A conserved feature of poxviruses is a protein, well characterized as E3L in vaccinia virus, that confers IFN resistance on the virus. This protein comprises two domains, an N-terminal Z-DNA-binding protein domain (Z␣) and a C-terminal double-stranded RNA-binding domain. Both are required for pathogenicity of vaccinia virus in mice infected by intracranial injection. Here, we describe the crystal structure of the Z␣ domain from the E3L-like protein of Yaba-like disease virus, a Yatapoxvirus, in a complex with Z-DNA, solved at a 2.0-Å resolution. The DNA contacting surface of Yaba-like disease virus Z␣ E3L closely resembles that of other structurally defined members of the Z␣ family, although some variability exists in the ␤-hairpin region. In contrast to the Z-DNA-contacting surface, the nonbinding surface of members of the Z␣ family are unrelated; this surface may effect protein-specific interactions. The presence of the conserved and tailored Z-DNA-binding surface, which interacts specifically with the zigzag backbone and syn base diagnostic of the Z-form, reinforces the importance to poxvirus infection of the ability of this protein to recognize the Z-conformation. The Yaba monkey tumor virus (YMTV) and the closely related Yaba-like disease virus (YLDV) are members of the Yatapoxvirus family. Both viruses have double-stranded DNA genomes of Ϸ150 kilobase pairs and code for Ͼ150 proteins (1). YMTV induces histiocytomas with characteristic microscopic morphology in soft tissue of monkeys and baboons (2, 3) and humans (4). YLDV, as well as a third Yatapoxvirus, Tanapox, causes vesicular skin lesions in monkeys and humans (5, 6). Furthermore, YMTV DNA has transforming activity in monkey cell lines (7). The high similarity between the YLDV and YMTV was revealed with the recent sequencing of the Yaba monkey tumor virus (8).All sequenced poxviruses have a gene, called E3L in vaccinia, that is required for IFN resistance. E3L has been shown to be oncogenic and antiapoptotic; NIH 3T3 cells expressing vaccinia E3L were found to grow faster than control cells, with increased expression of cyclin A and decreased levels of the suppressor molecule p26 (9). When the NIH 3T3 E3L-expressing cells were injected into nude mice, solid tumors were formed. The expression of E3L has been shown to be essential for these oncogenic and antiapoptotic activities.The E3L protein and its orthologues comprise two domains, an N-terminal Z-DNA-binding protein domain (Z␣) and a C-terminal double-stranded RNA-binding domain. A similar Z␣ motif is found in vertebrate ADAR1 (double-stranded RNA adenosine deaminase) and in the IFN-inducible DLM-1 (also known as ZBP-1) of mammals (10, 11). Extensive biochemical and biophysical studies of human Z␣ ADAR1 show that it binds tightly and specifically to Z-DNA (10,(12)(13)(14). Both domains of the mouse-adapted vaccinia virus E3L are essential for pathogenicity in mice (15, 16). The C-terminal domain is sufficient for viral replication in cultured cells in vitro. However, both domains are needed to infe...

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UDP-glucose pyrophosphorylases (UGPase; EC 2.7.7.9) catalyze the conversion of UTP and glucose-1-phosphate to UDP-glucose and pyrophosphate and vice versa. Prokaryotic UGPases are distinct from their eukaryotic counterparts and are considered appropriate targets for the development of novel antibacterial agents since their product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharides and capsular polysaccharides. In this study, the crystal structures of UGPase from Helicobacter pylori (HpUGPase) were determined in apo- and UDP-glucose/Mg(2+)-bound forms at 2.9 A and 2.3 A resolutions, respectively. HpUGPase is a homotetramer and its active site is located in a deep pocket of each subunit. Magnesium ion is coordinated by Asp130, two oxygen atoms of phosphoryl groups, and three water molecules with octahedral geometry. Isothermal titration calorimetry analyses demonstrated that Mg(2+) ion plays a key role in the enzymatic activity of UGPase by enhancing the binding of UGPase to UTP or UDP-glucose, suggesting that this reaction is catalyzed by an ordered sequential Bi Bi mechanism. Furthermore, the crystal structure explains the specificity for uracil bases. The current structural study combined with functional analyses provides essential information for understanding the reaction mechanism of bacterial UGPases, as well as a platform for the development of novel antibacterial agents.

Escherichia coli SdiA is a quorum-sensing (QS) receptor that responds to autoinducers produced by other bacterial species to control cell division and virulence. Crystal structures reveal that E. coli SdiA, which is composed of an N-terminal ligand-binding domain and a C-terminal DNA-binding domain (DBD), forms a symmetrical dimer. Although each domain shows structural similarity to other QS receptors, SdiA differs from them in the relative orientation of the two domains, suggesting that its ligand-binding and DNA-binding functions are independent. Consistently, in DNA gel-shift assays the binding affinity of SdiA for the ftsQP2 promoter appeared to be insensitive to the presence of autoinducers. These results suggest that autoinducers increase the functionality of SdiA by enhancing the protein stability rather than by directly affecting the DNA-binding affinity. Structural analyses of the ligand-binding pocket showed that SdiA cannot accommodate ligands with long acyl chains, which was corroborated by isothermal titration calorimetry and thermal stability analyses. The formation of an intersubunit disulfide bond that might be relevant to modulation of the DNA-binding activity was predicted from the proximal position of two Cys residues in the DBDs of dimeric SdiA. It was confirmed that the binding affinity of SdiA for the uvrY promoter was reduced under oxidizing conditions, which suggested the possibility of regulation of SdiA by multiple independent signals such as quorum-sensing inducers and the oxidation state of the cell.