Transcriptional regulation allows rapid adaptation of protein synthesis to environmental
conditions. In prokaryotes, the clustering of genes into operons allows coordination of the
regulation of functionally related genes in response to different growth conditions. Toxin-antitoxin
(TA) operons are ubiquitous on bacterial chromosomes. They are believed to play a major role in
bacterial stress physiology and may be at the basis of the multi-drug resistance phenomenon
where a small amount of bacterial cells defies any antibiotic even at very high doses, leading to
re-emergence of infection.
Regulation of the phage P1 phd/doc addiction operon, involves the toxin Doc as co- or derepressor depending on the ratio between Phd and Doc. This unexplained transcriptional
regulatory phenomenon is known as conditional co-operativity. Binding of Doc to the intrinsically
disordered C-terminal domain of Phd structures the DNA binding domain of Phd, for the first time
illustrating allostery between two distinct disordered protein domains.
In this work we determined the molecular basis of the inhibition of the toxin Doc by the antitoxin
Phd. The neutralization event involves the binding of Phd to a pronounced crevice located in the
central region of Doc. Upon binding, the antitoxin domain of Phd, unstructured in solution, folds
into an -helix completing the FIC-like fold of Doc.
Based on a combination of X-ray crystallography and SAXS, we proposed a model for
transcriptional regulation of the phd/doc operon by conditional co-operativity. We show that the
transcription factor Phd of the phd/doc TA module exists as an equilibrium between two
conformational states : one where its N-terminal domain is fully folded and capable of operator
DNA binding and one where this domain is largely unstructured and the DNA binding site
disrupted. The C-terminal Doc-binding region of Phd is intrinsically disordered in both states.
Binding of both operator DNA or Doc induce a similar structuring of the N-terminal DNA binding
domain, as evidenced through a series of DSC and CD measurements and through the crystal
structure of the Phd-Doc complex. Electrophoretic mobility shift assays show that Doc enhances
the operator DNA binding of wild-type Phd but not of certain Phd mutants that nevertheless
retain wild-type like binding of Doc. These mutations that uncouple Doc binding (which remains
indistinguishable from wild-type) from DNA binding are located at the C-terminus of the DNA
binding domain and do not overlap with either the DNA or the Doc binding sites.
The crystal structure of the Phd-Doc complex further shows that the intrinsically disordered Cterminal segment of PhD binds to two distinct and structurally unrelated sites on the monomeric
Doc protein, thereby adopting two distinct conformations. These interactions create a Phd2-DocPhd2 sandwich that allows Phd to interact cooperatively with two binding sites on the phd/doc
operator DNA. The two orders of magnitude difference in binding affinity of the two Doc
interaction sites for Phd mediate a competition between a high/low a_nity sites which depends
on the toxin:antitoxin ratio. This feature constitutes the basis of the conditional co-operativity
regulation of transcription and allows us to understand this elusive regulatory process (whereby
Doc acts as a co-repressor or as an anti-repressor depending on the ratio between Phd and
Doc) in terms of a site-exchange mechanism.
This mechanism is further corroborated by in vivo and in vitro experiments using point mutants
of Doc showing that both binding sites are required for transcriptional regulation but only a single
one for neutralization of the ribosome-poisoning activity of Doc. We thus show for the first time
how binding of a co-repressor to an intrinsically unstructured segment can induce order in a
distant domain. Our example further illustrates a novel regulatory mechanism of transcriptional
regulation that is likely general for all toxin-antitoxin operons.