Fuzzy interactions maintain cognate pairings in TA systems
Christina R. Bourne, Kevin J. Snead, Felipe Avelino da Costa Ferreira · Structural Dynamics · 2025
Type II toxin-antitoxin (TA) ParDE systems are widespread in bacteria. The non-secreted protein toxin, ParE, potently inhibits DNA gyrase unless it is neutralized via direct interaction with the ParD antitoxin protein. ParDE operons encode cognate pairs, insulating from cross-interactions between structurally homologous ParE toxins that can reside in the same bacterial cell. These nano- to picomolar interactions are extensive and a model for "fuzzy" interactions, wherein the ParD antitoxin contains an unstructured region that is induced to fold by interactions with the ParE toxin. A major open question is if this strict cognate insulation can be overcome such that, for example, a plasmid-derived ParE toxin might be effectively neutralized by a chromosomal ParD antitoxin. Our studies have decomposed the sites of interaction between a canonical ParD antitoxin with its cognate ParE toxin. These identified a hotspot for the pairing that we hypothesize is crucial for the induced folding of the antitoxin. This is supported by data demonstrating a specific seed region of antitoxin needed to initiate the interaction, followed by induced secondary structure formation. We are using structural, biochemical, and molecular dynamics approaches to further probe our hypothesis, with an over- arching goal of identifying the crucial contacts needed to discern cognate versus non-cognate pairings. This will allow database mining for the applications of machine learning, providing insights into the potential prevalence and capacity for cross-pairing. Answering these open questions will provide blueprints for intentional interruption of pairings, such that the ParE toxin could be liberated to control bacterial growth, or supplementation with excess ParD antitoxin to maintain pairings, such as can be applied to cure bacterial cells of plasmid harboring antibacterial resistance genes. These studies also inform models for how specificity is encoded in the disorder to order transitions, a common yet understudied mediator of many important biochemical interactions.