RESUMO
The glycoprotein spikes of membrane-enveloped viruses include a subunit that catalyzes fusion (joining) of the viral and target cell membranes. For influenza virus, this is subunit 2 of hemagglutinin which has a â¼ 20-residue N-terminal fusion peptide (Fp) region that binds target membrane. An outstanding question is whether there are associated membrane changes important for fusion. Several computational studies have found increased "protrusion" of lipid acyl chains near Fp, i.e. one or more chain carbons are closer to the aqueous region than the headgroup phosphorus. Protrusion may accelerate initial joining of outer leaflets of the two membranes into a stalk intermediate. In this study, higher protrusion probability in membrane with vs. without Fp is convincingly detected by larger Mn2+-associated increases in chain 13C NMR transverse relaxation rates (Γ2's). Data analysis provides a ratio Γ2,neighbor/Γ2,distant for lipids neighboring vs. more distant from the Fp. The calculated ratio depends on the number of Fp-neighboring lipids and the experimentally-derived range of 4 to 24 matches the range of increased protrusion probabilities from different simulations. For samples either with or without Fp, the Γ2 values are well-fitted by an exponential decay as the 13C site moves closer to the chain terminus. The decays correlate with free-energy of protrusion proportional to the number of protruded -CH2 groups, with free energy per -CH2 of â¼0.25 kBT. The NMR data support one major fusion role of the Fp to be much greater protrusion of lipid chains, with highest protrusion probability for chain regions closest to the headgroups.
Assuntos
Hemaglutininas , Orthomyxoviridae , Hemaglutininas/análise , Hemaglutininas/metabolismo , Membrana Celular/química , Peptídeos/química , Orthomyxoviridae/metabolismo , Lipídeos/química , Fusão de MembranaRESUMO
There is complete attenuation of fusion and infection mediated by HIV gp160 with gp41 subunit with V2E mutation, and also V2E dominance with WT/V2E mixtures. V2E is at the N-terminus of the â¼25-residue fusion peptide (Fp) which likely binds the target membrane. In this study, large V2E attenuation and dominance were observed for vesicle fusion induced by FP_HM, a large gp41 ectodomain construct with Fp followed by hyperthermostable hairpin with N- and C-helices, and membrane-proximal external region (Mper). FP_HM is a trimer-of-hairpins, the final gp41 structure during fusion. Vesicle fusion and helicity were measured for FP_HM using trimers with different fractions (f's) of WT and V2E proteins. Reductions in FP_HM fusion and helicity vs. fV2E were quantitatively-similar to those for gp160-mediated fusion and infection. Global fitting of all V2E data supports 6 WT gp41 (2 trimers) required for fusion. These data are understood by a model in which the â¼25 kcal/mol free energy for initial membrane apposition is compensated by the thermostable hairpin between the Fp in target membrane and Mper/transmembrane domain in virus membrane. The data support a structural model for V2E dominance with a membrane-bound Fp with antiparallel ß sheet and interleaved strands from the two trimers. Relative to fV2E = 0, a longer Fp sheet is stabilized with small fV2E because of salt-bridge and/or hydrogen bonds between E2 on one strand and C-terminal Fp residues on adjacent strands, like R22. A longer Fp sheet results in shorter N- and C-helices, and larger separation during membrane apposition which hinders fusion.
Assuntos
Infecções por HIV , Fusão de Membrana , Humanos , Conformação Proteica em Folha beta , Sequência de Aminoácidos , Peptídeos/genética , Infecções por HIV/metabolismo , Mutação , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismoRESUMO
An early step in cellular infection by a membrane-enveloped virus like HIV or influenza is joining (fusion) of the viral and cell membranes. Fusion is catalyzed by a viral protein that typically includes an apolar "fusion peptide" (fp) segment that binds the target membrane prior to fusion. In this study, the effects of nonhomologous HIV and influenza fp's on lipid acyl chain motion are probed with 2H NMR transverse relaxation rates (R2's) of a perdeuterated DMPC membrane. Measurements were made between 35 and 0 °C, which brackets the membrane liquid-crystalline-to-gel phase transitions. Samples were made with either HIV "GPfp" at pH 7 or influenza "HAfp" at pH 5 or 7. GPfp induces vesicle fusion at pH 7, and HAfp induces more fusion at pH 5 vs 7. GPfp bound to DMPC adopts an intermolecular antiparallel ß sheet structure, whereas HAfp is a monomer helical hairpin. The R2's of the no peptide and HAfp, pH 7, samples increase gradually as temperature is lowered. The R2's of GPfp and HAfp, pH 5, samples have very different temperature dependence, with a â¼10× increase in R2CD2 when temperature is reduced from 25 to 20 °C and smaller but still substantial R2's at 10 and 0 °C. The large R2's with GPfp and HAfp, pH 5, are consistent with large-amplitude motions of lipid acyl chains that can aid fusion catalysis by increasing the population of chains near the aqueous phase, which is the chain location for transition states between membrane fusion intermediates.
