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Andrew H-J Wang

Publications and source records attributed to Andrew H-J Wang.

At least 19 recordsLinked to original sources
PubMed2007

Enzyme-substrate interactions revealed by the crystal structures of the archaeal Sulfolobus PTP-fold phosphatase and its phosphopeptide complexes.

The P-loop-containing protein phos-phatases are important regulators in signal transduction. These enzymes have structural and functional similarity with a conserved sequence of Dx(25-41)HCxxGxxR(T/S) essential for catalysis. The singular protein tyrosine phosphatase (PTP) from archaeal Sulfolobus solfataricus is one of the smallest known PTPs with extreme thermostability. Here, we report the crystal structure of this phosphatase and its complexes with two tyrosyl phosphopeptides A-(p)Y-R and N-K-(p)Y-G-N. The structure suggests the minimal structural motif of the PTP family, having two variable sequences inserted between the beta2-beta3 and beta3-beta4 strands, respectively. The phosphate of both phosphopeptide substrates is bound to the P-loop through several hydrogen bonds. Comparison of several phosphatase-substrate complexes revealed that Gln135 on the Q-loop has different modes of recognition toward phosphopeptides. The substrate specificity of SsoPTP is primarily localized at the phosphotyrosine, suggesting that this phosphatase may be a prototypical PTP.

Amino Acid Sequence↗
PubMed2006

Preliminary X-ray analysis of XC5848, a hypothetical ORFan protein with an Sm-like motif from Xanthomonas campestris.

XC5848, a hypothetical protein from the pathogenic bacterium Xanthomonas campestris that causes black rot, has been chosen as a potential target for the discovery of novel folds. It is unique to the Xanthomonas genus and has significant sequence identity mainly to corresponding proteins from the Xanthomonas genus. In this paper, the cloning, overexpression, purification and crystallization of the XC5848 protein are reported. The XC5848 crystals diffracted to a resolution of at least 1.68 A. They belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 48.13, b = 51.62, c = 82.32 A. Two molecules were found in each asymmetric unit. Preliminary structural studies nevertheless indicate that XC5848 belongs to the highly conserved Sm-like alpha-beta-beta-beta-beta fold. However, significant differences in sequence and structure were observed. It therefore represents a novel variant of the crucial Sm-like motif that is heavily involved in mRNA splicing and degradation.

Amino Acid Motifs↗
PubMed2006

Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection.

To better understand the pathogenesis of white spot syndrome virus (WSSV) and to determine which cell pathways might be affected after WSSV infection, two-dimensional gel electrophoresis (2-DE) was used to produce protein expression profiles from samples taken at 48 h post-infection (hpi) from the stomachs of Litopenaeus vannamei (also called Penaeus vannamei) that were either specific pathogen free or else infected with WSSV. Seventy-five protein spots that consistently showed either a marked change (>50%) in accumulated levels or else were highly expressed throughout the course of WSSV infection were selected for further study. After in-gel trypsin digestion followed by LC-nanoESI-MS/MS, bioinformatics databases were searched for matches. A total of 53 proteins were identified, with functions that included energy production, calcium homeostasis, nucleic acid synthesis, signaling/communication, oxygen carrier/transportation, and SUMO-related modification. 2-DE results were shown to be consistent with relative EST database data from a previously developed EST database of two Penaeus monodon cDNA libraries. For seven selected genes, 2-DE and EST data were also compared with transcriptional time-course RT-PCR data. This study is the first global analysis of differentially expressed proteins in WSSV-infected shrimp, and in addition to increasing our understanding of the molecular pathogenesis of this virus-associated shrimp disease, the results presented here should be useful both for identifying potential biomarkers and for developing antiviral measures.

Animals↗
PubMed2006

Crystallization and preliminary X-ray analysis of XC1015, a histidine triad-like protein from Xanthomonas campestris.

