Effect of Nonpolar Substitutions of the Conserved Phe<sup>11</sup> in the Fusion Peptide of HIV-1 gp41 on Its Function, Structure, and Organization in Membranes

Pritsker, Moshe; Rucker, Joseph; Hoffman, Trevor L.; Doms, Robert W.; Shai, Yechiel

doi:10.1021/bi990232e

Cited by 69 publications

(98 citation statements)

References 83 publications

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“…Fluorescence and ESR data also suggest that the nonhelical HIV-1 peptides form oligomeric structures (16,20,35). The possible significance of fusion peptide oligomerization is suggested both by envelope protein trimerization and by experiments and modeling studies which indicate that the fusion site contains multiple trimers and a corresponding high fusion peptide concentration (31,32,(59)(60)(61).…”

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confidence: 96%

Solid-State Nuclear Magnetic Resonance Evidence for Parallel and Antiparallel Strand Arrangements in the Membrane-Associated HIV-1 Fusion Peptide

Yang

Weliky

2003

Biochemistry

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The HIV-1 fusion peptide serves as a useful model system for understanding viral/target cell fusion, at least to the lipid-mixing stage. Previous solid-state NMR studies have shown that the membranebound HIV-1 fusion peptide adopts an extended conformation in a lipid mixture close to that of host cells of the virus. In the present study, solid-state NMR REDOR methods were applied for detection of oligomeric strand structure. The samples were prepared under fusogenic conditions and contained equimolar amounts of two peptides, one with selective [ 13 C]carbonyl labeling and the other with selective [ 15 N]amide labeling. In the REDOR measurements, observation of reduced 13 C intensity due to hydrogen-bonded amide 15 N provides strong experimental evidence of oligomer formation by the membrane-associated peptide.Comparison of REDOR spectra on samples that were labeled at different residue positions suggests that there are both parallel and antiparallel arrangements of peptide strands. In the parallel arrangement, interpeptide hydrogen bonding decreases toward the C-terminus, while in the antiparallel arrangement, hydrogen bonds are observed along the entire length of residues which was probed (Gly-5 to Gly-16). For the parallel arrangement, these observations are consistent with the model in which the apolar N-terminal and central regions of the peptides penetrate into the membrane and hydrogen bond with one another while the polar C-terminus of the peptide is outside the membrane and hydrogen bonds with water. These measurements show that, at least at the end state of fusion, the peptide can adopt an oligomeric strand structure.Fusion between the membranes of enveloped viruses such as HIV-1 1 and influenza and the membranes of their target host cells is an essential step in infection (1-4). For these viruses, this process is mediated by integral membrane viral envelope proteins which contain N-terminal ∼20-residue apolar fusion peptide domains. For HIV-1 and influenza, the free fusion peptide has also been shown to be a useful model to understand fusion, at least to the lipid-mixing stage. The free peptide causes fusion of liposomes and erythrocytes, and numerous mutational studies have shown strong correlations between fusion peptide-induced liposome fusion and viral/host cell fusion (5-20). Recent studies suggest that envelope protein regions other than the fusion peptide also interact with membranes and play a role in fusion (21-26).There is a crystal structure of the part of the influenza hemagglutinin envelope protein which lies outside the virus and which contains the fusion peptide (27). This "ectodomain" structure corresponds to a prefusogenic conformation. For both the hemagglutinin and the HIV-1 gp41 envelope proteins, there are also atomic resolution structures of the "soluble ectodomains" which do not contain the fusion peptide (28-34). These structures are believed to correspond to the protein conformations after fusion has occurred and perhaps during fusion as well. In each of these soluble...

