Lipid Binding of the Amphipathic Helix Serving as Membrane Anchor of Pestivirus Glycoprotein Erns

Pestiviruses express a peculiar protein named E^rns representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of E^rns from the infected cell. Retention/secretion of E^rns is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane. Here we present results of experiments conducted with a lipid vesicle sedimentation assay able to separate lipid-bound from unbound protein dissolved in the water phase. Using this technique we show that a protein composed of tag sequences and the carboxyterminal 65 residues of E^rns binds specifically to membrane vesicles with a clear preference for compositions containing negatively charged lipids. Mutations disturbing the helical folding and/or amphipathic character of the anchor as well as diverse truncations and exchange of amino acids important for intracellular retention of E^rns had no or only small effects on the proteins membrane binding. This result contrasts the dramatically increased secretion rates observed for E^rns proteins with equivalent mutations within cells. Accordingly, the ratio of secreted versus cell retained E^rns is not determined by the lipid affinity of the membrane anchor.     

Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry

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Highlights? Structure of the major antigenically dominant protein of BVDV ? The overall fold of BVDV E2 shows no similarity to the class II fusion proteins ? At low pH, BVDV E2 N-terminal domain is disordered ? Entry mechanism of BVDV is probably applicable to hepatitis C virus     

Role of Amphipathic Helix of a Herpesviral Protein in Membrane Deformation and T Cell Receptor Downregulation

Lipid rafts are membrane microdomains that function as platforms for signal transduction and membrane trafficking. Tyrosine kinase interacting protein (Tip) of T lymphotropic Herpesvirus saimiri (HVS) is targeted to lipid rafts in T cells and downregulates TCR and CD4 surface expression. Here, we report that the membrane-proximal amphipathic helix preceding Tip''s transmembrane (TM) domain mediates lipid raft localization and membrane deformation. In turn, this motif directs Tip''s lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation.     

Membrane Association via an Amino-terminal Amphipathic Helix Is Required for the Cellular Organization and Function of RNase II

The subcellular localization of the exoribonuclease RNase II is not known despite the advanced biochemical characterization of the enzyme. Here we report that RNase II is organized into cellular structures that appear to coil around the Escherichia coli cell periphery and that RNase II is associated with the cytoplasmic membrane by its amino-terminal amphipathic helix. The helix also acts as an autonomous transplantable membrane binding domain capable of directing normally cytoplasmic proteins to the membrane. Assembly of the organized cellular structures of RNase II required the RNase II amphipathic membrane binding domain. Co-immunoprecipitation of the protein from cell extracts indicated that RNase II interacts with itself. The RNase II self-interaction and the ability of the protein to assemble into organized cellular structures required the membrane binding domain. The ability of RNase II to maintain cell viability in the absence of the exoribonuclease polynucleotide phosphorylase was markedly diminished when the RNase II cellular structures were lost due to changes in the amphipathicity of the amino-terminal helix, suggesting that membrane association and assembly of RNase II into organized cellular structures play an important role in the normal function of the protein within the bacterial cell.     

Membrane Tethering of SepF,a Membrane Anchor for the Mycobacterium tuberculosis Z-ring

Bacterial cell division begins with the formation of the Z-ring via polymerization of FtsZ and the localization of Z-ring beneath the inner membrane through membrane anchors. In Mycobacterium tuberculosis (Mtb), SepF is one such membrane anchor, but our understanding of the underlying mechanism is very limited. Here we used molecular dynamics simulations to characterize how SepF itself, a water-soluble protein, tethers to acidic membranes that mimic the Mtb inner membrane. In addition to an amphipathic helix (residues 1–12) at the N-terminus, membrane binding also occurs through two stretches of positively charged residues (Arg27-Arg37 and Arg95-Arg107) in the long linker preceding the FtsZ-binding core domain (residues 128–218). The additional interactions via the disordered linker stabilize the membrane tethering of SepF, and keep the core domain of SepF and hence the attached Z-ring close to the membrane. The resulting membrane proximity of the Z-ring in turn enables its interactions with and thus recruitment of two membrane proteins, FtsW and CrgA, at the late stage of cell division.     

