The Crystal Structure of the Intact E. coli RelBE Toxin-Antitoxin Complex Provides the Structural Basis for Conditional Cooperativity

Abstract

SummaryThe bacterial relBE locus encodes a toxin-antitoxin complex in which the toxin, RelE, is capable of cleaving mRNA in the ribosomal A site cotranslationally. The antitoxin, RelB, both binds and inhibits RelE, and regulates transcription through operator binding and conditional cooperativity controlled by RelE. Here, we present the crystal structure of the intact Escherichia coli RelB2E2 complex at 2.8 Å resolution, comprising both the RelB-inhibited RelE and the RelB dimerization domain that binds DNA. RelE and RelB associate into a V-shaped heterotetrameric complex with the ribbon-helix-helix (RHH) dimerization domain at the apex. Our structure supports a model in which relO is optimally bound by two adjacent RelB2E heterotrimeric units, and is not compatible with concomitant binding of two RelB2E2 heterotetramers. The results thus provide a firm basis for understanding the model of conditional cooperativity at the molecular level.

Defining the mRNA recognition signature of a bacterial toxin protein

“…S2). Although other toxins are proposed to undergo allosteric regulation after release from the antitoxin (32,37,38), our data suggest that HigB has a preformed tertiary structure primed for interaction with the ribosome. HigB binds the A site between the head and body of the small subunit, similar to where tRNA interacts with mRNA ( Fig.…”

Mechanisms of Toxin Inhibition and Transcriptional Repression by Escherichia coli DinJ-YafQ

“…This regulation has been suggested to be controlled by conditional cooperativity although the exact details differ. In one interpretation, one TA complex can cooperatively enhance binding at the second inverted repeat; a slightly different interpretation is that the toxin enhances avidity for the inverted repeat DNA, in the context of the complex (25,50). Modeling tetrameric TA complexes such as RelBE to its operator sequence shows there is a steric clash when two RelBE tetramers are bound to two adjacent inverted repeats.…”

“…4C). This is in contrast to when two structurally homologous RelBE tetramers are modeled onto the same Arc-DNA complex (50). In our study, we adjusted our model to allow for a 10-bp spacing between inverted repeats, which is consistent with the endogenous spacing of the dinJ operon (Fig.…”

“…1C). Similar inhibitory interactions that bury the toxin active site when bound to its cognate antitoxin include YefM-YoeB and RelBE complexes (16,50,51). For example, the proposed general base in RelE, Lys-52, forms an interaction with Asp-55 of RelB (59).…”

“…Transcriptional Autorepression-Our structure reveals that DinJ adopts an RHH DNA-binding motif consistent with structurally homologous antitoxins such as RelB (50). To test whether the intact N terminus of DinJ is required for transcriptional repression, we designed two N-terminal DinJ truncations based upon our structure and tested for their ability to repress lacZ transcription from the dinJ promoter (PdinJ) using ␤-galactosidase activity assays (Fig.…”