SUMMARY Dengue is the most common vector-borne viral disease, causing nearly 400 million infections yearly. Currently there are no approved therapies. Antibody epitopes that elicit weak humoral responses may not be accessible by conventional B cell panning methods. To demonstrate an alternative strategy to generating a therapeutic antibody, we employed a non-immunodominant, but functionally relevant, epitope in domain III of the E protein, and engineered by structure-guided methods an antibody directed to it. The resulting antibody, Ab513, exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes. To assess therapeutic relevance of Ab513, activity against important human clinical features of dengue was investigated. Ab513 mitigates thrombocytopenia in a humanized mouse model, resolves vascular leakage, reduces viremia to nearly undetectable levels, and protects mice in a maternal transfer model of lethal antibody-mediated enhancement. The results demonstrate that Ab513 may reduce the public health burden from dengue.

cWe report a highly reproducible method to crystallize the RNA-dependent RNA polymerase (RdRp) domain of dengue virus serotype 3 (DENV-3), allowing structure refinement to a 1.79-Å resolution and revealing amino acids not seen previously. We also present a DENV-3 polymerase/inhibitor cocrystal structure at a 2.1-Å resolution. The inhibitor binds to the RdRp as a dimer and causes conformational changes in the protein. The improved crystallization conditions and new structural information should accelerate structure-based drug discovery. Many flaviviruses are significant human pathogens. However, no antiviral therapy is currently available for the treatment of flavivirus infections. The flavivirus RNA-dependent RNA polymerase (RdRp), located at the C-terminal two-thirds of nonstructural protein 5 (NS5), is an attractive target for antiviral development (1-3). Like other polymerases, the flavivirus RdRp adopts a right-hand configuration composed of the fingers, palm, and thumb subdomains (4-6). Unfortunately, because of their flexibility, several segments of the protein are missing in the current flavivirus RdRp unliganded crystal structures, including several loops at the interface between the fingers and thumb subdomains (5, 6). Moreover, crystals of the dengue virus serotype 3 (DENV-3) RdRp were obtained at 4°C following a tedious dehydration procedure (7), making high-throughput structure-based drug discovery very inconvenient. It is therefore critical to develop a robust crystallization protocol to allow structural determination of DENV RdRp in complex with small-molecule inhibitors. A simplified crystallization protocol will be invaluable for structurebased rational design of inhibitors of DENV RdRp.Here we report such a procedure to grow crystals of DENV-3 RdRp at the temperature of 18°C that routinely diffract to a resolution higher than 2.0 Å. This allowed an improved refinement of the original RdRp structure (PDB code 2J7U [6]), revealing several amino acids hitherto not seen. Using these improved conditions for crystallization, we present the first cocrystal structure of a flavivirus RdRp with an inhibitor. This cocrystal structure shows that the inhibitor induces major conformational changes in the DENV-3 RdRp.An improved protein purification protocol for the reproducible crystallization of DENV-3 RdRp. The previous conditions used to grow DENV-3 RdRp crystals required a complicated dehydration protocol through gradual transfer of the crystals into increasing concentrations of polyethylene glycol (PEG) (7). This method was difficult to routinely reproduce because crystals were obtained only after a few weeks at 4°C. To improve crystallization, we established a new purification protocol of the DENV-3 RdRp that avoided the use of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), EDTA, or ␤-mercaptoethanol. Instead, we included Tris (2-carboxyethyl)-phosphine (TCEP; Thermo Scientific) in the final RdRp solution to prevent protein oxidation. The addition of TCEP minimizes protein precipita...

