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Amyloidogenesis of a Type III-dependent Hpa1 and Structural analysis of a drug-pump repressor DNA complex

ZHANG, BING (2010) Amyloidogenesis of a Type III-dependent Hpa1 and Structural analysis of a drug-pump repressor DNA complex. Doctoral thesis, Durham University.




Part I Amyloidogenesis of Type III-dependent Hpa1 from Xanthomonas oryzae pv. oryzicola

The first harpin, HrpN from Erwinia amylovora, the bacterium that causes fire blight of pear, apple and other rosaceous plants, was reported as an HR (hypersensitive response) elicitor in 1992. Similar harpins were found in Pseudomonas syringae, Ralstonia solanacearum, Xanthomonas campestris, X. axopnpodis and X. oryzae, and secreted through a Type III secretion system (TTSS), which is utilized by bacterial pathogens to invade the host. The wide presence and secretion pathway of harpins in plant pathogens indicate the important function of harpins in plant pathogenicity.

Herein, I report that Hpa1, the harpin of X. oryzae pv. oryzicola, assembles into fibrils at acidic pHs. As determined by analytical gel filtration chromatography, purified Hpa1 is a decamer. Hpa1 has two predicted  helical domains, located towards its N- and C-terminus, respectively. The C-terminal  helix domain is responsible for the fibril assembly, since the synthesized polypeptide (ASPLTQMLMNIVGEILQAQ), corresponding to the C-terminal  helix domain of Hpa1, could form fibrils. The mature fibrils of this peptide consist of three strands of protofibrils. We propose that the fibril formation by full length Hpa1 involves a conformational transition from -helix to β-sheet structure, which exposes the C-terminal  helix domain for fibril assembly. In contrast to the assembly of fibrils by full length Hpa1 at acidic pH, that of synthesized polypeptide (ASPLTQMLMNIVGEILQAQ) is at acidic and basic pH, which may be an inherent characteristic of the sequence component. Interestingly, the N-terminal  helix domain (ISEKQLDQLLCQLIQALL) of Hpa1 inhibits the assembly of fibrils by the C-terminal domain polypeptide, suggesting that the intramolecular interaction of Hpa1 between the two  helix domains keeps Hpa1 in a non-fibrillar state.

In addition, the fibrils formed by full length Hpa1, the N-terminal truncated Hpa1 (55-138) and synthesized polypeptide (ASPLTQMLMNIVGEILQAQ) disrupted the membrane of liposomes prepared from extracted lipids from E. coli, which suggests a new function for Hpa1- targeting and damaging the cell membrane.

Part II Crystallization and structure analysis of the Vibrio cholerae VceR drug-pump repressor with bound DNA

The VceR protein, from Vibrio cholerae, which is encoded by the vceR gene, represses transcription of the vceCAB operon, which encodes a multidrug efflux pump in this microorganism. This type of pump, composed of a tripartite protein complex that spans the inner and outer membranes, resembles type I secretion systems. VceR, which is a member of the TetR family of transcriptional repressors, binds specifically to a 28 bp inverted repeat (IR) located in the 107 bp vceR-vceCAB intergenic region.
In order to completely understand the structural basis and functional mechanism by which VceR binds DNA, we performed crystallization trials on the VceR/DNA complex, with the aim of determining its crystal structure. A number of oligos were screened, including 30 bp, 28 bp, 27 bp, 26 bp and 24 bp duplex, depending on the published operator sequence bound by VceR, to obtain X-ray quality VceR/DNA complex crystals for structure analysis. These crystals have space group I 4122, with a=b=90.2 Å, c=298.1 Å, α=β=γ=90°.
To retrieve phase information, Se-Met substituted VceR and Br labelled DNA oligonucleotides were prepared and crystallized with unlabelled DNA and native VceR, respectively. Several three-wavelength MAD data sets from these crystals were collected and analyzed. A merged Se-Met data set, obtained from 2 data sets of three wavelengths and one peak data set, yielded the correct positions of 12 Se by ShelxD. Sharp/Autosharp was used to refine these 12 Se positions and for calculating phases. A preliminary electron density map shows, after solvent flattening, clear helical characteristics in the VceR dimer and a partial DNA backbone. A model with one VceR dimer and an 11 base pair duplex was built in the preliminary electron density map.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Faculty and Department:Faculty of Science > Biological and Biomedical Sciences, School of
Thesis Date:2010
Copyright:Copyright of this thesis is held by the author
Deposited On:13 May 2010 11:28

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