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Structure, membrane topology and influence of cholesterol of the membrane proximal region: transmembrane helical anchor sequence of gp41 from HIV

ABSTRACT: During the first steps of HIV infection the Env subunit gp41 is thought to establish contact between the membranes and to be the main driver of fusion. Here we investigated in liquid crystalline membranes the structure and cholesterol recognition of constructs made of a gp41 external region carrying a cholesterol recognition amino acid consensus (CRAC) motif and a hydrophobic membrane anchoring sequence. CD- und ATR-FTIR spectroscopies indicate that the constructs adopt a high degree of helical secondary structure in membrane environments. Furthermore, 15N and 2H solid-state NMR spectra of gp41 polypeptides reconstituted into uniaxially oriented bilayers agree with the CRAC domain being an extension of the transmembrane helix. Upon addition of cholesterol the CRAC NMR spectra remain largely unaffected when being associated with the native gp41 transmembrane sequence but its topology changes when anchored in the membrane by a hydrophobic model sequence. The 2H solid-state NMR spectra of deuterated cholesterol are indicative of a stronger influence of the model sequence on this lipid when compared to the native gp41 sequence. These observations are suggestive of a strong coupling between the transmembrane and the membrane proximal region of gp41 possibly enforced by oligomerization of the transmembrane helical region.

SUBMITTER: Aisenbrey C

PROVIDER: S-EPMC7746737 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:The HIV-1 glycoprotein 41 promotes fusion of the viral membrane with that of the target cell. Structural, biochemical, and biophysical studies suggest that its membrane-proximal external region (MPER) may interact with the HIV-1 membrane and induce its disruption and/or deformation during the process. However, the high cholesterol content of the envelope (ca. 40 to 50 mol%) imparts high rigidity, thereby acting against lipid bilayer restructuring. Here, based on the outcome of vesicle stability assays, all-atom molecular dynamics simulations, and atomic force microscopy observations, we propose that the conserved sequence connecting the MPER with the N-terminal residues of the transmembrane domain (TMD) is involved in HIV-1 fusion. This junction would function by inducing phospholipid protrusion and acyl-chain splay in the cholesterol-enriched rigid envelope. Supporting the functional relevance of such a mechanism, membrane fusion was inhibited by the broadly neutralizing 4E10 antibody but not by a nonneutralizing variant with the CDR-H3 loop deleted. We conclude that the MPER-TMD junction embodies an envelope-disrupting C-terminal fusion peptide that can be targeted by broadly neutralizing antibodies.Fusion of the cholesterol-enriched viral envelope with the cell membrane marks the beginning of the infectious HIV-1 replicative cycle. Consequently, the Env glycoprotein-mediated fusion function constitutes an important clinical target for inhibitors and preventive vaccines. Antibodies 4E10 and 10E8 bind to one Env vulnerability site located at the gp41 membrane-proximal external region (MPER)-transmembrane domain (TMD) junction and block infection. These antibodies display broad viral neutralization, which underscores the conservation and functionality of the MPER-TMD region. In this work, we combined biochemical assays with molecular dynamics simulations and microscopy observations to characterize the unprecedented fusogenic activity of the MPER-TMD junction. The fact that such activity is dependent on cholesterol and inhibited by the broadly neutralizing 4E10 antibody emphasizes its physiological relevance. Discovery of this functional element adds to our understanding of the mechanisms underlying HIV-1 infection and its blocking by antibodies.

Project description:The HIV-1 glycoprotein, gp41, mediates fusion of the virus lipid envelope with the target cell membrane during virus entry into cells. Despite extensive studies of this protein, inconsistent and contradictory structural information abounds in the literature about the C-terminal membrane-interacting region of gp41. This C-terminal region contains the membrane-proximal external region (MPER), which harbors the epitopes for four broadly neutralizing antibodies, and the transmembrane domain (TMD), which anchors the protein to the virus lipid envelope. Due to the difficulty of crystallizing and solubilizing the MPER-TMD, most structural studies of this functionally important domain were carried out using truncated peptides either in the absence of membrane-mimetic solvents or bound to detergents and lipid bicelles. To determine the structural architecture of the MPER-TMD in the native environment of lipid membranes, we have now carried out a solid-state NMR study of the full MPER-TMD segment bound to cholesterol-containing phospholipid bilayers. 13C chemical shifts indicate that the majority of the peptide is α-helical, except for the C-terminus of the TMD, which has moderate β-sheet character. Intermolecular 19F-19F distance measurements of singly fluorinated peptides indicate that the MPER-TMD is trimerized in the virus-envelope mimetic lipid membrane. Intramolecular 13C-19F distance measurements indicate the presence of a turn between the MPER helix and the TMD helix. This is supported by lipid-peptide and water-peptide 2D 1H-13C correlation spectra, which indicate that the MPER binds to the membrane surface whereas the TMD spans the bilayer. Together, these data indicate that full-length MPER-TMD assembles into a trimeric helix-turn-helix structure in lipid membranes. We propose that the turn between the MPER and TMD may be important for inducing membrane defects in concert with negative-curvature lipid components such as cholesterol and phosphatidylethanolamine, while the surface-bound MPER helix may interact with N-terminal segments of the protein during late stages of membrane fusion.

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Structure, membrane topology and influence of cholesterol of the membrane proximal region: transmembrane helical anchor sequence of gp41 from HIV

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