Assuntos
Membrana Celular/metabolismo , Proteína gp41 do Envelope de HIV/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Lipídeos/química , Fusão de Membrana , Peptídeos/química , Fenômenos Biofísicos , Catálise , Membrana Celular/química , Proteína gp41 do Envelope de HIV/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Estrutura Secundária de ProteínaRESUMO
Viral fusion is a critical step in the entry pathway of enveloped viruses and remains a viable target for antiviral exploration. The current approaches for studying fusion mechanisms include ensemble fusion assays, high-resolution cryo-TEM, and single-molecule fluorescence-based methods. While these methods have provided invaluable insights into the dynamic events underlying fusion processes, they come with their own limitations. These often include extensive data and image analysis in addition to experimental time and technical requirements. This work proposes the use of the spin-spin T2 relaxation technique as a sensitive bioanalytical method for the rapid quantification of interactions between viral fusion proteins and lipids in real time. In this study, new liposome-coated iron oxide nanosensors (LIONs), which mimic as magnetic-labeled host membranes, are reported to detect minute interactions occurring between the membrane and influenza's fusion glycoprotein, hemagglutinin (HA). The influenza fusion protein's interaction with the LION membrane is detected by measuring changes in the sensitive spin-spin T2 magnetic relaxation time using a bench-top NMR instrument. More data is gleaned from including the fluorescent dye DiI into the LION membrane. In addition, the effects of environmental factors on protein-lipid interaction that affect fusion such as pH, time of incubation, trypsin, and cholesterol were also examined. Furthermore, the efficacy and sensitivity of the spin-spin T2 relaxation assay in quantifying similar protein/lipid interactions with more native configurations of HA were demonstrated using virus-like particles (VLPs). Shorter domains derived from HA were used to start a reductionist path to identify the parts of HA responsible for the NMR changes observed. Finally, the known fusion inhibitor Arbidol was employed in our spin-spin T2 relaxation-based fusion assay to demonstrate the application of LIONs in real-time monitoring of this aspect of fusion for evaluation of potential fusion inhibitors.
Assuntos
Influenza Humana , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Humanos , Lipossomos , Fenômenos MagnéticosRESUMO
Enveloped viruses are surrounded by a membrane which is obtained from an infected host cell during budding. Infection of a new cell requires joining (fusion) of the virus and cell membranes. This process is mediated by a monotopic viral fusion protein with a large ectodomain outside the virus. The ectodomains of class I enveloped viruses have a N-terminal "fusion peptide" (fp) domain that is critical for fusion and binds to the cell membrane. In this study, 2H NMR spectra are analyzed for deuterated membrane with fp from either HIV gp41 (GP) or influenza hemagglutinin (HA) fusion proteins. In addition, the HAfp samples are studied at more fusogenic pH 5 and less fusogenic pH 7. GPfp adopts intermolecular antiparallel ß sheet structure whereas HAfp is a monomeric helical hairpin. The data are obtained for a set of temperatures between 35 and 0 °C using DMPC-d54 lipid with perdeuterated acyl chains. The DMPC has liquid-crystalline (Lα) phase with disordered chains at higher temperature and rippled gel (Pß') or gel phase (Lß') with ordered chains at lower temperature. At given temperature T, the no peptide and HAfp, pH 7 samples exhibit similar spectral lineshapes. Spectral broadening with reduced temperature correlates with the transition from Lα to Pß' and then Lß' phases. At given T, the lineshapes are narrower for HAfp, pH 5 vs. no peptide and HAfp, pH 7 samples, and even narrower for the GPfp sample. These data support larger-amplitude fast (>105 Hz) lipid acyl chain motion for samples with fusogenic peptides, and peptide interference with chain ordering. The NMR data of the present paper correlate with insertion of these peptides into the hydrocarbon core of the membrane and support a significant fusion contribution from the resultant lipid acyl chain disorder, perhaps because of reduced barriers between the different membrane topologies in the fusion pathway. Membrane insertion and lipid perturbation appear common to both ß sheet and helical hairpin peptides.
