CRYSTAL STRUCTURE OFCcdg, A PROTEIN THAT POISONS GYRASE

Remy Loris^l, Minh-Hoa Dao-Thi^l, Martine Couturier² and Lode Wyns¹

¹Laboratorium voor Ultrastruktuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium
²Laboratoire de Genetique, University Libre de Bruxelles, Paardenstraat 67, B-1640 Sint-Genesius-Rode, Belgium

Maintenance of low copy number plasmids in bacterial populations is ensured by plasmid addiction systems that are responsible for the killing of plasmid-free segregants. The proteic systems such as ccd of E. coli encode for a stable toxin (CcdB) and an unstable antidote (CcdA). CcdB poisons covalent DNA-gyrase complexes, and under normal conditions its action is prevented by complex formation with its antidote CcdA. This complex also functions as a repressor for the ccd operon. Upon loss of the F-plasmid, the continuous supply of freshly synthesised CcdA stops. As CcdA is an efficient substrate for the Lon protease, CcdB is released, resulting in filamentation and ultimately cell death.

In order to gain a better understanding of the action of CcdB at a molecular level, we determined its three-dimensional structure by X-ray crystallography. The protein consists of a 5-stranded anti-parallel b-pleated sheet followed by a C- terminal a-helix. In one of the loops of the sheet, a second small 3-stranded antiparallel b-sheet is inserted that sticks out of the molecule as a wing. A dimer is formed by sheet extension and by extensive hydrophobic contacts involving three of the five methionines and the C-terminus of the a-helix. The surface of the dimer containing the a-helix is overall negatively charged, while the opposite side as well as the wing sheet is dominated by positive charges. A model for the CcdB-GyrA interaction is proposed

Loris, R., Dao-Thi, M.H., Bahassi, E.M., Van Melderen, L., Poortmans, F., Liddington, R. , Couturier, M. & Wyns, L. ( 1998) Crystal Structure of CcdB, a Topoisomerase Poison from E. coli Nature Struct. Biol. (submitted).