Histidine-triad (HIT) proteins are a superfamily of nucleotide hydrolases and transferases that contain a conserved Hphi Hphi Hphi phi motif (where phi is a hydrophobic amino acid) and are found in a variety of organisms. In addition to binding to a variety of nucleotides, other biological functions of the HIT superfamily proteins have been discovered and HIT malfunction has been implicated in several human diseases. Structural studies of HIT superfamily proteins are thus of particular interest. In this manuscript, the cloning, expression, crystallization and preliminary X-ray analysis of XC1015, a HIT protein present in the plant pathogen Xanthomonas campestris pathovar campestris, are reported. The XC1015 crystals diffracted to a resolution of 1.3 A. They are tetragonal and belong to space group P4(3)2(1)2, with unit-cell parameters a = 40.52, b = 40.52, c = 126.89 A.

Bacterial Proteins↗
PubMed2006

Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase.

Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.

Adenosine Triphosphate↗
PubMed2006

White spot syndrome virus annexes a shrimp STAT to enhance expression of the immediate-early gene ie1.

Although the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is part of the antiviral response in arthropods such as Drosophila, here we show that white spot syndrome virus (WSSV) uses a shrimp STAT as a transcription factor to enhance viral gene expression in host cells. In a series of deletion and mutation assays using the WSSV immediate-early gene ie1 promoter, which is active in shrimp cells and also in insect Sf9 cells, an element containing a STAT binding motif was shown to be important for the overall level of WSSV ie1 promoter activity. In the Sf9 insect cell line, a specific protein-DNA complex was detected by using electrophoresis mobility shift assays (EMSA) with the 32P-labeled STAT binding motif of the WSSV ie1 promoter as the probe. When recombinant Penaeus monodon STAT (rPmSTAT) was overexpressed in Sf9 cells, EMSA with specific antibodies confirmed that the STAT was responsible for the formation of the specific protein-DNA complex. Another EMSA showed that in WSSV-infected P. monodon, levels of activated PmSTAT were higher than in WSSV-free P. monodon. A transactivation assay of the WSSV ie1 promoter demonstrated that increasing the level of rPmSTAT led to dose-dependent increases in ie1 promoter activity. These results show that STAT directly transactivates WSSV ie1 gene expression and contributes to its high promoter activity. We conclude that WSSV successfully annexes a putative shrimp defense mechanism, which it uses to enhance the expression of viral immediate-early genes.

Animals↗
PubMed2006

Photoreactivation of DNA by an archaeal nucleoprotein Sso7d.

Sso7d is a chromosomal protein of hyperthermophilic Archaea. The crystal structure of Sso7d-d(GTAAT(I)UAC)(2) has been clarified at high resolution, showing that the protein binds in the minor groove of DNA, causing a sharp kink of approximately 60 degrees. Recently, we found that photoirradiation of Sso7d and 5-iodouracil-((I)U)-containing 5'-d(GTAAT(I)UAC)-3' efficiently induced the abstraction of hydrogen from the methyl group of T(5) at the kink. In the present study, we found that the photoreactivity of 5-bromouracil ((Br)U)-containing 5'-d(GTAAT(Br)UAC)-3' was enhanced in the presence of Sso7d. Using hypoxanthine (I)-containing 5'-d(ITAAT(Br)UAC)-3', we demonstrated that electron transfer occurs efficiently from Sso7d to DNA. Product analysis showed that Trp-24 of Sso7d, located at the surface of the DNA, is consumed to produce N'-formylkynurenine during photoirradiation, indicating that Trp-24 acts as an electron source. To explore the possibility of electron transfer between Sso7d and other DNA substrates, we examined the photochemical repair of the thymine dimer 5'-d(GTAAT<>TAC)-3' by Sso7d. Sso7d effectively repaired 5'-d(GTAAT<>TAC)-3' to 5'-d(GTAATTAC)-3' under irradiation conditions. During this reaction, Trp-24 was not oxidized significantly, indicating that the anion radical of the repaired TT sequence is oxidized by the cation radical of Trp-24, and that a so-called "circular electron transfer" mechanism is operating in this system.

