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Discovery and Characterization of Novel ADP-Ribosylating Toxins

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Title: Discovery and Characterization of Novel ADP-Ribosylating Toxins
Author: Fieldhouse, Robert John
Department: Department of Molecular and Cellular Biology
Program: Biophysics
Advisor: Merrill, A. Rod
Abstract: This thesis is an investigation of novel mono-ADP-ribosylating toxins. In the current data-rich era, making the leap from sequence data to knowledge is a task that requires an elegant bioinformatics toolset to pinpoint questions. A strategy to expand important protein-family knowledge is required, particularly in cases in which primary sequence identity is low but structural conservation is high. For example, the mono-ADP-ribosylating toxins fit these criteria and several approaches have been used to accelerate the discovery of new family members. A newly developed tactic for detecting remote members of this family -- in which fold recognition dominates -- reduces reliance on sequence similarity and advances us toward a true structure-based protein-family expansion methodology. Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins identified and characterized using in silico and cell-based techniques. Medically relevant toxins from Mycobacterium avium and Enterococcus faecalis were also uncovered. Agriculturally relevant toxins were found in Photorhabdus luminescens and Vibrio splendidus. Computer software was used to build models and analyze each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. Yeast-based activity tests have since confirmed activity. Vibrio cholerae produces cholix – a potent protein toxin of particular interest that has diphthamide-specific ADP-ribosyltransferase activity against eukaryotic elongation factor 2. Here we present a 2.1Å apo X-ray structure as well as a 1.8Å X-ray structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide (NAD+). Hallmark catalytic residues were substituted and analyzed both for NAD+ binding and ADP-ribosyltransferase activity using a fluorescence-based assay. These new toxins serve as a reference for ongoing inhibitor development for this important class of virulence factors. In addition to using toxins as targets for antivirulence compounds, they can be used to make vaccines and new cancer therapies.
Date: 2011-12
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