Assuntos
Proteína gp41 do Envelope de HIV/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Concentração de Íons de Hidrogênio , Lipídeos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Sequência de Aminoácidos , Deutério/química , Conformação Proteica em Folha betaRESUMO
The influenza virus hemagglutinin (HA) protein has HA1 and HA2 subunits, which form an initial complex. HA1's bind host cell sialic acids, which triggers endocytosis, HA1/HA2 separation, and HA2-mediated fusion between virus and endosome membranes. We report hydrogen-deuterium exchange mass spectrometry (HDX-MS) on the HA2 subunit without HA1. HA2 contains the fusion peptide (FP), soluble ectodomain (SE), transmembrane domain (TM), and endodomain. FP is a monomer by itself, while SE is a trimer of hairpins that includes an interior bundle of residue 38-105 helices, turns, and residue 154-178 strands packed antiparallel to the bundle. FP and TM extend from the same side of the SE hairpin, and fusion models often depict a FP/TM complex with membrane traversal of both domains that is important for membrane pore expansion. The HDX-MS data of this study do not support this complex and instead support independent FP and TM with respective membrane-interfacial and traversal locations. The data also show a low level of aqueous exposure of the 22-38 segment, consistent with retention of the 23-35 antiparallel ß sheet observed in the initial HA1/HA2 complex. We propose the ß sheet as a semirigid connector between FP and SE that enables close membrane apposition prior to fusion. The I173E mutant exhibits greater exchange for residues 22-69 and 150-191, consistent with dissociation of SE C-terminal strands from interior N-helices. Similar trends are observed for the G1E mutant as well as less exchange for G1E FP. Fusion is highly impaired with either mutant, which correlates with reduced membrane apposition and, for G1E, FP binding to SE rather than the target membrane.
Assuntos
Membrana Celular/metabolismo , Deutério/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Hemaglutininas Virais/metabolismo , Hidrogênio/metabolismo , Peptídeos/metabolismo , Domínios Proteicos , Sequência de Aminoácidos , Medição da Troca de Deutério/métodos , Humanos , Concentração de Íons de Hidrogênio , Influenza Humana/metabolismo , Influenza Humana/virologia , Fusão de Membrana/fisiologia , Orthomyxoviridae/metabolismo , Conformação Proteica em Folha betaRESUMO
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.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Hemaglutininas Virais/química , Hemaglutininas/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Hemaglutininas Virais/metabolismo , Humanos , Conformação Proteica , Domínios Proteicos , Subunidades ProteicasRESUMO
Influenza virus is a class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5-6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of â¼ 25, â¼ 160, â¼ 25, and â¼ 10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP+SE, and SHA2-TM ≡ SE+TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm>90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM.
Assuntos
Hemaglutininas Virais/química , Vírus da Influenza A/química , Sequência de Aminoácidos , Hemaglutininas Virais/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Vírus da Influenza A/genética , Estabilidade Proteica , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
The â¼25 N-terminal "HAfp" residues of the HA2 subunit of the influenza virus hemagglutinin protein are critical for fusion between the viral and endosomal membranes at low pH. Earlier studies of HAfp in detergent support (1) N-helix/turn/C-helix structure at pH 5 with open interhelical geometry and N-helix/turn/C-coil structure at pH 7; or (2) N-helix/turn/C-helix at both pHs with closed interhelical geometry. These different structures led to very different models of HAfp membrane location and different models of catalysis of membrane fusion by HAfp. In this study, the interhelical geometry of membrane-associated HAfp is probed by solid-state NMR. The data are well-fitted to a population mixture of closed and semiclosed structures. The two structures have similar interhelical geometries and are planar with hydrophobic and hydrophilic faces. The different structures of HAfp in detergent vs membrane could be due to the differences in interaction with the curved micelle vs flat membrane with better geometric matching between the closed and semiclosed structures and the membrane. The higher fusogenicity of longer sequences and low pH is correlated with hydrophobic surface area and consequent increased membrane perturbation.
