Discrete Structural Dynamics of Pseudo‐Palindromic Motifs Control DNA Binding of Bacterial Toxin‐Antitoxin Complexes

Toxin‐antitoxin (TA) systems have been strongly implicated in bacterial cell survival during antibiotic stress and are likely to play important roles in persister cell formation1,2. Understanding the function and regulation of TA systems will thus be vital for combatting chronic and recurrent human infections in the future. Expression of type II TA systems is regulated at the transcriptional level through direct binding of the antitoxin to pseudo‐palindromic sequences on operator DNA. In this process, the toxin functions as a co‐repressor by stimulating DNA binding through direct interaction with the antitoxin and formation of a higher‐order protein complex3. In the VapBC TA class, the VapC toxin is inhibited by direct protein interaction with the VapB antitoxin, which also contains a DNA binding domain of the AbrB type3. Since TA complexes show a great structural variety and no structures have been determined of the same system both on and off DNA, the structural changes occurring upon DNA binding are not known. Furthermore, details of how the flexible C‐terminus of the antitoxin becomes available for degradation when the toxin is activated, are scarce. Here, we present high‐resolution crystal structures of the complete 90 kDa heterooctameric VapBC1 complex from Caulobacter crescentus CB15 both in isolation and bound to a cognate 27‐bp DNA operator sequence at 1.6 and 2.7 Å resolution, respectively. The structures reveal that DNA binding is associated with a dramatic architectural rearrangement of conserved TA interactions involving VapB antitoxin C‐termini. We find that the C‐terminal tails of the antitoxin VapB1 that inhibit the VapC toxin by directly interacting with its active site, swap positions to interlock the complex in the DNA‐bound state. We further show that 2:1 interaction between toxin and antitoxin depends on a pseudo‐palindromic protein sequence in the antitoxin, which is able to bind and inactivate both molecules of a VapC1 toxin dimer. Sequence analysis of 4,127 orthologous VapB sequences reveals that such palindromic protein sequences are widespread among bacterial and archaeal VapB antitoxins suggesting that we have uncovered a general principle governing regulation of VapBC TA systems. Finally, a structure of C‐terminally truncated VapB1 bound to VapC1 reveals discrete states of the TA interaction that suggest a structural basis for antitoxin degradation and thus toxin activation in vivo . Support or Funding Information This work was funded by the Lundbeck Foundation [R173‐2014‐1182 to D.E.B.]; the Danish National Research Foundation's Centre for Bacterial Stress Response and Persistence, BASP [DNRF120 to D.E.B]; and a Chinese Scholarship Council postgraduate [K.X.].