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Background: Der f 7 is the group 7 allergen from the dust mite Dermatophagoides farinae, homologous to the major allergen Der p 7 from D. pteronyssinus. Monoclonal antibody that bind to residues Leu48 and Phe50 was found to inhibit IgE binding to residue Asp159, which is important for the cross-reactivity between Der f 7 and Der p 7. Methodology/Principal Findings: Here, we report the crystal structure of Der f 7 that shows an elongated and curved molecule consisting of two anti-parallel β-sheets - one 4-stranded and the other 5-stranded - that wrap around a long Cterminal helix. The overall fold of Der f 7 is similar to Der p 7 but key difference was found in the β1-β2 loop region. In Der f 7, Leu48 and Phe50 are in close proximity to Asp159, explaining why monoclonal antibody binding to Leu48 and Phe50 can inhibit IgE binding to Asp159. Both Der f 7 and Der p 7 bind weakly to polymyxin B via a similar binding site that is formed by the N-terminal helix, the 4-stranded b-sheet and the C-terminal helix. The thermal stability of Der f 7 is significantly lower than that of Der p 7, and the stabilities of both allergens are highly depend on pH. Conclusion/Significance:Der f 7 is homologous to Der p 7 in terms of the amino acid sequence and overall 3D structure but with significant differences in the region proximal to the IgE epitope and in thermal stability. The crystal structure of Der f 7 provides a basis for studying the function and allergenicity of this group of allergens. [ABSTRACT FROM AUTHOR]

Background: The putative needle complex subunit AscF forms a ternary complex with the chaperones AscE and AscG in the type III secretion system of Aeromonas hydrophila so as to avoid premature assembly. Previously, we demonstrated that the C-terminal region of AscG (residues 62-116) in the hetero-molecular chaperone, AscE-AscG, is disordered and susceptible to limited protease digestion. Methodology/Principal Findings: Here, we report the crystal structure of the ordered AscG1-61 region in complex with AscE at 2.4 Å resolution. Helices α2 and α3 of AscE in the AscE-AscG1-61 complex assumes a helix-turn-helix conformation in an anti-parallel fashion similar to that in apo AscE. However, in the presence of AscG, an additional N-terminal helix α1 in AscE (residues 4-12) is observed. PscG or YscG in the crystal structures of PscE-PscF-PscG or YscE-YscF-YscG, respectively, assumes a typical tetratricopeptide repeat (TPR) fold with three TPR repeats and one C-terminal capping helix. By comparison, AscG in AscE-AscG1-61 comprises three anti-parallel helices that resembles the N-terminal TPR repeats in the corresponding region of PscG or YscG in PscE-PscF-PscG or YscE-YscF-YscG. Thermal denaturation of AscE-AscG and AscEAscG1- 61 complexes demonstrates that the C-terminal disordered region does not contribute to the thermal stability of the overall complex. Conclusion/Significance: The N-terminal region of the AscG in the AscE-AscG complex is ordered and assumes a structure similar to those in the corresponding regions of PscE-PscG-PscF or YscE-YscF-YscG complexes. While the C-terminal region of AscG in the AscE-AscG complex is disordered and will assume its structure only in the presence of the substrate AscF. We hypothesize that AscE act as a chaperone of the chaperone to keep AscG in a stable but partially disordered state for interaction with AscF. [ABSTRACT FROM AUTHOR]

The recently identified type VI secretion system (T6SS) is implicated in the virulence of many Gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E. tarda virulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 Å resolution, along with functional studies of the protein. EvpC has a β-barrel domain with extended loops. The β-barrel consists of 11 anti-parallel β-strands with an a-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in Gram-negative pathogens. [ABSTRACT FROM AUTHOR]