Coemergence of the Amphipathic Helix on Ameloblastin With Mammalian Prismatic Enamel

To investigate correlation between the ameloblastin (Ambn) amino acid sequence and the emergence of prismatic enamel, a notable event in the evolution of ectodermal hard tissues, we analyzed Ambn sequences of 53 species for which enamel microstructures have been previously reported. We found that a potential amphipathic helix (AH) within the sequence encoded by Exon 5 of Ambn appeared in species with prismatic enamel, with a few exceptions. We studied this correlation by investigating synthetic peptides from different species. A blue shift in fluorescence spectroscopy suggested that the peptides derived from mammalian Ambn interacted with liposomes. A downward shift at 222 nm in circular dichroism spectroscopy of the peptides in the presence of liposomes suggested that the peptides of mammals with prismatic enamel underwent a transition from disordered to helical structure. The peptides of species without prismatic enamel did not show similar secondary structural changes in the presence of liposomes. Peptides of mammals with prismatic enamel caused liposome leakage and inhibited LS8 and ALC cell spreading regulated by full-length Ambn. RT-PCR showed that AH is involved in Ambn’s regulation of cell polarization genes: Vangl2, Vangl1, Prickle1, ROCK1, ROCK2, and Par3. Our comprehensive sequence analysis clearly demonstrates that AH motif is closely related to the emergence of enamel prismatic structure, providing insight into the evolution of complex enamel microstructure. We speculate that the AH motif evolved in mammals to interact with cell membrane, triggering signaling pathways required for specific changes in cell morphology associated with the formation of enamel prismatic structure.     

The Atlastin C-terminal Tail Is an Amphipathic Helix That Perturbs the Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion

Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.     

Cholesterol Binds the Amphipathic Helix of IFITM3 and Regulates Antiviral Activity

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.     

Inactivation of the RNase Activity of Glycoprotein Erns of Classical Swine Fever Virus Results in a Cytopathogenic Virus

Envelope glycoprotein E^rns of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E^rns are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E^rns was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E^rns, the mutated proteins had lost their RNase activity. The mutated proteins reacted with E^rns-specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E^rns. This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E^rns (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E^rns gene and by neutralizing polyclonal antibodies directed against E^rns, indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E^rns plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.Classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV), and border disease virus belong to the genus Pestivirus within the family Flaviviridae (10). The viruses are structurally, antigenically, and genetically closely related. BVDV and border disease virus can infect ruminants and pigs. CSFV infections are restricted to pigs (6). Pestiviruses are small, enveloped, positive-stranded RNA viruses (23). The genome of pestiviruses varies in length from 12.5 to 16.5 kb (1, 2, 7, 17, 19, 25, 26, 28, 32) and contains a single large open reading frame (ORF) (1, 7, 8, 17, 26). The ORF is translated into a polyprotein which is processed into mature proteins by viral and host cell proteases (30). The envelope of the pestivirus virion contains three glycoproteins, E^rns, E1, and E2 (35). Animals infected with pestiviruses raise antibodies against at least two viral glycoproteins, namely, E^rns and E2 (16, 34, 42). Inhibition studies with E2 and E^rns produced in insect cells showed that both envelope proteins are indispensable for viral attachment and entry of pestiviruses into susceptible cells (13). In the virion, E^rns is present as a homodimer with a molecular mass of about 100 kDa (35). E^rns lacks a membrane anchor, and association with the envelope is accomplished by an as-yet-unknown mechanism. Significant amounts of E^rns are secreted from infected cells (30). A unique feature is that E^rns, besides being an envelope protein, possesses RNase activity (12, 31). E^rns belongs to the family of extracellular RNases consisting of several fungal (e.g., RNase T₂ and Rh) and plant (e.g., S glycoproteins of Nicotiana alata) RNases (12, 31). These RNases contain two homologous regions of 8 amino acids each which are spaced by 38 (E^rns) nonhomologous amino acids and which form the RNase active site. Histidine residues in both regions appear to be essential for RNase catalysis (15).The role of this RNase activity in the replication of pestiviruses or in the pathogenesis of a pestivirus infection is an interesting issue that, as yet, has not been studied. The availability of a recently generated infectious DNA copy of CSFV strain C (24) has given us the opportunity to study the effect of defined mutations in a pestivirus genome. In this paper, we report the inactivation of the RNase activity of E^rns by mutagenesis. To characterize the mutated proteins, we produced large amounts of them in insect cells (12). By reverse genetics, we generated an RNase-negative CSFV recombinant. The effect of the inactivation of the RNase activity of E^rns on the replication of CSFV in vitro was studied.     

