Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the "pH sensor" that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.Dengue virus, a member of the flavivirus family, imposes one of the largest social and economic burdens of any mosquitoborne viral pathogen (6, 11). Three structural proteins (C, M, and E) and a lipid bilayer package the positive-strand RNA genomes of flaviviruses (13). The core nucleocapsid protein, C, binds directly to genomic RNA, while the major envelope glycoprotein, E, and the membrane protein, M, form the outer protein shell (9). C-terminal ␣-helical hairpins anchor E and M in the lipid membrane. E binds a receptor on the host cell surface during infection. Receptor binding directs the virion to the endocytic pathway. E responds to the reduced pH of the endosome with a large conformational rearrangement (17). This rearrangement delivers the energy required to bend the host cell membrane toward the viral membrane, inducing the two membranes to fuse (17). The fusogenic conformational rearrangement is a critical step in viral entry, as it delivers the viral genome into the cytoplasm. Crystal structures of the E protein ectodomains from dengue virus type 2 (DEN-2) and from tick-borne encephalitis (TBE) virus have been determined both before and after their fusogenic conformational rearrangements (3,16,17,22,26). The structures of DEN-3 virus E and of West Nile virus E in the prefusion conformation have also been determined (8,18,19). These structures provide us with a detailed molecular picture of the fusion mechanism of flaviviruses (15). First, E inserts a hydrophobic anchor, the so-called fusion loop, into the outer bilayer leaflet of the host cell membrane. Second, E folds back on itself, directing its C-terminal transmembrane anchor toward the fusion loop. This fold-back forces the host cell membrane (held by the fusion loop) and the viral membrane (held by the C-terminal transmembrane anchor) against each other, resulting in fusion of the two membranes. Here we report the crystal structure of a soluble fragment of the E protein (sE) from DEN-1 containing residues 1 to 400, that is, all but the last 50 residues of the ectodomain (Fig. 1). The protein is in the postfusion conformation even though it was never ex...

The Snf1/AMPK kinases are intracellular energy sensors, and the AMPK pathway has been implicated in a variety of metabolic human disorders. Here we report the crystal structure of the kinase domain from yeast Snf1, revealing a bilobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution, as demonstrated by equilibrium sedimentation, and in yeast cells, as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved alphaC helix, and the burial of the activation segment and substrate binding site within the dimer, suggests that it represents an inactive form of the kinase. Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family, and an avenue for development of AMPK-specific activating compounds.

SignificanceTransporters isomerize between conformations to shuttle cargo across membranes, but the mechanism is not understood. Double electron–electron resonance measurements on the sodium-dependent sugar transporter (vSGLT) were used to explore the conformational state of the transporter under specific ligand conditions. Although sugar transport by vSGLT is driven by sodium gradients, vSGLT adopts an inward-open conformation irrespective of the presence of sodium. In the presence of sodium and galactose, the transporter transitions to an occluded conformation. We propose that the cell’s negative membrane potential aids in driving vSGLT toward the outward-facing state to bind sugar and begin the transport cycle. These findings could be applicable to other transporters whereby the inherent cellular membrane potential is integrated into the transport cycle.