The Interaction between Influenza HA Fusion Peptide and Transmembrane Domain Affects Membrane Structure.
ABSTRACT: Viral glycoproteins, such as influenza hemagglutinin (HA) and human immunodeficiency virus gp41, are anchored by a single helical segment transmembrane domain (TMD) on the viral envelope membrane. The fusion peptides (FP) of the glycoproteins insert into the host membrane and initiate membrane fusion. Our previous study showed that the FP or TMD alone perturbs membrane structure. Interaction between the influenza HA FP and TMD has previously been shown, but its role is unclear. We used PC spin labels dipalmitoylphospatidyl-tempo-choline (on the headgroup), 5PC and 14PC (5-C and 14-C positions on the acyl chain) to detect the combined effect of FP-TMD interaction by titrating HA FP to TMD-reconstituted 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol)/cholesterol lipid bilayers using electron spin resonance. We found that the FP-TMD increases the lipid order at all positions, which has a greater lipid ordering effect than the sum of the FP or TMD alone, and this effect reaches deeper into the membranes. Although HA-mediated membrane fusion is pH dependent, this combined effect is observed at both pH 5 and pH 7. In addition to increasing lipid order, multiple components are found for 5PC at increased concentration of FP-TMD, indicating that distinct domains are induced. However, the mutation of Gly1 in the FP and L187 in the TMD eliminates the perturbations, consistent with their fusogenic phenotypes. Electron spin resonance on spin-labeled peptides confirms these observations. We suggest that this interaction may provide a driving force in different stages of membrane fusion: initialization, transition from hemifusion stalk to transmembrane contact, and fusion pore formation.
Project description:The HIV envelope glycoprotein mediates virus entry into target cells by fusing the virus lipid envelope with the cell membrane. This process requires large-scale conformational changes of the fusion protein gp41. Current understanding of the mechanisms with which gp41 induces membrane merger is limited by the fact that the hydrophobic N-terminal fusion peptide (FP) and C-terminal transmembrane domain (TMD) of the protein are challenging to characterize structurally in the lipid bilayer. Here we have expressed a gp41 construct that contains both termini, including the FP, the fusion peptide-proximal region (FPPR), the membrane-proximal external region (MPER), and the TMD. These hydrophobic domains are linked together by a shortened water-soluble ectodomain. We reconstituted this "short NC" gp41 into a virus-mimetic lipid membrane and conducted solid-state NMR experiments to probe the membrane-bound conformation and topology of the protein. 13C chemical shifts indicate that the C-terminal MPER-TMD is predominantly ?-helical, whereas the N-terminal FP-FPPR exhibits ?-sheet character. Water and lipid 1H polarization transfer to the protein revealed that the TMD is well-inserted into the lipid bilayer, whereas the FPPR and MPER are exposed to the membrane surface. Importantly, correlation signals between the FP-FPPR and the MPER are observed, providing evidence that the ectodomain is sufficiently collapsed to bring the N- and C-terminal hydrophobic domains into close proximity. These results support a hemifusion-like model of the short NC gp41 in which the ectodomain forms a partially folded hairpin that places the FPPR and MPER on the opposing surfaces of two lipid membranes.
Project description:BACKGROUND: To study the organization and interaction with the fusion domain (or fusion peptide, FP) of the transmembrane domain (TMD) of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion. RESULTS: The fluorescence and gel electrophoresis experiments revealed a tight self-assembly of TMD in the model membrane. A weak but non-random interaction between TMD and FP in the membrane was found. In the complex, the central TMD oligomer was packed by FP in an antiparallel fashion. FP insertion into the membrane was altered by binding to TMD. An infrared study exhibited an enhanced membrane perturbation by the complex formation. A model was built to illustrate the role of TMD in the late stages of influenza virus-mediated membrane fusion reaction. CONCLUSION: The TMD oligomer anchors the fusion protein in the membrane with minimal destabilization to the membrane. Upon associating with FP, the complex exerts a synergistic effect on the membrane perturbation. This effect is likely to contribute to the complete membrane fusion during the late phase of fusion protein-induced fusion cascade. The results presented in the work characterize the nature of the interaction of TMD with the membrane and TMD in a complex with FP in the steps leading to pore initiation and dilation during virus-induced fusion. Our data and proposed fusion model highlight the key role of TMD-FP interaction and have implications on the fusion reaction mediated by other type I viral fusion proteins. Understanding the molecular mechanism of membrane fusion may assist in the design of anti-viral drugs.
Project description:Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an ?-helical or ?-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the ?-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed.
Project description:Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the C-terminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus-cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.
