Model for RNA Binding and the Catalytic Site of the RNase Kid of the Bacterial parD Toxin–Antitoxin System

Abstract

The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5 0 side of the A residue in the nucleotide sequence 5 0 -UA(A/C)-3 0 of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2 0 :3 0 -cyclic phosphate group and a free 5 0 -OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2 0 -OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.

Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci

“…As discussed below, mazEF and homologous systems (parD and chpB) have been most extensively studied on their plasmid and chromosomal origins in enterobacteria (34)(35)(36). Because 100% of the VRE strains obtained for this study harbored genes for a plasmidencoded mazEF TA system (Fig.…”

Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot

“…7C). ToxN enzymes are proposed to cleave their substrates through a metal-independent RNase T1-like mechanism, based on their active-site architectures, the presence of the 2′-3′-cyclic phosphate product in the active site, and their structural homology to Kid, whose reaction mechanism has been studied in detail (21,25). The RNA fragment patterns produced by ToxN Pa degradation in vitro (Fig.…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…In this case, the complex formation was in the fast exchange regime. NMR titration experiments indicated that one dimer binds a single RNA molecule and the stoichiometry of the complex was further confirmed by native mass spectrometry [47]. The RNA binding protein RsmE also folds into a symmetrical homodimer [157,158].…”

“…Of these structures, 52 were determined using classical NMR methodology (Fig. 1) and two consist of structural models generated by docking using sparse NMR data [46,47]. This results in an estimated weight for the contribution of NMR in the structure elucidation of protein-RNA complexes to approach 40%.…”