Structural Insights into the PrpTA Toxin–antitoxin System in Pseudoalteromonas Rubra

Bacteria could survive stresses by a poorly understood mechanism that contributes to the emergence of bacterial persisters exhibiting multidrug tolerance (MDT). Recently, Pseudoalteromonas rubra prpAT module was found to encode a toxin PrpT and corresponding cognate antidote PrpA. In this study, we first reported multiple individual and complex structures of PrpA and PrpT, which uncovered the high-resolution three-dimensional structure of the PrpT:PrpA(2):PrpT heterotetramer with the aid of size exclusion chromatography-multi-angle light scattering experiments (SEC-MALS). PrpT:PrpA(2):PrpT is composed of a PrpA homodimer and two PrpT monomers which are relatively isolated from each other and from ParE family. The superposition of antitoxin monomer structures from these structures highlighted the flexible C-terminal domain (CTD). A striking conformational change in the CTDs of PrpA homodimer depolymerized from homotetramer was provoked upon PrpT binding, which accounts for the unique PrpT-PrpA(RHH) mutual interactions and further neutralizes the toxin PrpT. PrpA(2-54)-form I and II crystal structures both contain a doughnut-shaped hexadecamer formed by eight homodimers organized in a cogwheel-like form via inter-dimer interface dominated by salt bridges and hydrogen bonds. Moreover, PrpA tends to exist in solution as a homodimer other than a homotetramer (SEC-MALS) in the absence of flexible CTD. Multiple multi-dimers, tetramer and hexamer included, of PrpA(2-54) mediated by the symmetric homodimer interface and the complicated inter-dimer interface could be observed in the solution. SEC-MALS assays highlighted that phosphate buffer (PB) and the increase in the concentration appear to be favorable for the PrpA(2-54) oligomerization in the solution. Taken together with previous research, a model of PrpA(2-54) homotetramer in complex with prpAT promoter and the improved mechanism underlying how PrpTA controls the plasmid replication were proposed here.