Assuntos
Detergentes/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Hemaglutininas Virais/química , Orthomyxoviridae/química , Sequência de Aminoácidos , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Influenza Humana/virologia , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Infecções por Orthomyxoviridae/virologia , Estrutura Secundária de ProteínaRESUMO
Rotational-echo double-resonance (REDOR) solid-state NMR is applied to probe the membrane locations of specific residues of membrane proteins. Couplings are measured between protein (13)CO nuclei and membrane lipid or cholesterol (2)H and (31)P nuclei. Specific (13)CO labeling is used to enable unambiguous assignment and (2)H labeling covers a small region of the lipid or cholesterol molecule. The (13)CO-(31)P and (13)CO-(2)H REDOR respectively probe proximity to the membrane headgroup region and proximity to specific insertion depths within the membrane hydrocarbon core. One strength of the REDOR approach is use of chemically-native proteins and membrane components. The conventional REDOR pulse sequence with 100 kHz (2)H π pulses is robust with respect to the (2)H quadrupolar anisotropy. The (2)H T1's are comparable to the longer dephasing times (τ's) and this leads to exponential rather than sigmoidal REDOR buildups. The (13)CO-(2)H buildups are well-fitted to A×(1-e(-γτ)) where A and γ are fitting parameters that are correlated as the fraction of molecules (A) with effective (13)CO-(2)H coupling d=3γ/2. The REDOR approach is applied to probe the membrane locations of the "fusion peptide" regions of the HIV gp41 and influenza virus hemagglutinin proteins which both catalyze joining of the viral and host cell membranes during initial infection of the cell. The HIV fusion peptide forms an intermolecular antiparallel ß sheet and the REDOR data support major deeply-inserted and minor shallowly-inserted molecular populations. A significant fraction of the influenza fusion peptide molecules form a tight hairpin with antiparallel N- and C-α helices and the REDOR data support a single peptide population with a deeply-inserted N-helix. The shared feature of deep insertion of the ß and α fusion peptide structures may be relevant for fusion catalysis via the resultant local perturbation of the membrane bilayer. Future applications of the REDOR approach may include samples that contain cell membrane extracts and use of lower temperatures and dynamic nuclear polarization to reduce data acquisition times.
Assuntos
Membrana Celular/química , Membrana Celular/ultraestrutura , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Ressonância Magnética Nuclear Biomolecular/métodos , Sítios de Ligação , Técnicas de Sonda Molecular , Ligação ProteicaRESUMO
Membrane locations of peptides and proteins are often critical to their functions. Solid-state rotational-echo double-resonance (REDOR) nuclear magnetic resonance is applied to probe the locations of two peptides via peptide (13)CO to lipid (2)H distance measurements. The peptides are KALP, an α-helical membrane-spanning peptide, and HFP, the ß-sheet N-terminal fusion peptide of the HIV gp41 fusion protein that plays an important role in HIV-host cell membrane fusion. Both peptides are shown to have at least two distinct locations within the hydrocarbon core of gel-phase membranes. The multiple locations are attributed to snorkeling of lysine side chains for KALP and to the distribution of antiparallel ß-sheet registries for HFP. The relative population of each location is also quantitated. To the best of our knowledge, this is the first clear experimental support of multiple peptide locations within the membrane hydrocarbon core. These data are for gel-phase membranes, but the approach should work for liquid-ordered membranes containing cholesterol and may be applicable to liquid-disordered membranes with appropriate additional analysis to take into account protein and lipid motion. This paper also describes the methodological development of (13)CO-(2)H REDOR using the lyophilized I4 peptide that is α-helical and (13)CO-labeled at A9 and (2)Hα-labeled at A8. The I4 spins are well-approximated as an ensemble of isolated (13)CO-(2)H spin pairs each separated by 5.0 Å with a 37 Hz dipolar coupling. A pulse sequence with rectangular 100 kHz (2)H π pulses results in rapid and extensive buildup of REDOR (ΔS/S0) with a dephasing time (τ). The buildup is well-fit by a simple exponential function with a rate of 24 Hz and an extent close to 1. These parameter values reflect nonradiative transitions between the (2)H spin states during the dephasing period. Each spin pair spends approximately two-thirds of its time in the (13)CO-(2)H (m = ±1) states and approximately one-third of its time in the (13)CO-(2)H (m = 0) state and contributes to the ΔS/S0 buildup during the former but not the latter time segments.
