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1.
Zhu J Zhang CW Qi Y Tien P Gao GF 《Biochemical and biophysical research communications》2002,299(5):897-902
Recent studies have shown that paramyxovirus might adopt a similar molecular mechanism of virus entry and fusion in which the attachment glycoprotein binds receptor/s and triggers the conformational changes of the fusion protein. There are two conserved regions of heptad repeat (HR1 and HR2) in the fusion protein and they were shown with fusion-inhibition effects in many paramyxoviruses, including measles virus. They also appear to show characteristic structure in the fusion core: the HR1/HR2 forms stable six-helix coiled-coil centered by HR1 and is surrounded by HR2 (trimer of HR1/HR2), which represents the post-fusion conformational structure. In this study, we expressed the HR1 and HR2 of measles virus fusion protein as a single chain (named 2-Helix) and subsequently tested its formation of trimer. Indeed, the results do show that the HR1 and HR2 interact with each other and form stable six-helix coiled-coil bundle. This is the first member in genus Morbillivirus of family Paramyxoviridae to be confirmed with this characteristic structure and provides the basis for the HR2-inhibition effects on virus fusion/entry for measles virus. 相似文献
2.
Dwyer JJ Wilson KL Martin K Seedorff JE Hasan A Medinas RJ Davison DK Feese MD Richter HT Kim H Matthews TJ Delmedico MK 《Protein science : a publication of the Protein Society》2008,17(4):633-643
HIV fusion is mediated by a conformational transition in which the C-terminal region (HR2) of gp41 interacts with the N-terminal region (HR1) to form a six-helix bundle. Peptides derived from the HR1 form a well-characterized, trimeric coiled-coil bundle in the presence of HR2 peptides, but there is little structural information on the isolated HR1 trimer. Using protein design, we have designed synthetic HR1 peptides that form soluble, thermostable HR1 trimers. In vitro binding of HR2 peptides to the engineered trimer suggests that the design strategy has not significantly impacted the ability to form the six-helix bundle. The peptides have enhanced antiviral activity compared to wild type, with up to 30-fold greater potency against certain viral isolates. In vitro passaging was used to generate HR1-resistant virus and the observed resistance mutations map to the HR2 region of gp41, demonstrating that the peptides block the fusion process by binding to the viral HR2 domain. Interestingly, the activity of the HR2 fusion inhibitor, enfuvirtide (ENF), against these resistant viruses is maintained or improved up to fivefold. The 1.5 A crystal structure of one of these designs has been determined, and we show that the isolated HR1 is very similar to the conformation of the HR1 in the six-helix bundle. These results provide an initial model of the pre-fusogenic state, are attractive starting points for identifying novel fusion inhibitors, and offer new opportunities for developing HIV therapeutics based on HR1 peptides. 相似文献
3.
The gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergent n-octyl-beta-D-glucopyranoside (betaOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly alpha-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion. 相似文献
4.
The HIV-1 gp41 envelope protein mediates membrane fusion that leads to virus entry into the cell. The core structure of fusion-active gp41 is a six-helix bundle in which an N-terminal three-stranded coiled coil is surrounded by a sheath of antiparallel C-terminal helices. A conserved glutamine (Gln 652) buried in this helical interface replaced by leucine increases HIV-1 infectivity. To define the basis for this enhanced membrane fusion activity, we investigate the role of the Gln 652 to Leu substitution on the conformation, stability, and biological activity of the N34(L6)C28 model of the gp41 ectodomain core. The 2.0 A resolution crystal structure of the mutant molecule shows that the Leu 652 side chains make prominent contacts with hydrophobic grooves on the surface of the central coiled coil. The Gln 652 to Leu mutation leads to a marginal stabilization of the six-helix bundle by -0.8 kcal/mol, evaluated from thermal unfolding experiments. Strikingly, the mutant N34(L6)C28 peptide is a potent inhibitor of HIV-1 infection, with 10-fold greater activity than the wild-type molecule. This inhibitory potency can be traced to the corresponding C-terminal mutant peptide that likely has greater potential to interact with the coiled-coil trimer. These results provide strong evidence that conserved interhelical packing interactions in the gp41 core are important determinants of HIV-1 entry and its inhibition. These interactions also offer a test-bed for the development of more potent analogues of gp41 peptide inhibitors. 相似文献
5.