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confidence: 96%

Solid-State Nuclear Magnetic Resonance Evidence for Parallel and Antiparallel Strand Arrangements in the Membrane-Associated HIV-1 Fusion Peptide

Yang

Weliky

2003

Biochemistry

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“…Models for the helical structure have been developed based on nuclear magnetic resonance (NMR), electron spin resonance (ESR), infrared (IR), and circular dichroism (CD) data, as well as computer simulations (33)(34)(35)(36)(37)(38)(39). A β hairpin model for non-helical structure has been proposed based on IR and surface activity measurements in membranes (40).Fluorescence, ESR, IR, and solid-state NMR data also suggest that the nonhelical HFPs in membranes form oligomeric structures (10,(41)(42)(43)(44). These oligomeric structures may be important as evidenced by envelope protein trimerization and by experiments and modeling which indicate that the fusion site contains multiple trimers and a corresponding high HFP concentration (17,18,45).…”

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Conformational Flexibility and Strand Arrangements of the Membrane-Associated HIV Fusion Peptide Trimer Probed by Solid-State NMR Spectroscopy

et al. 2006

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The human immunodeficiency virus (HIV) fusion peptide (HFP) is the N-terminal apolar region of the HIV gp41 fusion protein and interacts with target cell membranes and promotes membrane fusion. The free peptide catalyzes vesicle fusion at least to the lipid mixing stage and serves as a useful model fusion system. For gp41 constructs which lack the HFP, high-resolution structures show trimeric protein and suggest that at least three HFPs interact with the membrane with their C-termini in close proximity. In addition, previous studies have demonstrated that HFPs which are cross-linked at their C-termini to form trimers (HFPtr) catalyze fusion at a rate which is 15−40 times greater than noncross-linked HFP. In the present study, the structure of membrane-associated HFPtr was probed with solid-state nuclear magnetic resonance (NMR) methods. Chemical shift and intramolecular 13 CO-15 N distance measurements show that the conformation of the Leu-7 to Phe-11 region of HFPtr has predominant helical conformation in membranes without cholesterol and β strand conformation in membranes containing ∼30 mol% cholesterol. Interstrand 13 CO-13 CO and 13 CO-15 N distance measurements were not consistent with an in-register parallel strand arrangement but were consistent with either: (1) parallel arrangement with adjacent strands tworesidues out-of-register; or (2) antiparallel arrangement with adjacent strand crossing between Phe-8 and Leu-9. Arrangement (1) could support the rapid fusion rate of HFPtr because of placement of the apolar N-terminal regions of all strands on the same side of the oligomer while arrangement (2) could support the assembly of multiple fusion protein trimers. KeywordsHIV; fusion peptide; cholesterol; membranes; NMR; trimer Enveloped viruses such as human immunodeficiency virus (HIV) are surrounded by a membrane and infect cells through fusion between the viral membrane and the target cell membrane (1,2). This process is mediated by envelope proteins which traverse the viral membrane (3). For HIV, the gp41 envelope protein has a ∼170-residue ectodomain region † This work was supported by NIH award AI47153 to D. P. W. * To whom correspondence should be addressed. Telephone: 517−355−9715. Fax: 517−353−1793. Email: weliky@chemistry.msu.edu.. SUPPORTING INFORMATION AVAILABLE Equations are derived for determination of (ΔS/S 0 ) cor for REDOR and fpCTDQBU analyses. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 October 20. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript which lies outside the viral membrane and contains a N-terminal ∼20-residue apolar "fusion peptide" (HFP). The HFP interacts with the target cell membrane and plays an essential role in membrane fusion (4). Peptides composed of the HFP sequence induce fusion between large unilamellar vesicles (LUVs) and between erythrocytes (5,6). There are similar mutation/fusion activity relationships...

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“…The free peptide causes fusion of liposomes and erythrocytes, and numerous mutational studies have shown strong correlations between fusion peptide-induced liposome fusion and viral/host cell fusion (5,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

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Synthesis, Enhanced Fusogenicity, and Solid State NMR Measurements of Cross-Linked HIV-1 Fusion Peptides

