Structural and biophysical characterization of the Vibrio cholerae parDE2 toxin-antitoxin module.

Abstract

Most, if not all, bacteria contain a number of toxin-antitoxin (TA) operons in their chromosomes. Such operons encode (i) a toxin that inhibits cell grow by acting on a target and (ii) an antitoxin that is able to neutralize the toxin activity. In type II TA systems, neutralization occurs by formation of a TA complex between the antitoxin and the toxin.
Binding of the TA complex to its own operator region appears to suppress transcription in many TA systems. For parDE present on plasmid RK2 this is not the case, since in this system ParD can individually auto-repress transcription by binding the DNA with its ribbon-helix-helix (RHH) motif. In this thesis, the biophysics of the V. cholerae parDE2 module is studied in vitro. The latter is one of the three type II TA systems homologous to the plasmid RK2 parDE system and is present in the superintegron of chromosome II.
The ParD2:ParE2-His complex of the parDE2 system is present in solution in an unexpected 6:2 antitoxin:toxin ratio. Crystals of ParD2:ParE2-His bound to a nanobody were obtained. Via denaturant-induced dissociation, the complex can be separated into its components: ParD2 and ParE2-His. After subsequent purification, both are well-folded and occur as a higher oligomer and a monomer in solution respectively.
In vitro binding studies involving complex, toxin and antitoxin show binding between ParD2:ParE2-His and the parDE2 putative operator region. In contrast to the RK2 homologue, no binding of the operator DNA with the purified ParD2 is observed. ParD2:ParE2-His protects a 44 bp fragment on the parDE putative operator region, on which a 2x9 bp split near-palindromic site (5’-ATACCAWAA-3’) is located, of which all bases are important for binding. That palindromic region therefore most likely is the (main) ParD2:ParE2-His binding site.

Date of Award	Jul 2019
Original language	English
Supervisor	Remy Loris (Promotor) & Gabriela García (Advisor)