The C-terminal transmembrane domain of human phospholipid scramblase 1 is essential for the protein flip-flop activity and Ca2+-binding

Human phospholipid scramblase 1 (SCR) is a 318 amino acid protein that was originally described as catalyzing phospholipid transbilayer (flip-flop) motion in plasma membranes in a Ca²⁺-dependent, ATP-independent way. Further studies have suggested an intranuclear role for this protein in addition. A putative transmembrane domain located at the C terminus (aa 291–309) has been related to the flip-flop catalysis. In order to clarify the role of the C-terminal region of SCR, a mutant was produced (SCRΔ) in which the last 28 amino acid residues were lacking, including the α-helix. SCRΔ had lost the scramblase activity and its affinity for Ca²⁺ was decreased by one order of magnitude. Fluorescence and IR spectroscopic studies revealed that the C-terminal region of SCR was essential for the proper folding of the protein. Moreover, it was found that Ca²⁺ exerted an overall destabilizing effect on SCR, which might facilitate its binding to membranes.     

Farnesylation of the SNARE protein Ykt6 increases its stability and helical folding

The evolutionarily conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in the fusion of vesicles with their target membranes. While most SNAREs are permanently anchored to membranes by their transmembrane domains, the vesicle-associated SNARE Ykt6 has been found both in soluble and in membrane-bound pools. The R-SNARE Ykt6 is thought to mediate interactions between various Q-SNAREs by a reversible membrane-targeting cycle. Membrane attachment of Ykt6 is achieved by its C-terminal prenylation and palmitoylation motif succeeding the SNARE motif. In this study, we have analyzed full-length farnesylated Ykt6 from yeast and humans by biochemical and structural means. In vitro farnesylation of the C-terminal CAAX box of recombinant full-length Ykt6 resulted in stabilization of the native protein and a more compactly folded structure, as shown by size exclusion chromatography and limited proteolysis. Circular dichroism spectroscopy indicated a specific increase in the helical content of the farnesylated Ykt6 compared to the nonlipidated form or the single-longin domain, which correlated with a marked increase in stability as observed by heat denaturation experiments. Although highly soluble, farnesylated Ykt6 is capable of lipid membrane binding independent of the membrane charge, as shown by surface plasmon resonance. The crystal structure of the N-terminal longin domain of yeast Ykt6 (1-140) was determined at 2.5 Å resolution. As similarly found in a previous NMR structure, the Ykt6 longin domain contains a hydrophobic patch at its surface that may accommodate the lipid moiety. In the crystal structure, this hydrophobic surface is buried in a crystallographic homomeric dimer interface. Together, these observations support a previously suggested closed conformation of cytosolic Ykt6, where the C-terminal farnesyl moiety folds onto a hydrophobic groove in the N-terminal longin domain.     

A Cholesterol Recognition Motif in Human Phospholipid Scramblase 1

Human phospholipid scramblase 1 (SCR) catalyzes phospholipid transmembrane (flip-flop) motion. This protein is assumed to bind the membrane hydrophobic core through a transmembrane domain (TMD) as well as via covalently bound palmitoyl residues. Here, we explore the possible interaction of the SCR TMD with cholesterol by using a variety of experimental and computational biophysical approaches. Our findings indicate that SCR contains an amino acid segment at the C-terminal region that shows a remarkable affinity for cholesterol, although it lacks the CRAC sequence. Other 3-OH sterols, but not steroids lacking the 3-OH group, also bind this region of the protein. The newly identified cholesterol-binding region is located partly at the C-terminal portion of the TMD and partly in the first amino acid residues in the SCR C-terminal extracellular coil. This finding could be related to the previously described affinity of SCR for cholesterol-rich domains in membranes.     

A Cholesterol Recognition Motif in Human Phospholipid Scramblase 1

Human phospholipid scramblase 1 (SCR) catalyzes phospholipid transmembrane (flip-flop) motion. This protein is assumed to bind the membrane hydrophobic core through a transmembrane domain (TMD) as well as via covalently bound palmitoyl residues. Here, we explore the possible interaction of the SCR TMD with cholesterol by using a variety of experimental and computational biophysical approaches. Our findings indicate that SCR contains an amino acid segment at the C-terminal region that shows a remarkable affinity for cholesterol, although it lacks the CRAC sequence. Other 3-OH sterols, but not steroids lacking the 3-OH group, also bind this region of the protein. The newly identified cholesterol-binding region is located partly at the C-terminal portion of the TMD and partly in the first amino acid residues in the SCR C-terminal extracellular coil. This finding could be related to the previously described affinity of SCR for cholesterol-rich domains in membranes.     

