Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae.

Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while "hovering" over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

Toxin:antitoxin ratio sensing autoregulation of the Vibrio cholerae parDE2 module.

The parDE family of toxin-antitoxin (TA) operons is ubiquitous in bacterial genomes and, in Vibrio cholerae, is an essential component to maintain the presence of chromosome II. Here, we show that transcription of the V. cholerae parDE2 (VcparDE) operon is regulated in a toxin:antitoxin ratio-dependent manner using a molecular mechanism distinct from other type II TA systems. The repressor of the operon is identified as an assembly with a 6:2 stoichiometry with three interacting ParD2 dimers bridged by two ParE2 monomers. This assembly docks to a three-site operator containing 5'- GGTA-3' motifs. Saturation of this TA complex with ParE2 toxin results in disruption of the interface between ParD2 dimers and the formation of a TA complex of 2:2 stoichiometry. The latter is operator binding-incompetent as it is incompatible with the required spacing of the ParD2 dimers on the operator.