Structure of Transmembrane Helix 8 and Possible Membrane Defects in CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that regulates the flow of anions across epithelia. Mutations in CFTR cause cystic fibrosis. CFTR belongs to the ATP-binding cassette transporter superfamily, and gating is controlled by phosphorylation and ATP binding and hydrolysis. Recently obtained ATP-free and ATP-bound structures of zebrafish CFTR revealed an unwound segment of transmembrane helix (TM) 8, which appears to be a unique feature of CFTR not present in other ATP-binding cassette transporter structures. Here, using μs-long molecular dynamics simulations, we investigate the interactions formed by this TM8 segment with nearby helices in both ATP-free and ATP-bound states. We highlight ATP-dependent interactions as well as the structural role of TM8 in maintaining the functional architecture of the pore via interactions common to both the ATP-bound and ATP-free state. The results of the molecular dynamics simulations are discussed in the context of the gating mechanism of CFTR.     

Phosphatidylserine Asymmetry Promotes the Membrane Insertion of a Transmembrane Helix

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer bilayer leaflets. A lipid of special interest in eukaryotic membranes is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM, but PS redistributes to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, the influence of PS asymmetry on membrane protein structure and folding are poorly understood. The pH low insertion peptide (pHLIP) adsorbs to the membrane surface at a neutral pH, but it inserts into the membrane at an acidic pH. We have previously observed that in symmetric vesicles, PS affects the membrane insertion of pHLIP by lowering the pH midpoint of insertion. Here, we studied the effect of PS asymmetry on the membrane interaction of pHLIP. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa Fluor 568 fluorophore as a new probe to quantify PS asymmetry. We observed that the membrane insertion of pHLIP was promoted by the asymmetric distribution of negatively charged PS, which causes a surface charge difference between bilayer leaflets. Our results indicate that lipid asymmetry can modulate the formation of an α-helix on the membrane. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.     

Understanding the Role of Amphipathic Helices in N-BAR Domain Driven Membrane Remodeling

Endophilin N-BAR (N-terminal helix and Bin/amphiphysin/Rvs) domain tubulates and vesiculates lipid membranes in vitro via its crescent-shaped dimer and four amphipathic helices that penetrate into membranes as wedges. Like F-BAR domains, endophilin N-BAR also forms a scaffold on membrane tubes. Unlike F-BARs, endophilin N-BARs have N-terminal H0 amphipathic helices that are proposed to interact with other N-BARs in oligomer lattices. Recent cryo-electron microscopy reconstructions shed light on the organization of the N-BAR lattice coats on a nanometer scale. However, because of the resolution of the reconstructions, the precise positioning of the amphipathic helices is still ambiguous. In this work, we applied a coarse-grained model to study various membrane remodeling scenarios induced by endophilin N-BARs. We found that H0 helices of N-BARs prefer to align in an antiparallel manner at two ends of the protein to form a stable lattice. The deletion of H0 helices causes disruption of the lattice. In addition, we analyzed the persistence lengths of the protein-coated tubes and found that the stiffness of endophilin N-BAR-coated tubules qualitatively agrees with previous experimental work studying N-BAR-coated tubules. Large-scale simulations on membrane liposomes revealed a systematic relation between H0 helix density and local membrane curvature fluctuations. The data also suggest that the H0 helix is required for BARs to form organized structures on the liposome, further illustrating its important function.     