Project description:Background:Phenylalanine (Phe) is involved in physiological and pathological processes in cell membranes in which expanded and condensed states coexist. In this direction, it was reported that surface hydration is important for the binding affinity of the amino acid which significantly perturbs 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer structure and morphology. A deeper insight showed that Phe inserts in DPPC monolayer defects as a monomer at pH 5 and forms aggregates that adsorb to the membrane surface generating a reconfiguration of the lipid arrangement in areas of higher packing. This new arrangement in the monolayer causes the reorientation of dipoles of lipid and water molecules which is congruent with the dehydration and surface tension changes reported above. With this background, this article studies the affinity of Phe in liquid-expanded 1,2-dimyristoyl-sn-glycero-3 phosphocholine (LE DMPC) and liquid-condensed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (LC DPPC) monolayers and their effects on membrane properties. Results:The adsorption of Phe can be described by a cooperative process in non-independent sites suggesting that Phe/lipid systems reorganize to form new structures at a high degree of coverage. Compressibility modulus and Brewster angle microscopy (BAM) images allow to propose that Phe causes a new phase in 1,2-dimyristoyl-sn-glycero-3 phosphocholine (DMPC) and DPPC. Conclusions:Phe imposes new arrangements in the lipid phase to form new structures with different compressibility behavior than lipid binary mixtures of DMPC and DPPC. Phe interaction with the LC and LE phases gives place to a process in which a synergistic effect between non-independent sites can be produced. These features of Phe/lipid interaction would be of great importance to understand the multiple effects of Phe on cell membranes.
Project description:Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. 13C chemical shifts indicate that the central leucine-rich segment of the TMD is ?-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the ?-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the ?-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the ?-helical core allowed us to measure interhelical distances using 19F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2-8.4Å for L493F and L504F and 10.5Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central ?-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a ?-sheet conformation.
Project description:Lipid modifications of proteins are widespread in nature and play an important role in numerous biological processes. The nonreceptor tyrosine kinase Src is equipped with an N-terminal myristoyl chain and a cluster of basic amino acids for the stable membrane association of the protein. We used (2)H NMR spectroscopy to investigate the structure and dynamics of the myristoyl chain of myr-Src(2-19), and compare them with the hydrocarbon chains of the surrounding phospholipids in bilayers of varying surface potentials and chain lengths. The myristoyl chain of Src was well inserted in all bilayers investigated. In zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes, the myristoyl chain of Src was significantly longer and appears "stiffer" than the phospholipid chains. This can be explained by an equilibrium between the attraction attributable to the insertion of the myristoyl chain and the Born repulsion. In a 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] membrane, where attractive electrostatic interactions come into play, the differences between the peptide and the phospholipid chain lengths were attenuated, and the molecular dynamics of all lipid chains were similar. In a much thicker 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine]/cholesterol membrane, the length of the myristoyl chain of Src was elongated nearly to its maximum, and the order parameters of the Src chain were comparable to those of the surrounding membrane.
Project description:Cellular entry of influenza virus is mediated by the viral protein hemagglutinin (HA), which forms an initial complex of three HA1 and three HA2 subunits. Each HA2 includes a fusion peptide (FP), a soluble ectodomain (SE), and a transmembrane domain. HA1 binds to cellular sialic acids, followed by virus endocytosis, pH reduction, dissociation of HA1, and structural rearrangement of HA2 into a final trimer-of-SE hairpins. A decrease in pH also triggers HA2-mediated virus/endosome membrane fusion. SE hairpins have an interior parallel helical bundle and C-terminal strands in the grooves of the exterior of the bundle. FPs are separate helical hairpins. This study compares wild-type HA2 (WT-HA2) with G1E(FP) and I173E(SE strand) mutants. WT-HA2 induces vesicle fusion at pH 5.0, whereas the extent of fusion is greatly reduced for both mutants. Circular dichroism for HA2 and FHA2?FP+SE constructs shows dramatic losses of stability for the mutants, including a Tm reduced by 40 °C for I173E-FHA2. This is evidence of destabilization of SE hairpins via dissociation of strands from the helical bundle, which is also supported by larger monomer fractions for mutant versus WT proteins. The G1E mutant may have disrupted FP hairpins, with consequent non-native FP binding to dissociated SE strands. It is commonly proposed that free energy released by the HA2 structural rearrangement catalyzes HA-mediated fusion. This study supports an alternate mechanistic model in which fusion is preceded by FP insertion in the target membrane and formation of the final SE hairpin. Less fusion by the mutants is due to the loss of hairpin stability and consequent reduced level of membrane apposition of the virus and target membranes.
Project description:The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive ?-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magic-angle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly ?-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the ?-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. (31)P NMR spectra and small-angle X-ray scattering (SAXS) data show that this ?-strand-rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. (1)H-(31)P 2D correlation spectra and (2)H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the ?-strand as the fusogenic conformation.
Project description:The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use (31)P and (13)C solid-state NMR to examine M2-induced membrane curvature. M2(22-46), which includes only the transmembrane (TM) helix, and M2(21-61), which contains an additional amphipathic helix, are studied. (31)P chemical shift lineshapes indicate that M2(21-61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22-46) has minimal effect. The lamellar and isotropic domains have distinct (31)P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. (31)P- and (13)C-detected (1)H T(2) relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21-61) preferentially binds to the high-curvature domain. (31)P linewidths indicate that the isotropic vesicles induced by M2(21-61) are 10-35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides.