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Cholesterol Alters the Orientation and Activity of the Influenza Virus M2 Amphipathic Helix in the Membrane.

ABSTRACT: The influenza virus M2 amphipathic helix (M2AH) alters membrane curvature in a cholesterol-dependent manner, mediating viral membrane scission during influenza virus budding. Here, we have investigated the biophysical effects of cholesterol on the ability of an M2AH peptide to manipulate membrane properties. We see that the ability of the M2AH to interact with membranes and form an ?-helix is independent of membrane cholesterol concentration; however, cholesterol affects the angle of the M2AH peptide within the membrane. This change in membrane orientation affects the ability of the M2AH to alter lipid order. In low-cholesterol membranes, the M2AH is inserted near the level of the lipid head groups, increasing lipid order, which may contribute to generation of the membrane curvature. As the cholesterol content increases, the M2AH insertion becomes flatter and slightly deeper in the membrane below the lipid headgroups, where the polar face can continue to interact with the headgroups while the hydrophobic face binds cholesterol. This changed orientation minimizes lipid packing defects and lipid order changes, likely reducing the generation of membrane curvature. Thus, cholesterol regulates M2 membrane scission by precisely modulating M2AH positioning within the membrane. This has implications for the understanding of many of amphipathic-helix-driven cellular budding processes that occur in specific lipid environments.

SUBMITTER: Martyna A

PROVIDER: S-EPMC7515559 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Cholesterol Alters the Orientation and Activity of the Influenza Virus M2 Amphipathic Helix in the Membrane.

Martyna Agnieszka A Bahsoun Basma B Madsen Jesper J JJ Jackson Frederic St J S FSJS Badham Matthew D MD Voth Gregory A GA Rossman Jeremy S JS

The journal of physical chemistry. B 20200723 31

The influenza virus M2 amphipathic helix (M2AH) alters membrane curvature in a cholesterol-dependent manner, mediating viral membrane scission during influenza virus budding. Here, we have investigated the biophysical effects of cholesterol on the ability of an M2AH peptide to manipulate membrane properties. We see that the ability of the M2AH to interact with membranes and form an α-helix is independent of membrane cholesterol concentration; however, cholesterol affects the angle of the M2AH pe ...[more]

PMID: 32644803

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Project description:The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22-46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45-62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21-61). (13)C-(2)H distance measurements of (13)C-labeled protein and (2)H-labeled amantadine showed that in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, the first equivalent of drug bound S31 inside the M2(21-61) pore, similar to the behavior of M2 transmembrane peptide (M2TM) in DMPC bilayers. The nonspecific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21-61) is S31 in the TM pore. Interestingly, when M2(21-61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, whereas M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state but did not rescue drug binding to M2(21-61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helix to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein.

Project description:One of the fundamental features of biomembranes is the ability to fuse or to separate. These processes called respectively membrane fusion and fission are central in the homeostasis of events such as those related to intracellular membrane traffic. Proteins that contain amphipathic helices (AHs) were suggested to mediate membrane fission via shallow insertion of these helices into the lipid bilayer. Here we analyze the AH-containing proteins that have been identified as essential for membrane fission and categorize them in few subfamilies, including small GTPases, Atg proteins, and proteins containing either the ENTH/ANTH- or the BAR-domain. AH-containing fission-inducing proteins may require cofactors such as additional proteins (e.g., lipid-modifying enzymes), or lipids (e.g., phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidic acid [PA], or cardiolipin). Both PA and cardiolipin possess a cone shape and a negative charge (-2) that favor the recruitment of the AHs of fission-inducing proteins. Instead, PtdIns(4,5)P2 is characterized by an high negative charge able to recruit basic residues of the AHs of fission-inducing proteins. Here we propose that the AHs of fission-inducing proteins contain sequence motifs that bind lipid cofactors; accordingly (K/R/H)(K/R/H)xx(K/R/H) is a PtdIns(4,5)P2-binding motif, (K/R)x6(F/Y) is a cardiolipin-binding motif, whereas KxK is a PA-binding motif. Following our analysis, we show that the AHs of many fission-inducing proteins possess five properties: (a) at least three basic residues on the hydrophilic side, (b) ability to oligomerize, (c) optimal (shallow) depth of insertion into the membrane, (d) positive cooperativity in membrane curvature generation, and (e) specific interaction with one of the lipids mentioned above. These lipid cofactors favor correct conformation, oligomeric state and optimal insertion depth. The most abundant lipid in a given organelle possessing high negative charge (more negative than -1) is usually the lipid cofactor in the fission event. Interestingly, naturally occurring mutations have been reported in AH-containing fission-inducing proteins and related to diseases such as centronuclear myopathy (amphiphysin 2), Charcot-Marie-Tooth disease (GDAP1), Parkinson's disease (?-synuclein). These findings add to the interest of the membrane fission process whose complete understanding will be instrumental for the elucidation of the pathogenesis of diseases involving mutations in the protein AHs.

Dataset Information

Cholesterol Alters the Orientation and Activity of the Influenza Virus M2 Amphipathic Helix in the Membrane.

Publications

Cholesterol Alters the Orientation and Activity of the Influenza Virus M2 Amphipathic Helix in the Membrane.

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