The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (gp120-gp41) promotes viral entry by mediating the fusion of viral and cellular membranes. Formation of a stable trimer-of-hairpins structure in the gp41 ectodomain brings the two membranes into proximity, leading to membrane fusion. The core of this hairpin structure is a six-helix bundle in which three carboxyl-terminal outer helices pack against an inner trimeric coiled coil. Here we investigate the role of these conserved interhelical interactions on the structure and function of both the envelope glycoprotein and the gp41 core. We have replaced each of the eight amino acids at the buried face of the carboxyl-terminal helix with a representative amino acid, alanine. Structural and physicochemical characterization of the alanine mutants shows that hydrophobic interactions are a dominant factor in the stabilization of the six-helix bundle. Alanine substitutions at the Trp628, Trp631, Ile635, and Ile642 residues also affected envelope processing and/or gp120-gp41 association and abrogated the ability of the envelope glycoprotein to mediate cell-cell fusion. These results suggest that the amino-terminal region of the gp41 outer-layer alpha-helix plays a key role in the sequence of events associated with HIV-1 entry and have implications for the development of antibodies and small-molecule inhibitors of this conserved element. 相似文献
6.
AIDS是严重危害人类健康的疾病,而HIV是导致这种疾病的病毒。gp41六螺旋在介导HIV-1病毒与靶细胞间的膜融合过程中起着重要作用。因此,对于gp41结合蛋白的研究有助于深入了解gp41在HIV-1感染整个过程中扮演的角色,解释gp41对靶细胞的调控机制,为寻找新的抗艾滋病药物靶点以及艾滋病抑制剂的设计提供有益的思路。作者的实验室相继发现了一批与gp41六螺旋结构相互作用的蛋白质,进而对HIV-1 gp41六螺旋介导的膜融合过程和HIV-1感染机理有了更深入的了解。 相似文献
7.
For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion. 相似文献
8.
Infection by human immunodeficiency virus type I requires the fusogenic activity of gp41, the transmembrane subunit of the viral envelope protein. Crystallographic studies have revealed that fusion-active gp41 is a "trimer-of-hairpins" in which three central N-terminal helices form a trimeric coiled coil surrounded by three antiparallel C-terminal helices. This structure is stabilized primarily by hydrophobic, interhelical interactions, and several critical contacts are made between residues that form a deep cavity in the N-terminal trimer and the C-helix residues that pack into this cavity. In addition, the trimer-of-hairpins structure has an extensive network of hydrogen bonds within a conserved glutamine-rich layer of poorly understood function. Formation of the trimer-of-hairpins structure is thought to directly force the viral and target membranes together, resulting in membrane fusion and viral entry. We test this hypothesis by constructing four series of gp41 mutants with disrupted interactions between the N- and C-helices. Notably, in the three series containing mutations within the cavity, gp41 activity correlates well with the stability of the N-C interhelical interaction. In contrast, a fourth series of mutants involving the glutamine layer residue Gln-653 show fusion defects even though the stability of the hairpin is close to wild-type. These results provide evidence that gp41 hairpin stability is critical for mediating fusion and suggest a novel role for the glutamine layer in gp41 function. 相似文献
9.
The structure of an HIV-1 specific cell entry inhibitor in complex with the HIV-1 gp41 trimeric core
Zhou G Ferrer M Chopra R Kapoor TM Strassmaier T Weissenhorn W Skehel JJ Oprian D Schreiber SL Harrison SC Wiley DC 《Bioorganic & medicinal chemistry》2000,8(9):2219-2227
The three-dimensional structure of the complex between an HIV-1 cell-entry inhibitor selected from screening a combinatorial library of non-natural building blocks and the central, trimeric, coiled-coil core of HIV-1 gp41 has been determined by X-ray crystallography. The biased combinatorial library was designed to identify ligands binding in nonpolar pockets on the surface of the coiled-coil core of gp41. The crystal structure shows that the non-peptide moiety of the inhibitor binds to the targeted cavity in two different binding modes. This result suggests a strategy for increasing inhibitor potency by use of a second-generation combinatorial library designed to give simultaneous occupancy of both binding sites. 相似文献
10.
