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Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion.

ABSTRACT: The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. The fusion domain formed an ?-helix in membranes containing less than 30 mol% cholesterol and formed ?-sheet secondary structure in membranes containing ?30 mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of ?-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its ?-sheet form. High cholesterol, which induced ?-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an ?-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, ?-helical and ?-sheet conformations contribute to membrane fusion.

SUBMITTER: Lai AL

PROVIDER: S-EPMC3654243 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion.

Lai Alex L AL Moorthy Anna Eswara AE Li Yinling Y Tamm Lukas K LK

Journal of molecular biology 20120217 1-2

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscop ...[more]

PMID: 22343048

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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 envelope glycoprotein (Env) composed of the receptor binding domain gp120 and the fusion protein subunit gp41 catalyzes virus entry and is a major target for therapeutic intervention and for neutralizing antibodies. Env interactions with cellular receptors trigger refolding of gp41, which induces close apposition of viral and cellular membranes leading to membrane fusion. The energy released during refolding is used to overcome the kinetic barrier and drives the fusion reaction. Here, we report the crystal structure at 2 A resolution of the complete extracellular domain of gp41 lacking the fusion peptide and the cystein-linked loop. Both the fusion peptide proximal region (FPPR) and the membrane proximal external region (MPER) form helical extensions from the gp41 six-helical bundle core structure. The lack of regular coiled-coil interactions within FPPR and MPER splay this end of the structure apart while positioning the fusion peptide towards the outside of the six-helical bundle and exposing conserved hydrophobic MPER residues. Unexpectedly, the section of the MPER, which is juxtaposed to the transmembrane region (TMR), bends in a 90 degrees-angle sideward positioning three aromatic side chains per monomer for membrane insertion. We calculate that this structural motif might facilitate the generation of membrane curvature on the viral membrane. The presence of FPPR and MPER increases the melting temperature of gp41 significantly in comparison to the core structure of gp41. Thus, our data indicate that the ordered assembly of FPPR and MPER beyond the core contributes energy to the membrane fusion reaction. Furthermore, we provide the first structural evidence that part of MPER will be membrane inserted within trimeric gp41. We propose that this framework has important implications for membrane bending on the viral membrane, which is required for fusion and could provide a platform for epitope and lipid bilayer recognition for broadly neutralizing gp41 antibodies.

Dataset Information

Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion.

Publications

Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion.

Similar Datasets

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