The biosynthesis of the enediyne moiety of the antitumor natural product calicheamicin involves an iterative polyketide synthase (CalE8) and other ancillary enzymes. In the proposed mechanism for the early stage of 10-membered enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the assistance of a putative thioesterase (CalE7). We have determined the x-ray crystal structure of CalE7 and found that the subunit adopts a hotdog fold with an elongated and kinked substrate-binding channel embedded between two subunits. The 1.75-Å crystal structure revealed that CalE7 does not contain a critical catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases. Based on biochemical and site-directed mutagenesis studies, we proposed a catalytic mechanism in which the conserved Arg 37 plays a crucial role in the hydrolysis of the thioester bond, and that Tyr 29 and a hydrogen-bonded water network assist the decarboxylation of the ␤-ketocarboxylic acid intermediate. Moreover, computational docking suggested that the substrate-binding channel binds a polyene substrate that contains a single cis double bond at the C4/C5 position, raising the possibility that the C4‫؍‬C5 double bond in the enediyne moiety could be generated by the iterative polyketide synthase. Together, the results revealed a hotdog fold thioesterase distinct from the common type I and type II thioesterases associated with polyketide biosynthesis and provided interesting insight into the enediyne biosynthetic mechanism.Enediyne natural products represent a family of structurally unique secondary metabolites with potent antitumor and antibiotic activities. Based on the structure of the bicyclic enediyne core, enediyne natural products are categorized into two groups with either a 9-or 10-membered enediyne moiety (1, 2). The antitumor activity of enediyne natural products derives from their capacity to induce chromosomal DNA cleavage through an oxidative radical mechanism (3). The biosynthetic mechanism for the enediyne moiety has been, however, elusive despite clues gleaned from early isotope-feeding experiments (4, 5). Pioneering genetic studies of the biosynthesis of calicheamicin and C-1027 from two research groups yielded major insights into the biosynthetic pathways, suggesting that an iterative polyketide synthase (PKS) 5 plays a central role in the assembly of both the 9-and 10-membered enediyne moieties (6, 7). The gene clusters also contain open reading frames encoding hypothetical proteins for the downstream processing of the PKS product. The involvement of similar genes in enediyne biosynthesis was later confirmed for neocarzinostatin, maduropeptin, dynemicin, and several putative enediyne natural products in soil and marine microorganisms (8 -11). Recently, based on the study on the 9-membered enediynecontaining C-1027, Shen and coworkers found that the iterative PKS (SgcE) and the putative thioesterase (SgcE10) generated a conjugated polyene (1,3,5,7,9,11,13-pentadecaheptaene) through an ACP-tethered 3-hydroxy-4...

Background: The NS5 protein from dengue virus comprises a methyltransferase and a polymerase domain connected by a linker region. Results: Linker residues enhance polymerase activity and thermostability. Conclusion: A crystal structure of the dengue virus polymerase reveals that linker residues contribute to protein stability. Significance: These results should accelerate the development of antivirals against dengue virus, a major human pathogen.

We have characterized a linear carbonyl-conjugated polyene generated by the iterative polyketide synthase (CalE8) involved in the biosynthesis of the 10-membered enediyne core of calicheamicin. The results provide insight into the mysterious biosynthetic mechanism of the unique enediyne. The carbonyl-conjugated polyene differs from the precursor for 9-membered enediyne, suggesting that the divergence of enediyne biosynthesis starts at the PKS stage.

RbdA is a positive egulator ofiofilm ispersal of Its cytoplasmic region (cRbdA) comprises a N-terminal PAS domain followed by a diguanylate cyclase (GGDEF) and an EAL domain, whose phosphodiesterase activity is allosterically stimulated by GTP binding to the GGDEF domain. We report crystal structures of cRbdA and of two binary complexes: with GTP/Mg bound to the GGDEF active site and with the EAL domain bound to the c-di-GMP substrate. These structures unveil a 2-fold symmetric dimer, stabilized by a closely packed N-terminal PAS domain and a non-canonical EAL dimer. The auto-inhibitory switch is formed by an alpha helix (S-helix) immediately N-terminal to the GGDEF domain that interacts with the EAL dimerization helix (α) of the other EAL monomer and maintains the protein in a locked conformation. We propose that local conformational changes in cRbdA upon GTP binding lead to a structure with the PAS domain and S-helix shifted away from the GGDEF-EAL domains, as suggested by SAXS experiments. Domain reorientation should be facilitated by the presence of a α-helical lever (H-helix) that tethers the GGDEF and EAL regions, allowing the EAL domain to rearrange into an active dimeric conformation.Biofilm formation by bacterial pathogens increases resistance to antibiotics. RbdA positively regulates biofilm dispersal of The crystal structures of the cytoplasmic region of RbdA protein presented here reveal that two evolutionary-conserved helices play an important role in regulating the activity of RbdA, with implications for other dual GGDEF-EAL domains that are abundant in the proteomes of several bacterial pathogens. Thus, this work could assist the development of small molecules that would promote bacterial biofilm dispersal.

The iterative polyketide synthases from the biosynthetic pathways of three enediyne natural products were examined. The results established the all-trans conjugated pentadecaheptanene as the only major product shared by the PKSs. The experiments further revealed some intrinsic differences among the PKSs by demonstrating the formation of different by-products.

Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNAMet(CAU) and tRNATrp(CCA) are substrates for Cm formation, tRNAGln(UUG), tRNAPro(UGG), tRNAPro(CGG) and tRNAHis(GUG) for Um, and tRNAPro(GGG) for Am. tRNASer(UGA), previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNAPro(GGG) and Um in tRNAGln(UUG) by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE. These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response.