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The Flavivirus nonstructural protein 1 (NS1) is a conserved, membrane-associated and secreted glycoprotein with replication and immune evasion functions. Secreted NS1 is a hexameric, barrel-shaped lipoprotein that can bind back to the plasma membrane of cells. Antibodies targeting cell surface-associated NS1 can be protective in vivo in a manner dependent on Fc effector functions. We describe here the crystal structure of a C-terminal fragment (residues 172-352) of West Nile (WNV) and Dengue virus NS1 proteins at 1.85 and 2.7 Å resolution, respectively. NS1(172-352) assembles as a unique rod-shaped dimer composed of a 16-stranded β-platform flanked on one face by protruding connecting loops. We also determined the 3.0 Å resolution structure of WNV NS1(172-352) with the protective 22NS1 antibody Fab, which engages the loop-face of the rod. The head-to-head NS1(172-352) dimer we observe in crystal lattices is supported by multiangle light and small-angle X-ray scattering studies. We used the available cryo-electron microscopy reconstruction to develop a pseudoatomic model of the NS1 hexamer. The model was constructed with the NS1(172-352) dimeric rod aligned with the long axis of the barrel, and with the loop-face oriented away from the core. Difference densities suggest that the N-terminal region of NS1 forms globular lobes that mediate lateral contacts between dimers in the hexamer. Our model also suggests that the N-terminal lobe forms the surface of the central cavity where lipid binding may occur.

Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of secreted and membrane proteins in bacteria. DsbG, a member of this family, has disulfide bond isomerase and chaperone activity. Here, we present two crystal structures of DsbG at 1.7and 2.0-A resolution that are meant to represent the reduced and oxidized forms, respectively. The oxidized structure, however, reveals a mixture of both redox forms, suggesting that oxidized DsbG is less stable than the reduced form. This trait would contribute to DsbG isomerase activity, which requires that the active-site Cys residues are kept reduced, regardless of the highly oxidative environment of the periplasm. We propose that a Thr residue that is conserved in the cis-Pro loop of DsbG and DsbC but not found in other Dsb proteins could play a role in this process. Also, the structure of DsbG reveals an unanticipated and surprising feature that may help define its specific role in oxidative protein folding. Thus, the dimensions and surface features of DsbG show a very large and charged binding surface that is consistent with interaction with globular protein substrates having charged surfaces. This finding suggests that, rather than catalyzing disulfide rearrangement in unfolded substrates, DsbG may preferentially act later in the folding process to catalyze disulfide rearrangement in folded or partially folded proteins.