Caulfield MJ Dudkin VY Ottinger EA Getty KL Zuck PD Kaufhold RM Hepler RW McGaughey GB Citron M Hrin RC Wang YJ Miller MD Joyce JG 《The Journal of biological chemistry》2010,285(52):40604-40611
We describe here a novel platform technology for the discovery of small molecule mimetics of conformational epitopes on protein antigens. As a model system, we selected mimetics of a conserved hydrophobic pocket within the N-heptad repeat region of the HIV-1 envelope protein, gp41. The human monoclonal antibody, D5, binds to this target and exhibits broadly neutralizing activity against HIV-1. We exploited the antigen-binding property of D5 to select complementary small molecules using a high throughput screen of a diverse chemical collection. The resulting small molecule leads were rendered immunogenic by linking them to a carrier protein and were shown to elicit N-heptad repeat-binding antibodies in a fraction of immunized mice. Plasma from HIV-1-infected subjects shown previously to contain broadly neutralizing antibodies was found to contain antibodies capable of binding to haptens represented in the benzylpiperidine leads identified as a result of the high throughput screen, further validating these molecules as vaccine leads. Our results suggest a new paradigm for vaccine discovery using a medicinal chemistry approach to identify lead molecules that, when optimized, could become vaccine candidates for infectious diseases that have been refractory to conventional vaccine development. 相似文献
11.
HIV-1跨膜蛋白gp41的截短及表达 总被引:2,自引:0,他引:2
将HIV-1跨膜蛋白gp41进行截短,在大肠杆菌中进行表达并纯化。PCR扩增gp41的部分编码基因,回收的PCR产物纯化后克隆到连接载体pGEM-T上,然后用EcoRI和Sal I切下目的基因,并构建到表达载体pGEX-4T3上,导入宿主细胞BL21(DE3),用IPTG诱导表达,表达产物用亲和层析进行纯化并作相应鉴定。截短的HIV-1跨膜蛋白gp41能直接在大肠杆菌内进行表达,利用亲和层析能方便地将目的蛋白进行纯化,为跨膜蛋白的进一步应用打下基础。 相似文献
12.
Triggered by receptor binding of gp120, the human immunodeficiency virus type 1 (HIV-1) gp41 changes its conformation to a fusogenic six-helix bundle structure. In the present study, this core conformation modeled by the peptides derived from the gp41 N- and C-terminal heptad repeat regions was determined by fluorescence native polyacrylamide gel electrophoresis and size exclusion high-performance liquid chromatography (HPLC). Two previously described small molecule HIV-1 fusion inhibitors significantly blocked the six-helix bundle formation. It suggests that these biophysical techniques can be used in a novel way to study the conformational change of gp41 during virus entry into cells and to identify HIV-1 fusion inhibitors. 相似文献
13.
Conformational partitioning of the fusion peptide of HIV-1 gp41 and its structural analogs in bilayer membranes
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Experiments have shown that the ability of the HIV-1 virus to infect cells can be greatly diminished by deactivation of the N-terminal (fusion) peptide of its glycoprotein gp41. Deactivation can be achieved by the deletion of several amino acid residues, or replacement of a hydrophobic residue with a polar residue, to form mutant variants of the wild-type peptide. We report Monte Carlo simulation studies of a simplified peptide/membrane model, representing the interaction of an HIV-1 fusion peptide (FP) and four closely related mutagens with a lipid bilayer. In agreement with experimental results, we show that FP inserts deeply into the bilayer at approximately 40 degrees to the bilayer normal. We also show a previously unreported behavior of membrane peptides, namely their equilibrium partitioning between several distinct conformations within the bilayer. We quantify this partitioning behavior and characterize each conformation in terms of its geometry, energy, and entropy. The diminished ability of FP mutagens to hemolyse and aggregate red blood cells due to their partitioning into unfavorable conformations, is also discussed. Our analysis supports a negative curvature mechanism for red blood cell hemolysis by FP. We also suggest that the small repulsive forces between surface-adsorbed peptides in opposing membrane surfaces may block aggregation. 相似文献
14.