A Highly Tilted Membrane Configuration for the Prefusion State of Synaptobrevin

The SNARE complex plays a vital role in vesicle fusion arising during neuronal exocytosis. Key components in the regulation of SNARE complex formation, and ultimately fusion, are the transmembrane and linker regions of the vesicle-associated protein, synaptobrevin. However, the membrane-embedded structure of synaptobrevin in its prefusion state, which determines its interaction with other SNARE proteins during fusion, is largely unknown. This study reports all-atom molecular-dynamics simulations of the prefusion configuration of synaptobrevin in a lipid bilayer, aimed at characterizing the insertion depth and the orientation of the protein in the membrane, as well as the nature of the amino acids involved in determining these properties. By characterizing the structural properties of both wild-type and mutant synaptobrevin, the effects of C-terminal additions on tilt and insertion depth of membrane-embedded synaptobrevin are determined. The simulations suggest a robust, highly tilted state for membrane-embedded synaptobrevin with a helical connection between the transmembrane and linker regions, leading to an apparently new characterization of structural elements in prefusion synaptobrevin and providing a framework for interpreting past mutation experiments.     

Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic proteinN-glycosylation are synthesized by a multistep pathway in which the biosyntheticlipid intermediate Man₅GlcNAc₂-PP-dolichol (M5-DLO) flips from the cytoplasmicto the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 isintimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLOflippase, but because biochemical tests challenged this assignment, the function of Rft1 remainsobscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis invivo in the complete absence of the protein. Rft1 is essential for yeast viability, and noRft1-null organisms are currently available. Here, we exploited Trypanosoma brucei(Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report thatTbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels ofmDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity.Remarkably, the mutant cells have 30–100-fold greater steady-state levels of M5-DLO thanwild-type cells. All N-glycans in the TbRft1-null cells originate from mDLOindicating that the M5-DLO excess is not available for glycosylation. These results suggest thatrather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by anothermechanism, possibly by acting as an M5-DLO chaperone.     

A Highly Tilted Membrane Configuration for the Prefusion State of Synaptobrevin

The SNARE complex plays a vital role in vesicle fusion arising during neuronal exocytosis. Key components in the regulation of SNARE complex formation, and ultimately fusion, are the transmembrane and linker regions of the vesicle-associated protein, synaptobrevin. However, the membrane-embedded structure of synaptobrevin in its prefusion state, which determines its interaction with other SNARE proteins during fusion, is largely unknown. This study reports all-atom molecular-dynamics simulations of the prefusion configuration of synaptobrevin in a lipid bilayer, aimed at characterizing the insertion depth and the orientation of the protein in the membrane, as well as the nature of the amino acids involved in determining these properties. By characterizing the structural properties of both wild-type and mutant synaptobrevin, the effects of C-terminal additions on tilt and insertion depth of membrane-embedded synaptobrevin are determined. The simulations suggest a robust, highly tilted state for membrane-embedded synaptobrevin with a helical connection between the transmembrane and linker regions, leading to an apparently new characterization of structural elements in prefusion synaptobrevin and providing a framework for interpreting past mutation experiments.     

The C-terminal Amphipathic Helix of Carboxypeptidase E Mediates Export from the ER and Secretion via Lysosomes

Carboxypeptidase E (CPE), an essential enzyme in the biosynthetic production line of most peptide hormones and neuropeptides, is predominantly expressed in endocrine tissues and in the nervous system. CPE is active in acidic environments where it cleaves the C’-terminal basic residues of peptide precursors to generate their bioactive form. Consequently, this highly conserved enzyme regulates numerous fundamental biological processes. Here, we combined live-cell microscopy and molecular analysis to examine the intracellular distribution and secretion dynamics of fluorescently tagged CPE. We show that, in non-endocrine cells, tagged-CPE is a soluble luminal protein that is efficiently exported from the ER via the Golgi apparatus to lysosomes. The C’-terminal conserved amphipathic helix serves as a lysosomal and secretory granule targeting and a secretion motif. Following secretion, CPE may be reinternalized into the lysosomes of neighboring cells.     

Delivery of a Secreted Soluble Protein to the Vacuole via a Membrane Anchor

To further understand how membrane proteins are sorted in the secretory system, we devised a strategy that involves the expression of a membrane-anchored yeast invertase in transgenic plants. The construct consisted of a signal peptide followed by the coding region of yeast invertase and the transmembrane domain and cytoplasmic tail of calnexin. The substitution of a lysine near the C terminus of calnexin with a glutamic acid residue ensured progression through the secretory system rather than retention in or return to the endoplasmic reticulum. In the transformed plants, invertase activity and a 70-kD cross-reacting protein were found in the vacuoles. This yeast invertase had plant-specific complex glycans, indicating that transport to the vacuole was mediated by the Golgi apparatus. The microsomal fraction contained a membrane-anchored 90-kD cross-reacting polypeptide, but was devoid of invertase activity. Our results indicate that this membrane-anchored protein proceeds in the secretory system beyond the point where soluble proteins are sorted for secretion, and is detached from its membrane anchor either just before or just after delivery to the vacuole.     

Lysosomal Targeting of the CLN7 Membrane Glycoprotein and Transport Via the Plasma Membrane Require a Dileucine Motif

CLN7 is a polytopic lysosomal membrane protein deficient in variant late infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder. In this study fluorescence protease protection assays and mutational analyses revealed the N‐ and C‐terminal tails of CLN7 in the cytosol and two N‐glycosylation sites at N371 and N376. Both partially and non‐glycosylated CLN7 were correctly transported to lysosomes. To identify lysosomal targeting motifs, we generated CD4‐chimera fused to the N‐ and C‐terminal domains of CLN7. Lysosomal localization of the chimeric proteins requires a consensus acidic dileucine‐based motif in the N‐terminus and two tandem tyrosine‐based signals in the C‐terminus. Mutation of these sorting motifs resulted in cell surface redistribution of CD4 chimeras. However, the dileucine‐based motif is of critical importance for lysosomal localization of the full‐length CLN7 in different cell lines. Cell surface biotinylation revealed that at equilibrium 22% of total CLN7 is localized at the plasma membrane. Mutation of the dileucine motif or the co‐expression of dominant‐negative mutant dynamin K44A led to a further increase of CLN7 at the plasma membrane. Our data demonstrate that CLN7 contains several cytoplasmic lysosomal targeting signals of which the N‐terminal dileucine‐based motif is required for the predominant lysosomal targeting along the indirect pathway and clathrin‐mediated endocytosis of CLN7.     

Location of the Glycoprotein in the Membrane of Sindbis Virus

SINDBIS virus, which is transmitted by arthropods, consists of a nucleoprotein core within a lipid-containing envelope. Its components assemble at a cellular membrane and virus particles form by an outfolding of this membrane. Thus, such viruses provide useful systems for studies of the structure and synthesis of membranes. The Sindbis virus particle contains only two proteins, one associated with the viral envelope and the other with the viral RNA in the core, or nucleocapsid¹. The protein associated with the membrane is a glycoprotein, whereas the core protein contains no carbohydrate². The exact location of the glycoprotein within the viral envelope has not been determined, nor has information been obtained about the function of the carbohydrate in the virion. The results described here indicate that the spikes which cover the surface of the virion are glycoprotein in nature.