Background: Nuclear Magnetic Resonance (NMR) spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, 15N Heteronuclear Single Quantum Correlation (HSQC) experiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of 15N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately. Methodology/Principal Findings: We have automated the mapping of binding sites for fast chemical exchange systems using information obtained from 15N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer) determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues. Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein hBclXL and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of hBclXL were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant (3:0 × 104) calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (12:8 × 104). Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker than the former one. Further NMR based model fitting for individual residues suggest single site model for residues present at these binding sites and two site model for residues present between these sites. This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event. Conclusion/Significance: Detail NMR chemical shift perturbation analysis enabled binding site residues to be distinguished from non-binding site residues for accurate mapping of interaction site in complex fast exchange system between small molecule and protein. The methodology is automated and implemented in a program called ''Auto-FACE'', which also allowed quantitative information of each interaction site and elucidation of binding mechanism. [ABSTRACT FROM AUTHOR]

Asthma is a chronic inflammatory disease characterized by airway inflammation, mucus hypersecretion and airway hyperresponsiveness. Mechanisms underlying the pathogenesis of asthma are not fully understood. In recent years, there are mounting evidences demonstrating that mammalian chitinases may play a key role in mediating the T-helper 2 cell-driven inflammatory response that is commonly associated with asthma. Chitinases (e.g., chitotriosidase and acidic mammalian chitinase) are enzymes that degrade chitin, the second most abundant biopolymer that can be found in the cell walls of fungi, microfilarial sheaths of helminths, and exoskeletons of insects and crustaceans. There are also chitinase-like proteins (e.g., YKL-40, Ym1 and Ym2) that lack chitinolytic activity but retain chitin-binding ability. Therefore, chitinases were originally believed to function in host defense against parasitic infections, but the first discovery of their role in inflammatory airway diseases came as a surprise. There is ample evidence to support an association of acidic mammalian chitinase and YKL-40 with allergic bronchial asthma in patients. Our recent studies in a mouse asthma model revealed that anti-inflammatory drugs like corticosteroid and cysteinyl leukotriene receptor antagonist were able to suppress elevated pulmonary levels of mammalian chitinases. Taken together, mammalian chitinases may be useful as biomarkers for asthma. Notwithstanding, large-scale multi-center association studies are required to confirm this hypothesis. Besides, substantially more works using knockout mice, recombinant chitinases and siRNA technology are required to investigate a potential role of chitinases in the pathogenesis of asthma. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Lipocalins are a broad family of proteins identified initially in eukaryotes and more recently in Gram-negative bacteria. The functions of lipocalin or lipid-binding proteins are often elusive and very diverse. Recently, we have determined the structure of GrlR (global regulator of LEE repressor), which plays a key role in the regulation of LEE (locus of enterocyte effacement) proteins. GrlR adopts a lipocalin-like fold that is composed of an eight-stranded β-barrel followed by an α-helix at the C-terminus. GrlR has a highly hydrophobic cavity region and could be a potential transporter of lipophilic molecules. To verify this hypothesis, we carried out structure-based analysis of GrlR, determined the structure of the lipid–GrlR complex and measured the binding of lipid to recombinant GrlR by ITC (isothermal titration calorimetry). In addition, we identified phosphatidylglycerol and phosphatidylethanolamine as the endogenously bound lipid species of GrlR using electrospray-ionization MS. Furthermore, we have shown that the lipid-binding property of GrlR is similar to that of its closest lipocalin structural homologue, β-lactoglobulin. Our studies demonstrate the hitherto unknown lipid-binding property of GrlR. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) is a common cause of severe hemorrhagic colitis. EHEC's virulence is dependent upon a type III secretion system (TTSS) encoded by 41 genes. These genes are organized in several operons clustered in the locus of enterocyte effacement. Most of the locus of enterocyte effacement genes, including grlA and grlR, are positively regulated by Ler, and Ler expression is positively and negatively modulated by GrlA and GrlR, respectively. However, the molecular basis for the GrlA and GrlR activity is still elusive. We have determined the crystal structure of GrlR at 1.9 Å resolution. It consists of a typical β-barrel fold with eight β-strands containing an internal hydrophobic cavity and a plug-like loop on one side of the barrel. Strong hydrophobic interactions between the two β-barrels maintain the dimeric architecture of GrlR. Furthermore, a unique surface-exposed EDED (Glu-Asp-Glu-Asp) motif is identified to be critical for GrlA-GrlR interaction and for the repressive activity of GrlR. This study contributes a novel molecular insight into the mechanism of GrlR function. [ABSTRACT FROM AUTHOR]