Model for RNA binding and the catalytic site of the RNase Kid of the bacterial parD toxin-antitoxin system.
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' side of the A residue in the nucleotide sequence 5'-UA(A/C)-3' of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2':3'-cyclic phosphate group and a free 5'-OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2'-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.
Kamphuis
Monique B
MB
Bijvoet Center for Biomolecular Research, Department of NMR Spectroscopy, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Bonvin
Alexandre M J J
AM
Monti
Maria Chiara
MC
Lemonnier
Marc
M
Muñoz-Gómez
Ana
A
van den Heuvel
Robert H H
RH
Díaz-Orejas
Ramón
R
Boelens
Rolf
R
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D003603:Cytotoxins / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D004268:DNA-Binding Proteins; C068353:Kid protein, Bacteria; C068354:Kis protein, Bacteria; D046911:Macromolecular Substances; C078613:parD protein, Bacteria; D012313:RNA