ESCHERICHIA COLI RNLB ANTITOXIN INHIBITS ITS COGNATE TOXIN VIA A NOVEL MECHANISM

Bacterial toxin:antitoxin (TA) modules are small operons encoding a toxin that interferes with vital cell processes and an antitoxin that counteracts this toxic activity. Escherichia coli rnlAB operon encodes a toxin:antitoxin module that is involved in protection against infection by bacteriophage T4. RnlA is the first endoribonuclease toxin with two known antitoxins, E. coli own antitoxin RnlB as well as phage antitoxin Dmd that binds and neutralizes the active RnlA during phage infection. The full length RnlA:RnlB toxin:antitoxin complex as well as the toxin RnlA were purified to homogeneity and crystallized. RnlA:RnlB complex containing stoichiometric amounts of both proteins could only be obtained with a His-tag placed C-terminal to RnlB. When the affinity tag is placed on RnlA, RnlB is largely lost during purification and the resulting crystals are of free RnlA. This free RnlA conformation shows a dimer that exposes the putative catalytic residues. The structure of the complex with its cognate antitoxin RnlB was solved using a combination of MR-Rosetta, Buccaneer and Manual Model Building to a resolution of 2.6 Å, with final R-work = 0.2084 and R-free = 0.2514. This new structure shows two RnlB monomers bound to a new dimeric form of RnlA where the catalytic residues are buried in a new dimerization interface hindering substrate binding and thus inhibiting the toxicity of RnlA. SAXS experiments suggested that single alanine mutations of two different catalytic arginines of RnlA lock the toxin in either of these two conformations. This structure shows a new mechanism of inhibition in which the antitoxin locks the toxin in an inactive dimeric conformation.