural and functional changes in a synthetic S5 segment of KvLQT1 channel as a result of a conserved amino acid substitution that occurs in LQT1 syndrome of human

Mutations in various voltage gated cardiac ion channels are the cause of different forms of long QT syndrome (LQTS), which is an inherited arrhythmic disorder marked as a prolonged QT interval on electrocardiogram. Of these LQTS1 is associated with mutations in the gene encoding KCNQ1 (KvLQT1) channel. One responsible mutation, G269S, in the S5 segment of KvLQT1, that affects the proper expression and function of channel protein leads to LQTS1. Our objective was to study how G269S mutation interferes with the structure and function of a synthetic S5 segment of KvLQT1 channel. One wild type 22-residue peptide and another mutant peptide of the same length with G269S mutation, derived from the S5 segment were synthesized and labeled with fluorescent probes. The mutant peptide exhibited lower affinity towards phospholipid vesicles as compared to the wild type peptide and showed impaired assembly and localization onto the lipid vesicles as evidenced by membrane-binding, energy transfer and proteolytic cleavage experiments. Loss in the helical content of S5 mutant peptide in membrane-mimetic environments was observed. Furthermore, it was observed that G269S mutation significantly inhibited the ability of S5 peptide to permeabilize the lipid vesicles. The present studies show the basis of change in function of the selected S5 segment as a result of G269S mutation which is associated with LQT1 syndrome. We speculate that the structural and functional changes related to the glycine to serine amino acid substitution in the S5 segment may also influence the activity of the whole KvLQT1 channel.     

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg²⁺ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg²⁺ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg²⁺ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the γ-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.     

P4 ATPases - Lipid flippases and their role in disease

P4 ATPases (type 4 P-type ATPases) are multispan transmembrane proteins that have been implicated in phospholipid translocation from the exoplasmic to the cytoplasmic leaflet of biological membranes. Studies in Saccharomyces cerevisiae have indicated that P4 ATPases are important in vesicle biogenesis and are required for vesicular trafficking along several intracellular vesicular transport routes. Although little is known about mammalian P4 ATPases, some members of this subfamily appear to be associated with human disease or mouse pathophysiology. ATP8B1, a phosphatidylserine translocase, is the most extensively studied mammalian P4 ATPase. This protein is important for maintaining the detergent resistant properties of the apical membrane of the hepatocyte. Mutations in ATP8B1 give rise to severe liver disease. Furthermore, a role for Atp8b3 in mouse sperm cell capacitation has been suggested, whereas deficiency of Atp10a and Atp10d leads to insulin resistance and obesity in mice. Here we review the present status on the pathophysiological consequences of P4 ATPase deficiency.     

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.     

Taenia solium: identification and preliminary characterization of a lipid binding protein with homology to the SEC14 catalytic domain

The objective of this work is to identify proteins of the human and porcine parasite, Taenia solium, which may be exploited for control of the parasite. Through screening a cDNA library of T. solium metacestodes, we have identified a novel Sec-14-like Taenia lipid-binding protein that may play an important role in membrane trafficking. The Sec14-like sequence is a single copy gene, encoding a putative polypeptide of 320 amino acids and 36.1 kDa (sec14Tsol protein). Secondary amino acid structural analysis suggested that the sec14Tsol protein might contain two distinct structural domains, an amino-terminal alpha-helix rich domain and a mixed alpha-helix/beta-stand carboxy-terminal zone, showing homology with the conserved SEC14 domain found in a great number of proteins that bind lipids, as the regulators of membrane trafficking between Golgi membrane bilayers. Significantly, therefore, in a phosphoinositide-binding assay, sec14Tsol purified recombinant protein specifically interacted with important lipid regulators of membrane trafficking, with a preference for PI(3)P(2), PI(3,4)P(2), PI(4,5)P(2) and phosphatidic acid. Moreover, the sec14Tsol protein was localized in the Golgi apparatus of transfected cells and in the spiral canal region of T. solium metacestode tegument. As sec14Tsol protein may play an important role in membrane trafficking, its demonstrated localisation in the intact parasite tegument suggests its involvement in the function of the tegument and thus perhaps interaction with the host.     

Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b-/- mouse model of Wilson's disease

Cellular copper overload as found in Wilson's disease may disturb mitochondrial function and integrity. Atp7b^−/− mice accumulate copper in the liver and serve as an animal model for this inherited disease. The molecular mechanism of copper toxicity in hepatocytes is poorly understood. Total mitochondrial lipids from liver of wild-type mice were subjected to oxidative stress by the Cu²⁺/H₂O₂/ascorbate system. Phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were detected as cardiolipin fragmentation products by thin-layer chromatography combined with MALDI-TOF mass spectrometry in oxidized samples, but not in unperturbed ones. The formation of PA and PHA in copper-treated model membrane correlated well with the decrease of cardiolipin. Mitochondrial lipids from Atp7b^−/− mice of different age were analyzed for the presence of PA. While 32-weeks old wild-type (control) and Atp7b^−/− mice did not show any PA, there was a steady increase in the amount of this lipid in Atp7b^−/− mice in contrast to control with increasing age. Hepatocytes from elder Atp7b^−/−mice contained morphologically changed mitochondria unlike cells from wild-type animals of the same age. We concluded that free-radical fragmentation of cardiolipin with the formation of PA is a likely mechanism that damages mitochondria under conditions of oxidative stress due to copper overload. Our findings are relevant for better understanding of molecular mechanisms for liver damage found in Wilson's disease.     

A retrospective: Use of Escherichia coli as a vehicle to study phospholipid synthesis and function

Although the study of individual phospholipids and their synthesis began in the 1920s first in plants and then mammals, it was not until the early 1960s that Eugene Kennedy using Escherichia coli initiated studies of bacterial phospholipid metabolism. With the base of information already available from studies of mammalian tissue, the basic blueprint of phospholipid biosynthesis in E. coli was worked out by the late 1960s. In 1970s and 1980s most of the enzymes responsible for phospholipid biosynthesis were purified and many of the genes encoding these enzymes were identified. By the late 1990s conditional and null mutants were available along with clones of the genes for every step of phospholipid biosynthesis. Most of these genes had been sequenced before the complete E. coli genome sequence was available. Strains of E. coli were developed in which phospholipid composition could be changed in a systematic manner while maintaining cell viability. Null mutants, strains in which phospholipid metabolism was artificially regulated, and strains synthesizing foreign lipids not found in E. coli have been used to this day to define specific roles for individual phospholipid. This review will trace the findings that have led to the development of E. coli as an excellent model system to study mechanisms underlying the synthesis and function of phospholipids that are widely applicable to other prokaryotic and eukaryotic systems. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10⁵ FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm² within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.     

Interaction with membrane mimics of transmembrane fragments 16 and 17 from the human multidrug resistance ABC transporter 1 (hMRP1/ABCC1) and two of their tryptophan variants

The human multidrug resistance-associated protein 1 (hMRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily. Together with P-glycoprotein (ABCB1) and the breast cancer resistance protein (BCRP/ABCG2), hMRP1 confers resistance to a large number of structurally diverse drugs. The current topological model of hMRP1 includes two cytosolic nucleotide-binding domains and 17 putative transmembrane (TM) helices forming three membrane-spanning domains. Mutagenesis and labeling studies have shown TM16 and TM17 to be important for function. We characterized the insertion of the TM16 fragment into dodecylphosphocholine (DPC) or n-dodecyl-β-d-maltoside (DM) micelles as membrane mimics and extended our previous work on TM17 (Vincent et al., 2007, Biochim. Biophys. Acta 1768, 538). We synthesized TM16 and TM17, with the Trp residues, W1198 in TM16 and W1246 in TM17, acting as an intrinsic fluorescent probe, and TM16 and TM17 Trp variants, to probe different positions in the peptide sequence. We assessed the interaction of peptides with membrane mimics by evaluating the increase in fluorescence intensity resulting from such interactions. In all micelle-bound peptides, the tryptophan residue appeared to be located, on average, in the head group micelle region, as shown by its fluorescence spectrum. Each tryptophan residue was partially accessible to both acrylamide and the brominated acyl chains of two DM analogs, as shown by fluorescence quenching. Tryptophan fluorescence lifetimes were found to depend on the position of the tryptophan residue in the various peptides, probably reflecting differences in local structures. Far UV CD spectra showed that TM16 contained significant β-strand structures. Together with the high Trp correlation times, the presence of these structures suggests that TM16 self-association may occur at the interface. In conclusion, this experimental study suggests an interfacial location for both TM16 and TM17 in membrane mimics. In terms of overall hMRP1 structure, the experimentally demonstrated amphipathic properties of these TM are consistent with a role in the lining of an at least partly hydrophilic transport pore, as suggested by the currently accepted structural model, the final structure being modified by interaction with other TM helices.