Assuntos
Membrana Celular/química , Deutério/química , Hidrocarbonetos/química , Lipídeos/química , Espectroscopia de Ressonância Magnética/métodos , Peptídeos/química , Transição de Fase , Sequência de Aminoácidos , Isótopos de Carbono , Géis , Dados de Sequência Molecular , RotaçãoRESUMO
The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region hypothesized to bind to the host cell membrane followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The present study focuses on the large gp41 ectodomain constructs "Hairpin" (HP) containing NHR+loop+CHR and "FP-Hairpin" (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH4 where the protein is positively-charged but do not induce fusion at pH7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. There are two kinetically distinct fusion processes at pH4: (1) a faster ~100 ms⻹ process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms⻹ process with extent strongly inversely correlated with this charge. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region between the two membranes. Previous solid-state NMR (SSNMR) of membrane-associated FP-HP has supported protein oligomers with FP's in an intermolecular antiparallel sheet. There was an additional population of molecules with α helical FPs and the samples likely contained a mixture of membrane-bound and -unbound proteins. For the present study, samples were prepared with fully membrane-bound FP-HP and subsequent SSNMR showed dominant ß FP conformation at both low and neutral pH. SSNMR also showed close contact of the FP with the lipid headgroups at both low and neutral pH whereas the NHR+CHR regions had contact at low pH and were more distant at neutral pH, consistent with the protein/membrane electrostatics.
Assuntos
Proteína gp41 do Envelope de HIV/química , Fusão de Membrana , Sequência de Aminoácidos , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequências Repetitivas de AminoácidosRESUMO
HIV is an enveloped virus and fusion between the HIV and host cell membranes is catalyzed by the ectodomain of the HIV gp41 membrane protein. Both the N-terminal fusion peptide (FP) and C-terminal membrane-proximal external region (MPER) are critical for fusion and are postulated to bind to the host cell and HIV membranes, respectively. Prior to fusion, the gp41 on the virion is a trimer in noncovalent complex with larger gp120 subunits. The gp120 bind host cell receptors and move away or dissociate from gp41 which subsequently catalyzes fusion. In the present work, large gp41 ectodomain constructs were produced and biophysically and structurally characterized. One significant finding is observation of synergy between the FP, hairpin, and MPER in vesicle fusion. The ectodomain-induced fusion can be very efficient with only â¼15 gp41 per vesicle, which is comparable to the number of gp41 on a virion. Conditions are found with predominant monomer or hexamer but not trimer and these may be oligomeric states during fusion. Monomer gp41 ectodomain is hyperthermostable and has helical hairpin structure. A new HIV fusion model is presented where (1) hemifusion is catalyzed by folding of gp41 ectodomain monomers into hairpins and (2) subsequent fusion steps are catalyzed by assembly into a hexamer with FPs in an antiparallel ß sheet. There is also significant interest in the gp41 MPER because it is the epitope of several broadly neutralizing antibodies. Two of these antibodies bind our gp41 ectodomain constructs and support investigation of the gp41 ectodomain as an immunogen in HIV vaccine development.
Assuntos
Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV/fisiologia , Interações Hospedeiro-Patógeno , Sequência de Aminoácidos , HIV/química , Infecções por HIV/metabolismo , Humanos , Fusão de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
The HIV gp41 protein catalyzes fusion between viral and target cell membranes. Although the ~20-residue N-terminal fusion peptide (FP) region is critical for fusion, the structure of this region is not well characterized in large gp41 constructs that model the gp41 state at different times during fusion. This paper describes solid-state NMR (SSNMR) studies of FP structure in a membrane-associated construct (FP-Hairpin), which likely models the final fusion state thought to be thermostable trimers with six-helix bundle structure in the region C-terminal of the FP. The SSNMR data show that there are populations of FP-Hairpin with either α helical or ß sheet FP conformation. For the ß sheet population, measurements of intermolecular (13)C-(13)C proximities in the FP are consistent with a significant fraction of intermolecular antiparallel ß sheet FP structure with adjacent strand crossing near L7 and F8. There appears to be negligible in-register parallel structure. These findings support assembly of membrane-associated gp41 trimers through interleaving of N-terminal FPs from different trimers. Similar SSNMR data are obtained for FP-Hairpin and a construct containing the 70 N-terminal residues of gp41 (N70), which is a model for part of the putative pre-hairpin intermediate state of gp41. FP assembly may therefore occur at an early fusion stage. On a more fundamental level, similar SSNMR data are obtained for FP-Hairpin and a construct containing the 34 N-terminal gp41 residues (FP34) and support the hypothesis that the FP is an autonomous folding domain.
