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Designed protein mimics of the Ebola virus glycoprotein GP2 α-helical bundle: stability and pH effects.

ABSTRACT: Ebola virus (EboV) belongs to the Filoviridae family of viruses that causes severe and fatal hemhorragic fever. Infection by EboV involves fusion between the virus and host cell membranes mediated by the envelope glycoprotein GP2 of the virus. Similar to the envelope glycoproteins of other viruses, the central feature of the GP2 ectodomain postfusion structure is a six-helix bundle formed by the protein's N- and C-heptad repeat regions (NHR and CHR, respectively). Folding of this six-helix bundle provides the energetic driving force for membrane fusion; in other viruses, designed agents that disrupt formation of the six-helix bundle act as potent fusion inhibitors. To interrogate determinants of EboV GP2-mediated membrane fusion, we designed model proteins that consist of the NHR and CHR segments linked by short protein linkers. Circular dichroism and gel filtration studies indicate that these proteins adopt stable α-helical folds consistent with design. Thermal denaturation indicated that the GP2 six-helix bundle is highly stable at pH 5.3 (melting temperature, T(m) , of 86.8 ± 2.0°C and van't Hoff enthalpy, ΔH(vH) , of -28.2 ± 1.0 kcal/mol) and comparable in stability to other viral membrane fusion six-helix bundles. We found that the stability of our designed α-helical bundle proteins was dependent on buffering conditions with increasing stability at lower pH. Small pH differences (5.3-6.1) had dramatic effects (ΔT(m) = 37°C) suggesting a mechanism for conformational control that is dependent on environmental pH. These results suggest a role for low pH in stabilizing six-helix bundle formation during the process of GP2-mediated viral membrane fusion.

SUBMITTER: Harrison JS

PROVIDER: S-EPMC3190153 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV requires fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein, GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔG(unf,H(2)O), of 33.4 ± 2.5 kcal/mol and melting temperature, T(m), of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH, with higher stability under lower-pH conditions (T(m) values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. On the basis of these results, we hypothesize that the pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism for controlling conformational preferences such that the six-helix bundle "postfusion" state is preferred under conditions of appropriately matured endosomes.

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Designed protein mimics of the Ebola virus glycoprotein GP2 α-helical bundle: stability and pH effects.

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