Characterization of the interaction of diacylglycerol acyltransferase-2 with the endoplasmic reticulum and lipid droplets

Acyl CoA:diacylglycerol acyltransferase-2 (DGAT2) is an integral membrane protein that catalyzes the synthesis of triacylglycerol (TG). DGAT2 is present in the endoplasmic reticulum (ER) and also localizes to lipid droplets when cells are stimulated with oleate. Previous studies have shown that DGAT2 can interact with membranes and lipid droplets independently of its two transmembrane domains, suggesting the presence of an additional membrane binding domain. In order to identify additional membrane binding regions, we confirmed that DGAT2 has only two transmembrane domains and demonstrated that the loop connecting them is present in the ER lumen. Increasing the length of this short loop from 5 to 27 amino acids impaired the ability of DGAT2 to localize to lipid droplets. Using a mutagenesis approach, we were able to identify a stretch of amino acids that appears to have a role in binding DGAT2 to the ER membrane. Our results confirm that murine DGAT2 has only two transmembrane domains but also can interact with membranes via a previously unidentified helical domain containing its active site.     

Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1

Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2-81). Vpu(2-37) encompasses the N-terminal transmembrane alpha-helix; Vpu(2-51) spans the N-terminal transmembrane helix and the first cytoplasmic alpha-helix; Vpu(28-81) includes the entire cytoplasmic domain containing the two C-terminal amphipathic alpha-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane alpha-helix. The C-terminal alpha-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

Carboxypeptidase E, a prohormone sorting receptor, is anchored to secretory granules via a C-terminal transmembrane insertion.

Carboxypeptidase E (CPE) is a sorting receptor that directs the prohormone pro-opiomelanocortin (POMC) to the regulated secretory pathway, and is also a prohormone processing enzyme in neuro/endocrine cells. It has been suggested that the 25 C-terminal amino acids are necessary for the binding of CPE to secretory granule membranes, but its orientation in the membrane is not known. In this study, we examined the structure and orientation of the membrane-binding domain at the C-terminus of CPE. In vitro experiments using model membranes demonstrated that the last 22 amino acids of CPE (CP peptide) insert in a shallow orientation into lipid bilayers at low pH. Circular dichroism analysis indicated that the CP peptide adopts a partial alpha-helical configuration at low pH, and helix content increases when it is bound to lipid. Protease protection experiments, immunolabeling, and immunoisolation of intact secretory granules with a C-terminal antibody revealed a cytoplasmic domain in CPE, consistent with a transmembrane orientation of this protein. We conclude that the membrane-binding domain of CPE must adopt an alpha-helical configuration to bind to lipids, and that CPE may require another integral membrane "chaperone" protein to insert through the lipid bilayer in a transmembrane fashion.     

The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin

Gram-negative bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a soluble catalytic domain. The crystal structure of the soluble domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been determined crystallographically and refined to 1.4 Å resolution. The structure reveals a core hydrolase fold similar to that of alkaline phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is observed. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chemistry; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.     

Structure of the C-terminal domain of the pro-apoptotic protein Hrk and its interaction with model membranes

The protein harakiri (Hrk) is a pro-apoptotic BH3-only protein which belongs to the Bcl-2 family. Hrk appears associated to the mitochondrial outer membrane, apparently by a putative transmembrane domain, where it exerts its function. In this work we have identified a 27mer peptide supposed to be the putative membrane domain of the protein at the C-terminal region, and used infrared and fluorescence spectroscopies to study its secondary structure as well as to characterize its effect on the physical properties of phospholipid model membranes. The results presented here showed that the C-terminal region of Hrk adopts a predominantly α-helical structure whose proportion and destabilization capability varied depending on phospholipid composition. Moreover it was found that the orientation of the α-helical component of this C-terminal Hrk peptide was nearly perpendicular to the plane of the membrane. These results indicate that this domain is able of inserting into membranes, where it adopts a transmembrane α-helical structure as well as it considerably perturbs the physical properties of the membrane.     

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Eukaryotic P-type plasma membrane H⁺-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H⁺-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H⁺-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules.     

