The Doc toxin and Phd antidote proteins of the bacteriophage P1 plasmid addiction system form a heterotrimeric complex

The toxin (Doc) and antidote (Phd) proteins of the plasmid addiction system of bacteriophage P1 were purified as a complex. Cocrystals of the complex contained a 2:1 molar ratio of Phd:Doc as assayed by dye binding following SDS-polyacrylamide gel electrophoresis and as determined by amino acid anal ysis. Gel filtration and analytical ultracentrifugation revealed that the t wo addiction proteins interact in solution to form a P2D trimer composed of one Doc and two Phd molecules, These results support a model in which Phd inhibits the toxic activity of Doc by direct binding, Circular dichroism ex periments showed that changes in secondary structure accompany formation of the heterotrimeric complex, raising the possibility that Phd may act by an allosteric mechanism. Studies of Phd and Doc molecules labeled with fluore scent energy donor and acceptor groups gave an equilibrium dissociation con stant of about 0.8 mu M-2 and a very short, sub second half-life of complex dissociation. As a consequence, low concentrations of free Doc toxin are l ikely to be present both transiently and in the steady state in cells conta ining the Phd antidote, making mechanisms of single-hit Doe toxicity improb able.