Biochemical and X-ray analyses of the players involved in the faRel2/aTfaRel2 toxin-antitoxin operon

The aTfaRel2/faRel2 operon from Coprobacillus sp. D7 encodes a bicistronic type II toxin-antitoxin (TA) module. The FaRel2 toxin is a toxic small alarmone synthetase (toxSAS) that inhibits translation through the pyrophosphorylation of uncharged tRNAs at the 3'-CCA end. The toxin is neutralized by the antitoxin ATfaRel2 through the formation of an inactive TA complex. Here, the production, biophysical analysis and crystallization of ATfaRel2 and FaRel2 as well as of the ATfaRel2-FaRel2 complex are reported. ATfaRel2 is monomeric in solution. The antitoxin crystallized in space group P2₁2₁2 with unit-cell parameters a = 53.3, b = 34.2, c = 37.6 Å, and the best crystal diffracted to a resolution of 1.24 Å. Crystals of FaRel2 in complex with APCPP, a nonhydrolysable ATP analogue, belonged to space group P2₁, with unit-cell parameters a = 31.5, b = 60.6, c = 177.2 Å, β = 90.6°, and diffracted to 2.6 Å resolution. The ATfaRel2-FaRel2^Y128F complex forms a heterotetramer in solution composed of two toxins and two antitoxins. This complex crystallized in two space groups: F4₁32, with unit-cell parameters a = b = c = 227.1 Å, and P2₁2₁2₁, with unit-cell parameters a = 51.7, b = 106.2, c = 135.1 Å. The crystals diffracted to 1.98 and 2.1 Å resolution, respectively.