Recent experimental study found that OLE (olive leaf extract) has anti-HIV activity by blocking the HIV virus entry to host cells [Lee-Huang, S., Zhang, L., Huang, P.L., Chang, Y. and Huang, P.L. (2003) Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochem. Biophys. Res. Commun. 307, 1029; Lee-Huang, S., Huang, P.L., Zhang, D., Lee, J.W., Bao, J., Sun, Y., Chang, Y.-Tae, Zhang, J.Z.H. and Huang, P.L. (2007) Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol. Biochem. Biophys. Res. Commun. 354, 872-878, 879-884]. As part of a joint experimental and theoretical effort, we report here computational study to help identify and characterize the binding complexes of several main compounds of OLE (olive leaf extract) to HIV-1 envelop protein gp41. A number of possible binding modes are found by docking oleuropein and its metabolites, aglycone, elenolic acid and hydroxytyrosol, onto the hydrophobic pocket on gp41. Detailed OLE-gp41 binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM-PBSA calculation. Specific molecular interactions in our predicted OLE/gp41 complexes are identified and hydroxytyrosol is identified to be the main moiety for binding to gp41. This computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of OLE-based gp41 inhibitors. 相似文献
15.
《Microbes and infection / Institut Pasteur》2015,17(9):665-670
We previously identified an HIV-1 fusion inhibitor P20A targeting HIV-1 gp41 6-HB fusion core. Using alanine scanning mutagenesis, we investigated the effect of 6-HB surface residue mutations on the binding affinity between P20A and 6-HB. Substitution of positively or negatively charged residues in the distal region of 6-HB with alanines resulted in significant decrease or increase of its binding affinity to P20A, respectively. The 6-HB with E630K, D632K, or E634K mutation exhibited enhanced binding affinity with P20A, suggesting that P20A blocks HIV-1 fusion through electrostatic interaction with the positively charged residues in the distal region of the gp41 fusion core. 相似文献
16.
The structure of a membrane fusion mutant of the influenza virus haemagglutinin. 总被引:4,自引:0,他引:4
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The haemagglutinin glycoprotein (HA) of influenza virus specifically mediates fusion of the viral and host cell endosomal membranes at the acidic pH of endosomes. The HAs from mutant viruses with raised fusion pH optima contain amino acid substitutions in regions of the HA structure thought to be involved in the fusion process [Daniels et al. (1985b) Cell, 40, 431-439]. We have determined the neutral pH crystal structure of one such mutant, HA2 112 Asp----Gly. A water molecule appears to partially replace the aspartate side chain, and no changes are observed in the surrounding structure. It appears that four intra-chain hydrogen bonds that stabilize the location of the N-terminus of HA2 are lost in the mutant, resulting in a local destabilization that facilitates the extrusion of the N-terminus at higher pH. 相似文献
17.
Venken T Krnavek D Münch J Kirchhoff F Henklein P De Maeyer M Voet A 《Proteins》2011,79(11):3221-3235
VIRus Inhibitory Peptide (VIRIP), a 20 amino acid peptide, binds to the fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) gp41 and blocks viral entry. VIRIP derivatives with improved antiviral activity have been developed, and one of those derivatives has recently proven effective and safe in a phase 1/2 clinical trial. Here, molecular dynamics were executed in combination with molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) free energy calculations to explore the binding interaction between VIRIP derivatives and gp41 FP. A promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations, and the use of multiple internal dielectric constants for the MM/PBSA calculations depending on the amino acid sequence. Based on these results, a virtual screening experiment was carried out to design VIRIP analogs with further improved antiretroviral activity. A selection of peptides was tested for inhibitory activity and several VIRIP derivatives were identified with significantly enhanced activity compared to the reference peptides. The results demonstrate that computational modeling strategies using an adapted MM/PBSA methodology improve the accuracy of binding free energy calculations of peptide complexes compared to the classic MM/PBSA protocol. As such, this virtual screening approach generated HIV-1 gp41 FP inhibitors with improved antiviral activity that could be useful for future clinical applications. 相似文献
18.
