Hidden States within Disordered Regions of the CcdA Antitoxin Protein

Abstract

The bacterial toxin-antitoxin system CcdB-CcdA provides a mechanism for the control of cell death and quiescence. The antitoxin CcdA protein is a homodimer composed of two monomers that each contain a folded N-terminal region and an intrinsically disordered C-terminal arm.Binding of the intrinsically disordered C-terminal arm of CcdA to the toxin CcdB prevents CcdB from inhibiting DNA Gyrase and thereby averts cell-death. Accurate models of the unfolded state of the partially disordered CcdA antitoxin can therefore provide insight into general mechanisms whereby protein disorder regulates events that are crucial to cell survival. Previous structural studies were able to model only two of three distinct structural states Ð a closed state and an open state Ð that were adopted by the C-terminal arm of CcdA. Using a combination of free energy simulations, single-pair Fšrster resonance energy transfer experiments, and existing NMR data, we developed structural models for all three states of the protein. Contrary to prior studies, we find that CcdA samples a previously unknown state where only one of the disordered C-terminal arms makes extensive contacts with the folded N-terminal domain. Moreover, our data suggest that previously unobserved conformational states play a role in regulating antitoxin concentrations and the activity of CcdAÕs cognate toxin. These data demonstrate that intrinsic disorder in CcdA provides a mechanism for regulating cell-fate.3

The Origins of Specificity in the Microcin-Processing Protease TldD/E

“…Full-length SGS-CcdA was stable in the presence of TldD/E in vitro ( Figure S2 A), but SGS-CcdA41 was readily degraded to small peptides ( Figure S2 B). CcdA41 is an intrinsically disordered C-terminal tail of CcdA, and previous studies have shown that it is more susceptible to cleavage by Lon protease compared with the full-length protein, presumably due to the lack of interactions with N-terminal folded domain ( Burger et al., 2017 , Van Melderen et al., 1996 ). Hence our results support earlier western blot data showing stabilization of Ccd41 in tld mutants ( Allali et al., 2002 ).…”

Mechanism of CcdA-Mediated Rejuvenation of DNA Gyrase

“…CcdA is one such molecular switch that restores gyrase function by facilitating extraction of CcdB from its complex with GyrA14. The conformational state of the disordered C-terminal domain of CcdA regulates its recognition by Lon protease, and is also involved in binding to CcdB (Burger et al, 2017;Drobnak et al, 2013;Madl et al, 2006). Binding of CcdA to CcdB inhibits CcdB toxicity as well as autoregulating transcription of the ccd operon (De Jonge et al, 2009).…”

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

“…In the present study, we have carried out detailed experimental studies of one such molecular switch, CcdA, which is known to restore the function of Gyrase by facilitating dissociation of CcdB from the CcdB:GyrA14:DNA complex (Dao-Thi et al, 2005; De Jonge et al, 2009). The disordered C-terminal domain of CcdA is involved in binding to CcdB, thereby inhibiting CcdB toxicity and autoregulating the transcription of the ccd operon (Burger et al, 2017; Drobnak et al, 2013; Madl et al, 2006).…”