Structural and functional studies on type III and type VI secretion system proteins

Ph.DDOCTOR OF PHILOSOPH

Structural Basis for the Secretion of EvpC: A Key Type VI Secretion System Protein from Edwardsiella tarda

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 α-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

Structure of GrlR and the Implication of Its EDED Motif in Mediating the Regulation of Type III Secretion System in EHEC

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

Crystal Structure of Der f 7, a Dust Mite Allergen from Dermatophagoides farinae

10.1371/journal.pone.0044850PLoS ONE79

Targeting cancer addiction for SALL4 by shifting its transcriptome with a pharmacologic peptide

Sal-like 4 (SALL4) is a nuclear factor central to the maintenance of stem cell pluripotency and is a key component in hepatocellular carcinoma, a malignancy with no effective treatment. In cancer cells, SALL4 associates with nucleosome remodeling deacetylase (NuRD) to silence tumor-suppressor genes, such as PTEN. Here, we determined the crystal structure of an amino-terminal peptide of SALL4(1-12) complexed to RBBp4, the chaperone subunit of NuRD, at 2.7 Å, and subsequent design of a potent therapeutic SALL4 peptide (FFW) capable of antagonizing the SALL4-NURD interaction using systematic truncation and amino acid substitution studies. FFW peptide disruption of the SALL4-NuRD complex resulted in unidirectional up-regulation of transcripts, turning SALL4 from a dual transcription repressor-activator mode to singular transcription activator mode. We demonstrate that FFW has a target affinity of 23 nM, and displays significant antitumor effects, inhibiting tumor growth by 85% in xenograft mouse models. Using transcriptome and survival analysis, we discovered that the peptide inhibits the transcription-repressor function of SALL4 and causes massive up-regulation of transcripts that are beneficial to patient survival. This study supports the SALL4-NuRD complex as a drug target and FFW as a viable drug candidate, showcasing an effective strategy to accurately target oncogenes previously considered undruggable

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

10.1371/journal.ppat.1003948PLoS Pathogens103

Identification and Structural Characterization of a New Three-Finger Toxin Hemachatoxin from Hemachatus haemachatus Venom

10.1371/journal.pone.0048112PLoS ONE710

Crystallization and preliminary X-ray diffraction analysis of hemextin A: a unique anticoagulant protein from Hemachatus haemachatus venom

Crystals of hemextin A, a three-finger toxin isolated and purified from African Ringhals cobra (H. haemachatus), are orthorhombic, space group P212121, with unit-cell parameters a = 49.27, b = 49.51, c = 57.87 Å, and diffract to 1.5 Å resolution

Crystallization of uracil phosphoribosyltransferase (MtUPRT) from Mycobacterium tuberculosis

Exploring new drug targets in parallel to designing strategies for rational use of existing drugs would greatly aid the Tuberculosis (TB) drug development program. The key enzymes involved in the essential metabolic and regulatory pathways are usually sought for in the pursuit of potential drug targets. Likewise, uracil phosphoribosyltransferase (UPRT) is a key enzyme in the synthesis uridine 5’-monophosphate (UMP), the precursor of the pyrimidine nucleotides. It has been recently shown to be the probable target of 5-fluorouracil in Mycobacterium tuberculosis (Mtb). Here we report the purification, characterization and crystallization of the full length UPRT from Mtb (MtUPRT) encoded by the gene upp (Rv3309c). The MtUPRT was overexpressed in BL21 (DE3) E.coli expression system followed by three step chromatographic purification procedures. The purified MtUPRT was concentrated to 8mg/ml; single crystals were obtained using the sitting drop vapour diffusion method. The crystals were diffracted to 3.0 Å resolution and belonged to the space group P32 with unit cell parameters a = b = 118.09, c = 77.88 Å and four monomers in the asymmetric unit. Understanding the three dimensional structure of this essential enzyme will greatly help in screening of appropriate inhibitors of MtUPRT and thus assist in TB drug development