Archaeal Proteins↗
PubMed2006

Cloning, crystallization and preliminary X-ray studies of XC2981 from Xanthomonas campestris, a putative CutA1 protein involved in copper-ion homeostasis.

Divalent metal ions play key roles in all living organisms, serving as cofactors for many proteins involved in a variety of electron-transfer activities. However, copper ions are highly toxic when an excessive amount is accumulated in a cell. CutA1 is a protein found in all kingdoms of life that is believed to participate in copper-ion tolerance in Escherichia coli, although its specific function remains unknown. Several crystal structures of multimeric CutA1 with different rotation angles and degrees of interaction between trimer interfaces have been reported. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2981, a possible CutA1 protein present in the plant pathogen Xanthomonas campestris, are reported. The XC2981 crystals diffracted to a resolution of 2.6 A. They are cubic and belong to space group I23, with unit-cell parameters a = b = c = 130.73 A.

Bacterial Proteins↗
PubMed2006

Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase.

Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a Mn(II)-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215-222, 1997; Gengenbach et al. in Biochemistry 38:11425-11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k (cat)/K (M)) than the variant in the original design, mostly due to a stronger K (M) of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with Co(II) at the designed Mn(II) site were also obtained. The metal ion in the engineered metal-binding site overlays well with Mn(II) bound in MnP, suggesting that this variant is the closest structural model of the Mn(II)-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal-ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (Co(II) rather than Mn(II)) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.

Binding Sites↗
PubMed2006

Cloning, crystallization and preliminary X-ray study of XC1258, a CN-hydrolase superfamily protein from Xanthomonas campestris.

CN-hydrolase superfamily proteins are involved in a wide variety of non-peptide carbon-nitrogen hydrolysis reactions, producing some important natural products such as auxin, biotin, precursors of antibiotics etc. These reactions all involve attack on a cyano or carbonyl carbon by a conserved novel catalytic triad Glu-Lys-Cys through a thiol acylenzyme intermediate. However, classification into the CN-hydrolase superfamily based on sequence similarity alone is not straightforward and further structural data are necessary to improve this categorization. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC1258, a CN-hydrolase superfamily protein from the plant pathogen Xanthomonas campestris (Xcc), are reported. The SeMet-substituted XC1258 crystals diffracted to a resolution of 1.73 A. They are orthorhombic and belong to space group P2(1)2(1)2, with unit-cell parameters a = 143.8, b = 154.63, c = 51.3 A, respectively.

Amino Acid Sequence↗
PubMed2006

The cloning, crystallization and preliminary X-ray analysis of XC2113, a YaeQ protein from Xanthomonas campestris.

Xanthomonas campestris is a Gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. It is therefore important to identify potential pathogenic factors involved in this plant disease. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2113, a YaeQ protein possibly involved in the production of virulence factors in Xanthomonas campestris pathovar campestris, are reported. The XC2113 crystals diffracted well to a resolution of at least 1.28 A. They are orthorhombic and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.86, b = 62.69, c = 79.96 A.

Bacterial Proteins↗
PubMed2006

Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor.

A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, Ki = 53 nM) has been developed. TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E replications by reducing the viral titer by 4.7 log (at 5 microM) for SARS CoV and 5.2 log (at 1.25 microM) for HCoV 229E. The crystal structure of TG-0205221 (resolution = 1.93 A) has revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information may be applied toward the design of other 3CL protease inhibitors.

Animals↗
PubMed2006

Purification, crystallization and preliminary X-ray crystallographic analysis of rice Bowman-Birk inhibitor from Oryza sativa.

Bowman-Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 A, the unit cell belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 74.37, b = 96.69, c = 100.36 A. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%.

Crystallization↗
PubMed2006

Crystal structure of infectious bursal disease virus VP2 subviral particle at 2.6A resolution: implications in virion assembly and immunogenicity.

The structural protein VP2 of infectious bursal disease virus (IBDV) spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6A resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. Our results of treatment of SVP with EGTA, a Ca(2+)-chelating reagent, indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca(2+)-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes.

Amino Acid Sequence↗