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Characterization of monomeric intermediates during VSV glycoprotein structural transition.

Albertini AA, Mérigoux C, Libersou S, Madiona K, Bressanelli S, Roche S, Lepault J, Melki R, Vachette P, Gaudin Y - PLoS Pathog. (2012)

Bottom Line: The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases.Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification.We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche de Gif, Laboratoire de Virologie Moléculaire et Structurale, CNRS (UPR 3296), Gif sur Yvette, France.

ABSTRACT
Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (G(th), aa residues 1-422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, G(th) is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.

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Related in: MedlinePlus

SAXS analysis of Gth in solution.(A) SAXS patterns of Gth at pH 8.8 (blue) and pH 7.5 (red). Scattering intensities are plotted against the momentum transfer q. The insets zoom on ranges of maximal differences between the two curves. Notice the color swap: blue curve above (resp. below) red one in the first (resp. second) inset. (B) Guinier plots calculated from the SAXS data shown in (A). (C) Distance distribution functions p(r) computed from SAXS experimental data at pH 8.8 (blue) and pH 7.5 (red).
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ppat-1002556-g003: SAXS analysis of Gth in solution.(A) SAXS patterns of Gth at pH 8.8 (blue) and pH 7.5 (red). Scattering intensities are plotted against the momentum transfer q. The insets zoom on ranges of maximal differences between the two curves. Notice the color swap: blue curve above (resp. below) red one in the first (resp. second) inset. (B) Guinier plots calculated from the SAXS data shown in (A). (C) Distance distribution functions p(r) computed from SAXS experimental data at pH 8.8 (blue) and pH 7.5 (red).

Mentions: The scattering patterns at pH 8.8 and 7.5 are shown in Figure 3A with their Guinier plots in Figure 3B and the distance distribution functions p(r) in Figure 3C. All structural parameters are presented in Table 2. At both pH values, the estimate of the molecular mass (53.5 kDa) is identical to the sequence derived value calculated for a monomer of Gth with associated sugar chains. Therefore, the protein appears to be monomeric in solution at pH 8.8 and 7.5 even at concentrations of a few mg/ml confirming and extending the observations made by analytical ultracentrifugation at lower protein concentration.


Characterization of monomeric intermediates during VSV glycoprotein structural transition.

Albertini AA, Mérigoux C, Libersou S, Madiona K, Bressanelli S, Roche S, Lepault J, Melki R, Vachette P, Gaudin Y - PLoS Pathog. (2012)

SAXS analysis of Gth in solution.(A) SAXS patterns of Gth at pH 8.8 (blue) and pH 7.5 (red). Scattering intensities are plotted against the momentum transfer q. The insets zoom on ranges of maximal differences between the two curves. Notice the color swap: blue curve above (resp. below) red one in the first (resp. second) inset. (B) Guinier plots calculated from the SAXS data shown in (A). (C) Distance distribution functions p(r) computed from SAXS experimental data at pH 8.8 (blue) and pH 7.5 (red).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285605&req=5

ppat-1002556-g003: SAXS analysis of Gth in solution.(A) SAXS patterns of Gth at pH 8.8 (blue) and pH 7.5 (red). Scattering intensities are plotted against the momentum transfer q. The insets zoom on ranges of maximal differences between the two curves. Notice the color swap: blue curve above (resp. below) red one in the first (resp. second) inset. (B) Guinier plots calculated from the SAXS data shown in (A). (C) Distance distribution functions p(r) computed from SAXS experimental data at pH 8.8 (blue) and pH 7.5 (red).
Mentions: The scattering patterns at pH 8.8 and 7.5 are shown in Figure 3A with their Guinier plots in Figure 3B and the distance distribution functions p(r) in Figure 3C. All structural parameters are presented in Table 2. At both pH values, the estimate of the molecular mass (53.5 kDa) is identical to the sequence derived value calculated for a monomer of Gth with associated sugar chains. Therefore, the protein appears to be monomeric in solution at pH 8.8 and 7.5 even at concentrations of a few mg/ml confirming and extending the observations made by analytical ultracentrifugation at lower protein concentration.

Bottom Line: The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases.Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification.We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche de Gif, Laboratoire de Virologie Moléculaire et Structurale, CNRS (UPR 3296), Gif sur Yvette, France.

ABSTRACT
Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (G(th), aa residues 1-422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, G(th) is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.

Show MeSH
Related in: MedlinePlus