Stability and DNA binding of the phd protein of the phage P1 plasmid addiction system.

The plasmid addiction module of bacteriophage P1 encodes two proteins, Doc, a toxin that is stable to proteolytic degradation, and Phd, the toxin's antidote that is proteolytically unstable. Phd has been shown to autoregulate its expression by specific DNA binding. Here, we investigate the secondary structure and thermal stability of Phd, the effect of operator DNA binding on the structure and stability of Phd, and the stoichiometry, affinity, and cooperativity of Phd binding to operator subsites and intact operator DNA. Phd folds as a monomer at low temperatures or in the presence of osmolytes but exists predominantly in an unfolded conformation at 37 degreesC. The native state of Phd is stabilized by operator binding. Two Phd monomers bind to each operator subsite, and four monomers bind to the intact operator. The subsite binding reaction shows a second-order dependence on protein concentration and monomer-bound DNA species are unpopulated, suggesting that two Phd molecules bind cooperatively to each operator subsite. In intact operator binding experiments, both dimer-bound and tetramer-bound DNA species are populated, and binding occurs at protein concentrations similar to those required for subsite binding, suggesting that there is no significant dimer-dimer cooperativity.