Several flaviviruses are important human pathogens, including dengue virus, a disease against which neither a vaccine nor specific antiviral therapies currently exist. During infection, the flavivirus RNA genome is translated into a polyprotein, which is cleaved into several components. Nonstructural protein 3 (NS3) carries out enzymatic reactions essential for viral replication, including proteolysis of the polyprotein through its serine protease N-terminal domain, with a segment of 40 residues from the NS2B protein acting as a cofactor. The ATPase/helicase domain is located at the C terminus of NS3. Atomic structures are available for these domains separately, but a molecular view of the full-length flavivirus NS3 polypeptide is still lacking. We report a crystallographic structure of a complete NS3 molecule fused to 18 residues of the NS2B cofactor at a resolution of 3.15 Å. The relative orientation between the protease and helicase domains is drastically different than the single-chain NS3-NS4A molecule from hepatitis C virus, which was caught in the act of cis cleavage at the NS3-NS4A junction. Here, the protease domain sits beneath the ATP binding site, giving the molecule an elongated shape. The domain arrangement found in the crystal structure fits nicely into an envelope determined ab initio using small-angle X-ray scattering experiments in solution, suggesting a stable molecular conformation. We propose that a basic patch located at the surface of the protease domain increases the affinity for nucleotides and could also participate in RNA binding, explaining the higher unwinding activity of the full-length enzyme compared to that of the isolated helicase domain.Several members of the flaviviruses are important human pathogens, including yellow fever virus (YFV), Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus (WNV), and dengue virus (25). For the latter virus, neither a specific therapy nor a vaccine exists, and treatment is currently limited to the use of analgesics and fluid replacement. Since dengue virus is endemic in most tropical and subtropical areas, causing several hundreds of thousands of severe cases (dengue shock syndrome or dengue hemorrhagic fever), with approximately 30,000 deaths per year, compounds with antiviral activity are actively sought (21, 46). The positive-sense flavivirus RNA genome of 11 kb forms a single open reading frame that is translated into a polyprotein precursor of ca. 370 kDa consisting of the structural proteins C, prM, and E and seven nonstructural proteins, nonstructural protein 1 (NS1), NS2A, NS2B, NS3, NS4A, NS4B, and NS5. During viral maturation, this polyprotein is cleaved by host cell proteases in the endoplasmic reticulum and by the NS3 protein in the cytoplasm (Fig. 1A) (25). Cleavage of the polyprotein is mediated by the serine protease N-terminal domain of NS3, with a hydrophilic segment of 40 residues from the transmembrane NS2B protein acting as a cofactor necessary for this activity. The domain required for ATPase/helicase ...

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dThe subunit of bacterial F 1 F O ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of of Mycobacterium tuberculosis (Mt) F 1 F O ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mt 103-120 ). Mt is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic ␤ subunit. The C-terminal segment displays an ␣-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mt. A model for the interaction of TMC207 with both and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of and the c-ring, respectively. T19 and R37 of provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F 1 F O ATP synthase in M. tuberculosis.

Background:Dengue virus surface proteins, envelope (E) and pre-membrane (prM), undergo rearrangement during the maturation process at acidic condition. Results: prM-stem region binds tighter to both E protein and lipid membrane when environment becomes acidic. Conclusion: At acidic condition, E proteins are attracted to the membrane-associated prM-stem. Significance: prM-stem region induces virus structural changes during maturation.

The mechanism by which a malaria merozoite recognizes a suitable host cell is mediated by a cascade of receptor-ligand interactions. In addition to the availability of the appropriate receptors, intracellular ATP plays an important role in determining whether erythrocytes are suitable for merozoite invasion. Recent work has shown that ATP secreted from erythrocytes signals a number of cellular processes. To determine whether ATP signaling might be involved in merozoite invasion, we investigated whether known plasmodium invasion proteins contain nucleotide binding motifs. Malaria is caused by unicellular protozoan parasites and is considered to be one of the most important infectious diseases still affecting mankind today. The complex life cycle of the parasite is characterized by three invasive forms: the sporozoite and merozoite that invade hepatocytes and erythrocytes in the vertebrate host, respectively, and the ookinetes inside the insect vector that penetrates the mosquito midgut epithelium (1-5). In the case of merozoites, specific organelles (rhoptries, micronemes, and dense granules) at the apical end of the parasite contain proteins that play an important role in the recognition and invasion of the host cell (6). Recognition of specific erythrocyte receptors by the merozoite is mediated by at least two gene families, the reticulocyte-binding protein homologues (RBL) 4 and the erythrocyte binding ligands (7,8). Like the erythrocyte binding ligands, the RBLs are also found in varying numbers in all plasmodium species with each member believed to play a role in recognizing a different receptor (7-16). In general, the members of RBL are large transmembrane proteins with molecular masses above 200 kDa that get proteolytically cleaved during the invasion process (14,17). In Plasmodium yoelii, members of the RBL termed Py235 (P. yoelii 235-kDa rhoptry protein) have been shown to be potential virulence factors that enable the parasite to invade a wider range of host erythrocytes (18 -20). In addition, Py235 is also involved in clonal phenotypic variation of merozoites (21) enabling the parasite to evade immune responses and adapt to changes in the host environment during the invasion step (22). Recently it has been demonstrated that variation in the amount of Py235 expressed in merozoites defines the host cell repertoire that a parasite can invade (20). Studies carried out on the RBLs of Plasmodium falciparum (PfRH1, (10, 14 -16, 23, 24) have provided additional evidence that different RBL members interact with different receptors on the erythrocyte and that these interactions are crucial for invasion. Previously work in Plasmodium vivax has indicated that RBLs may have an initial sensing role preceding and possibly enabling the subsequent interaction of the erythrocyte binding ligand member with its corresponding receptor (25). In a more recent study the erythrocyte binding region of PfRH1 and PfRH4 has been identified (26,71), showing that only a relatively small region of these proteins is actually invol...