Assuntos
Membrana Celular/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Espectroscopia de Ressonância Magnética , Fusão de Membrana/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , HIV/fisiologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Recombinant proteins (RPs) are commonly expressed in bacteria followed by solubilization and chromatography. Purified RP yield can be diminished by losses at any step with very different changes in methods that can improve the yield. Time and labor can therefore be saved by first identifying the specific reason for the low yield. This study describes a new solid-state nuclear magnetic resonance approach to RP quantitation in whole cells or cell extracts without solubilization or purification. The method is straightforward and inexpensive and requires only â¼50 mL culture and a low-field spectrometer.
Assuntos
Proteínas de Bactérias/química , Espectroscopia de Ressonância Magnética/métodos , Bactérias/química , Bactérias/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Extratos Celulares/química , DNA Recombinante/química , Temperatura Alta , Corpos de Inclusão/química , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , SolubilidadeRESUMO
Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the â¼25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of "HFP", i.e., a â¼25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was (13)CO backbone labeled. Samples were then prepared that each contained a singly (13)CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric ß sheet structure. Proximity between the HFP (13)CO nuclei and (31)P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct (13)CO shifts for the α helical and ß sheet structures so that the proximities to (31)P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the (13)CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. "HFPmn" was a linear peptide that contained the 23 N-terminal residues of gp41. "HFPmn_V2E" contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and infection. The present study shows that HFPmn_V2E induces much less vesicle fusion than HFPmn. "HFPtr" contained three strands with HFPmn sequence that were chemically cross-linked near their C-termini. HFPtr mimics the trimeric topology of gp41 and induces much more rapid and extensive vesicle fusion than HFPmn. For HFPmn and HFPtr, well-resolved α and ß peaks were observed for A6-, L9-, and L12-labeled samples. For each of these samples, there were similar HFP (13)CO to lipid (31)P proximities in the α and ß structures, which evidenced comparable membrane locations of the HFP in either structure including insertion into a single membrane leaflet. The data were also consistent with deeper insertion of HFPtr relative to HFPmn in both the α and ß structures. The results supported a strong correlation between the membrane insertion depth of the HFP and its fusogenicity. More generally, the results supported membrane location of the HFP as an important determinant of its fusogenicity. The deep insertion of HFPtr in both the α and ß structures provides the most relevant membrane location of the FP for HIV gp41-catalyzed membrane fusion because HIV gp41 is natively trimeric. Well-resolved α and ß signals were observed in the HFPmn_V2E samples with L9- and L12- but not A6-labeling. The α signals were much more dominant for L9- and L12-labeled HFPmn_V2E than the corresponding HFPmn or HFPtr. The structural model for the less fusogenic HFPmn_V2E includes a shorter helix and less membrane insertion than either HFPmn or HFPtr. This greater helical population and different helical structure and membrane location could result in less membrane perturbation and lower fusogenicity of HFPmn_V2E and suggest that the ß sheet fusion peptide is the most functionally relevant structure of HFPmn, HFPtr, and gp41.
Assuntos
Membrana Celular/química , Proteína gp41 do Envelope de HIV/química , HIV/química , Sequência de Aminoácidos , Isótopos de Carbono , Monóxido de Carbono/química , Proteína gp41 do Envelope de HIV/genética , Lipídeos/química , Fusão de Membrana , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Isótopos de Fósforo , Estrutura Secundária de Proteína , Internalização do VírusRESUMO
The influenza virus fusion peptide is the N-terminal ~20 residues of the HA2 subunit of the hemagglutinin protein and this peptide plays a key role in the fusion of the viral and endosomal membranes during initial infection of a cell. The fusion peptide adopts N-helix/turn/C-helix structure in both detergent and membranes with reports of both open and closed interhelical topologies. In the present study, backbone (13)CO-(15)N REDOR solid-state NMR was applied to the membrane-associated fusion peptide to detect the distribution of interhelical distances. The data clearly showed a large fraction of closed and semi-closed topologies and were best-fitted to a mixture of two structures that do not exchange. One of the earlier open structural models may have incorrect G13 dihedral angles derived from TALOS analysis of experimentally correct (13)C shifts.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Ressonância Magnética Nuclear Biomolecular/métodos , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Conformação ProteicaRESUMO
Residue-specific location of peptides in the hydrophobic core of membranes was examined using (13)C-(2)H REDOR and samples in which the lipids were selectively deuterated. The transmembrane topology of the KALP peptide was validated with this approach with substantial dephasing observed for deuteration in the bilayer center and reduced or no dephasing for deuteration closer to the headgroups. Insertion of ß sheet HIV and helical and ß sheet influenza virus fusion peptides into the hydrophobic core of the membrane was validated in samples with extensively deuterated lipids.