Toxin-antitoxin-DNA interactions

Low-copy-number plasmids often possess toxin-antitoxin systems, which guarantee preferential growth of plasmid-carrying cells in a bacterial population by killing newborn bacteria that have not inherited a plasmid at cell division. TA systems have also been found on bacterial chromosomes. Although the function of chromosomal TA systems is still under debate they have been postulated to help bacteria deal with nutritional stress by adjusting the rates of protein and DNA synthesis via down-regulation of translation and replication. In the presented project we plan to carry out a structural and dynamic investigation of the chromosomal antitoxin MazE bound to its cognate DNA. This protein-DNA complex will be the first of this fold since the N-terminal part of MazE forms a completely new DNA binding motif. In addition we will investigate the ternary interactions of toxin (CcdB), antitoxin (CcdA) and their cognate DNA with a new exchange quenched NMR technique. A novel NMR approach will also be established and used for the solution structure determination of the MazE-DNA complex. We will be using paramagnetic relaxation enhancements as structural restraints. By using these PRE values the number of necessary NOEs can be reduced significantly to obtain the protein fold. Besides the establishment of novel techniques the presented project will provide a deeper understanding of how plasmid stabilization and maintenance in bacteria is achieved and about differences between plasmidic and chromosomal toxin-antitoxin modules. Apart from these fundamental biological questions, killing systems play an increasing role in biotechnology due to their use for stabilizing autonomously replicating vectors employed in recombinant bacteria. In addition, structural studies on TA systems hold promise for the design of new antibiotics and might open a new route towards fighting antibiotic resistance, which uses TA systems to ensure their persistence during host replication.

The major goals of this project could all be achieved and partly surpassed: A method of using relaxation enhancements in a paramagnetic environment for the structure determination of proteins was carried out on ubiquitin and maltose-binding protein and published in the journal Angewandte Chemie Int. Ed. (T.Madl, W.Bermel and K.Zangger, Use of relaxation enhancements in a paramagnetic environment for the structure determination of proteins using NMR spectroscopy, Angew.Chem.Int.Ed.48(44), 8259-8262, 2009). This method is currently used by us and a few other groups on a routine bases for protein structure determinations. A manuscript showing an application of this technique on a small protein and its transient DNA binding is currently being prepared. Additionally, the toxin CcdB was NMR spectroscopically assigned and its structure determine in solution by NMR and in the solid state by X-ray diffraction by our collaborator at the University of Brussels. The structures were jointly published in the Journal of Biological Chemistry (N. De Jonge, W.Hohlweg, A. Garcia-Pino, M. Respondek, L. Buts, S. Haesaerts, J. Lah, K. Zangger and R. Loris, Structural and thermodynamic characterization of Vibrio fischeri CcdB, J.Biol.Chem. 285(8), 5606-5613, 2010). Together with this group we also studied the rejuvenation of CcdB poisoned gyrase by binding of an intrinsically unstructured CcdA-derived peptide (N. de Jonge, A. Garcia-Pino, L. buts, S. Haesaerts, D. Charlier, K. Zangger, L. Wyns, H. De Greve and R. Loris, Rejuvenation of CcdB-poisened gyrase by an intrinsically disordered protein domain., Mol. Cell, 35, 154-163 2009). Furthermore we determined the structure of the bacterial antitoxin MazE and its complex with DNA. A manuscript about that will be submitted within the next few months.