Yang

Weliky

2003

Biochemistry

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In the HIV-1 gp41 and other viral fusion proteins, the minimal oligomerization state is believed to be trimeric with three N-terminal fusion peptides inserting into the membrane in close proximity. Previous studies have demonstrated that the fusion peptide by itself serves as a useful model fusion system, at least to the hemifusion stage in which the viral and target cell lipids are mixed. In the present study, HIV-1 fusion peptides were chemically synthesized and cross-linked at their C-termini to form dimers or trimers. C-terminal trimerization is their likely topology in the fusogenic form of the intact gp41 protein. The fusogenicity of the peptides was then measured in an intervesicle lipid mixing assay, and the assay results were compared to those of the monomer. For monomer, dimer, and trimer at peptide strand/lipid mol ratios between 0.0050 and 0.010, the final extent of lipid mixing for the dimer and trimer was 2-3 times greater than for the monomer. These data suggest that the higher local concentration of peptide strands in the cross-linked peptides enhances fusogenicity and that oligomerization of the fusion peptide in gp41 may enhance the rate of viral/target cell membrane fusion. For gp41, this effect is in addition to the role of the trimeric coiled-coil structure in bringing about apposition of viral and target cell membranes. NMR measurements on the membrane-associated dimeric fusion peptide were consistent with an extended structure at Phe-8, which is the same as has been observed for the membrane-bound monomer in the same lipid composition.Membrane fusion plays an essential role in enveloped virus entry into target host cells (1-4). Fusion is mediated by viral envelope proteins that contain apolar fusion peptide domains. The interaction between fusion peptides and lipids is believed to be one key event in initiating membrane fusion (5). Recent studies suggest that fusion protein regions other than the fusion peptide also interact with membranes and play a role in fusion (6-10).For HIV-1 1 and other enveloped viruses, the free ∼20-residue fusion peptide has been shown to be a useful model fusion system, at least to the hemifusion stage in which there is significant mixing between the viral and the target cell lipids. The free peptide causes fusion of liposomes and erythrocytes, and numerous mutational studies have shown strong correlations between fusion peptide-induced liposome fusion and viral/host cell fusion (5,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).For both the influenza hemagglutinin and the HIV-1 gp41 envelope proteins, there are crystal and NMR structures of soluble ectodomains in their fusogenic/final fusion conformations (26-32). These trimeric coiled-coil structures represent ∼140-residue sequences that begin near the C-terminus of the fusion peptide and end near the N-terminus of the viral transmembrane domain. For the influenza viral fusion protein, a pre-fusion structure has also been observed and is different from the fusogenic/post-fusion coiled-co...

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Effect of Nonpolar Substitutions of the Conserved Phe¹¹ in the Fusion Peptide of HIV-1 gp41 on Its Function, Structure, and Organization in Membranes

Cited by 69 publications

References 83 publications

Solid-State Nuclear Magnetic Resonance Evidence for Parallel and Antiparallel Strand Arrangements in the Membrane-Associated HIV-1 Fusion Peptide

Solid-State Nuclear Magnetic Resonance Evidence for Parallel and Antiparallel Strand Arrangements in the Membrane-Associated HIV-1 Fusion Peptide

Conformational Flexibility and Strand Arrangements of the Membrane-Associated HIV Fusion Peptide Trimer Probed by Solid-State NMR Spectroscopy

Synthesis, Enhanced Fusogenicity, and Solid State NMR Measurements of Cross-Linked HIV-1 Fusion Peptides

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Effect of Nonpolar Substitutions of the Conserved Phe11 in the Fusion Peptide of HIV-1 gp41 on Its Function, Structure, and Organization in Membranes

Cited by 69 publications

References 83 publications

Solid-State Nuclear Magnetic Resonance Evidence for Parallel and Antiparallel Strand Arrangements in the Membrane-Associated HIV-1 Fusion Peptide

Solid-State Nuclear Magnetic Resonance Evidence for Parallel and Antiparallel Strand Arrangements in the Membrane-Associated HIV-1 Fusion Peptide

Conformational Flexibility and Strand Arrangements of the Membrane-Associated HIV Fusion Peptide Trimer Probed by Solid-State NMR Spectroscopy

Synthesis, Enhanced Fusogenicity, and Solid State NMR Measurements of Cross-Linked HIV-1 Fusion Peptides

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Effect of Nonpolar Substitutions of the Conserved Phe¹¹ in the Fusion Peptide of HIV-1 gp41 on Its Function, Structure, and Organization in Membranes