Sequence-independent targeting of transmembrane proteins synthesized within vaccinia virus factories to nascent viral membranes

The primary membrane of vaccinia virus, as well as those of other poxviruses, forms within a discrete cytoplasmic factory region. We recently determined the existence of an operative pathway from the endoplasmic reticulum within the virus factory to nascent viral membranes and demonstrated that a viral protein could be diverted from this pathway to Golgi membranes by the addition of COPII-binding sites (M. Husain, A. S. Weisberg, and B. Moss, Proc. Natl. Acad. Sci. USA, 103:19506-19511, 2006). Here we describe an investigation of the structural features that are required for transit of proteins to the viral membrane. Deletion of either the N-terminal domain or the C-terminal cytoplasmic tail from the conserved A9 protein did not prevent its incorporation into viral membranes, whereas deletion of the transmembrane domain resulted in its distribution throughout the cytoplasm. Nevertheless, replacement of the A9 transmembrane domain with the corresponding region of a nonpoxvirus transmembrane protein or of a vaccinia virus extracellular envelope protein allowed viral membrane targeting, indicating no requirement for a specific amino acid sequence. Remarkably, the epitope-tagged A9 transmembrane domain alone, as well as a heterologous transmembrane domain lacking a poxvirus sequence, was sufficient for viral membrane association. The data are consistent with a sequence-independent pathway in which transmembrane proteins that are synthesized within the virus factory and lack COPII or other binding sites that enable conventional endoplasmic reticulum exiting are incorporated into nascent viral membranes.     

Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer

Spontaneous membrane insertion and folding of beta-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either alpha-helical or beta-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane beta-barrel with an additional N-terminal alpha-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first beta-sheet secondary structure in aqueous solution, while the alpha-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial beta-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of beta-sheet and lower content of alpha-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of beta-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial beta-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target beta-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.     

The membrane environment modulates self-association of the human GpA TM domain—Implications for membrane protein folding and transmembrane signaling

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix-helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix-helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties.     

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling?

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB_1-23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB_1-23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.     

The calcium-binding C-terminal domain of Escherichia coli alpha-hemolysin is a major determinant in the surface-active properties of the protein

alpha-Hemolysin (HlyA) from Escherichia coli is a protein toxin (1024 amino acids) that targets eukaryotic cell membranes, causing loss of the permeability barrier. HlyA consists of two main regions, an N-terminal domain rich in amphipathic helices, and a C-terminal Ca(2+)-binding domain containing a Gly- and Asp-rich nonapeptide repeated in tandem 11-17 times. The latter is called the RTX domain and gives its name to the RTX protein family. It had been commonly assumed that membrane interaction occurred mainly if not exclusively through the amphipathic helix domain. However, we have cloned and expressed the C-terminal region of HlyA, containing the RTX domain plus a few stabilizing sequences, and found that it is a potent surface-active molecule. The isolated domain binds Ca(2+) with about the same affinity (apparent K(0.5) approximately 150 microM) as the parent protein HlyA, and Ca(2+) binding induces in turn a more compact folding with an increased proportion of beta-sheet structure. Both with and without Ca(2+) the C-terminal region of HlyA can interact with lipid monolayers spread at an air-water interface. However, the C-terminal domain by itself is devoid of membrane lytic properties. The present results can be interpreted in the light of our previous studies that involved in receptor binding a peptide in the C-terminal region of HlyA. We had also shown experimentally the distinction between reversible membrane adsorption and irreversible lytic insertion of the toxin. In this context, the present data allow us to propose that both major domains of HlyA are directly involved in membrane-toxin interaction, the nonapeptide repeat, calcium-binding RTX domain being responsible for the early stages of HlyA docking to the target membrane.     

Dimerization of the transmembrane domain of human tetherin in membrane mimetic environments

Tetherin/Bst-2 is a cell surface protein that can act as a restriction factor against a number of enveloped viruses, including HIV-1. It acts by tethering new virus particles to the host cell membrane, promoting their internalization and degradation. Tetherin is a type II membrane protein, with an N-terminal transmembrane domain, an extracellular coiled-coil domain, and a C-terminal GPI anchor. This double membrane anchor is important for anti-HIV activity, as is dimerization of the coiled-coil domain, but despite recent crystal structures of the coiled-coil ectodomains of human and mouse tetherin, the topology of tetherin with respect to host and viral membranes has yet to be determined. The tetherin transmembrane domain is also thought to mediate interactions with the HIV-1 encoded integral membrane protein Vpu, which is an antagonist of tetherin, through direct binding to the transmembrane region of Vpu. Using a combination of SDS-PAGE, size exclusion chromatography, and pyrene excimer fluorescence, we show that in the absence of the coiled-coil domain the transmembrane domain of human tetherin forms parallel homodimers in membrane mimetic environments. Transmembrane domain dimerization does not require disulfide bond formation and is favored in TFE, SDS micelles, and POPC liposomes. This observation has implications for functional models of tetherin, suggesting that both transmembrane domains in the dimeric molecule are inserted into the same lipid bilayer, rather than into opposing membranes.     