Lu L Zhu Y Huang J Chen X Yang H Jiang S Chen YH 《The Journal of biological chemistry》2008,283(24):16723-16731
HIV-1 gp41 cytoplasmic tail (CT) is highly conserved among HIV-1 isolates, particularly the region designated lentivirus lytic peptide (LLP1-2), which includes two alpha-helical domains LLP1 and LLP2. Although the gp41 CT is recognized as a modulator of viral fusogenicity, little is known about the regulatory mechanism of this region in the viral fusion process. Here we report that anti-LLP1-2 and anti-LLP2 antibodies (IgG) inhibited HIV-1 Env-mediated cell fusion and bound to the interface between effector and target cells at a suboptimal temperature (31.5 degrees C), which slows down the fusion process and prolongs the fusion intermediate state. This suggests that LLP1-2, especially the LLP2 region located inside the viral membrane, is transiently exposed on the membrane surface during the fusion process. Synthetic LLP2 peptide could bind to the gp41 six-helix bundle core with high binding affinity. These results suggest that the gp41 CT may interact with the gp41 core, via the surface-exposed LLP2 domain, to regulate Env-mediated membrane fusion. 相似文献
19.
HIV-1 envelope glycoprotein-mediated fusion is driven by the concerted coalescence of the HIV-1 gp41 N- and C-helical regions, which results in the formation of 6-helix bundles. These two regions are considered prime targets for peptides and antibodies that inhibit HIV-1 entry. However, the parameters that govern this inhibition have yet to be elucidated. We address this issue by monitoring the temporal sequence of conformational states of HIV-1 gp41 during the course of HIV-1-mediated cell-cell fusion by quantitative video microscopy using reagents that bind to N- and C-helical regions, respectively. Env-expressing cells were primed by incubation with target cells at different times at 37 degrees C followed by washing. The reactivity of triggered gp41 to the NC-1 monoclonal antibody, which we demonstrate here to bind to N-helical gp41 trimers, increased rapidly to a maximal level in the primed state but decreased once stable fusion junctions had formed. In contrast, reactivity with 5-helix, which binds to the C-helical region of gp41, increased continuously as a function of time following the priming. The peptide N36(Mut(e,g)) reduced NC-1 monoclonal antibody binding and enhanced 5-helix binding, consistent with the notion that this molecule promotes dissociation of gp41 trimers. This inactivation pathway may be important for the design of entry inhibitors and vaccine candidates. 相似文献
20.
The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide. 总被引:9,自引:1,他引:8
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W Weissenhorn S A Wharton L J Calder P L Earl B Moss E Aliprandis J J Skehel D C Wiley 《The EMBO journal》1996,15(7):1507-1514
The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein is composed of a soluble glycopolypeptide gp120 and a transmembrane glycopolypeptide gp41. These subunits form non-covalently linked oligomers on the surface of infected cells, virions and cells transfected with the complete env gene. Two length variants of the extracellular domain of gp41 (aa 21-166 and aa 39-166), that both lack the N-terminal fusion peptide and the C-terminal membrane anchor and cytoplasmic domain, have been expressed in insect cells to yield soluble oligomeric gp41 proteins. Oligomerization was confirmed by chemical cross-linking and gel filtration. Electron microscopy and circular dichroism measurements indicate a rod-like molecule with a high alpha-helical content and a high melting temperature (78 degrees C). The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs. We propose that gp41 folds into its membrane fusion-active conformation, when expressed alone. 相似文献