Characterization of the myristoyl lipid modification of membrane-bound GCAP-2 by H solid-state NMR spectroscopy

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca²⁺ sensor protein. It is responsible for the regulation of both isoforms of the transmembrane photoreceptor guanylate cyclase, a key enzyme of vertebrate phototransduction. GCAP-2 is N-terminally myristoylated and full activation of its target proteins requires the presence of this lipid modification. The structural role of the myristoyl moiety in the interaction of GCAP-2 with the guanylate cyclases and the lipid membrane is currently not well understood. In the present work, we studied the binding of Ca²⁺-free myristoylated and non-myristoylated GCAP-2 to phospholipid vesicles consisting of dimyristoylphosphatidylcholine or of a lipid mixture resembling the physiological membrane composition by a biochemical binding assay and ²H solid-state NMR. The NMR results clearly demonstrate the full-length insertion of the aliphatic chain of the myristoyl group into the membrane. Very similar geometrical parameters were determined from the ²H NMR spectra of the myristoyl group of GCAP-2 and the acyl chains of the host membranes, respectively. The myristoyl chain shows a moderate mobility within the lipid environment, comparable to the acyl chains of the host membrane lipids. This is in marked contrast to the behavior of other lipid-modified model proteins. Strikingly, the contribution of the myristoyl group to the free energy of membrane binding of GCAP-2 is only on the order of − 0.5 kJ/mol, and the electrostatic contribution is slightly unfavorable, which implies that the main driving forces for membrane localization arises through other, mainly hydrophobic, protein side chain-lipid interactions. These results suggest a role of the myristoyl group in the direct interaction of GCAP-2 with its target proteins, the retinal guanylate cyclases.     

Interaction of peptides spanning the transmembrane domain of caveolin‐1 with model membranes

Caveolin‐1 has an atypical membrane‐spanning domain comprising of 34 residues. Caveolin‐1 targets to lipid droplets under certain conditions, where they are involved in signaling and cholesterol balance. In the present study, membrane association of synthetic peptides corresponding to the membrane‐spanning domain of caveolin‐1 has been investigated to obtain an insight into the topology of transmembrane region in the lipid bilayer and the effect of truncations in this sequence, as observed in the targeting to lipid droplets, by using model membranes. Fluorescence studies revealed strong association of the peptide corresponding to the membrane‐spanning domain of caveolin‐1 with anionic lipids as compared with zwitterionic lipids, which is consistent with the location of this domain in the cytoplasmic side of the plasma membrane. Association of a short 9 residue peptide corresponding to the C‐terminus of caveolin‐1 membrane‐spanning domain with lipid vesicles revealed the importance of this region for association with model membranes. Our investigations indicate that the peptide corresponding to the membrane‐spanning domain of caveolin‐1 does not span the lipid bilayer. We propose that both caveolin scaffolding domain and transmembrane segment of caveolin‐1 contribute to the strong association with the plasma membrane rendering the protein highly detergent resistant. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction with membranes of the full C-terminal domain of protein NS4B from hepatitis C virus

Hepatitis C virus (HCV) NS4B protein is a transmembrane highly hydrophobic protein responsible for many key aspects of the viral replication process. The C-terminal part of NS4B is essential for replication and is a potential target for HCV replication inhibitors. In this work we have carried out a study of the binding to and interaction with model biomembranes of a peptide corresponding to the C-terminal domain of NS4B, NS4B(Cter). We show that NS4B(Cter) partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. NS4B(Cter) is located in a shallow position in the membrane but it is able to affect the lipid environment from the membrane surface down to the hydrophobic core. Our results identify the C-terminal region of the HCV NS4B protein as a membrane interacting domain, and therefore directly implicated in the HCV life cycle and possibly in the formation of the membranous web.