PhoP-PhoP interaction at adjacent PhoP binding sites is influenced by protein phosphorylation.

Mycobacterium tuberculosis PhoP regulates the expression of unknown virulence determinants and the biosynthesis of complex lipids. PhoP, like other members of the OmpR family, comprises a phosphorylation domain at the amino-terminal half and a DNA-binding domain at the carboxy-terminal half of the protein. To explore structural effect of protein phosphorylation and to examine effect of phosphorylation on DNA binding, purified PhoP was phosphorylated by acetyl phosphate in a reaction that was dependent on Mg2+ and Asp-71. Protein phosphorylation was not required for DNA binding; however, phosphorylation enhanced in vitro DNA binding through protein-protein interaction(s). Evidence is presented here that the protein-protein interface is different in the unphosphorylated and phosphorylated forms of PhoP and that specific DNA binding plays a critical role in changing the nature of the protein-protein interface. We show that phosphorylation switches the transactivation domain to a different conformation, which specifies additional protein-protein contacts between PhoP protomers bound to adjacent cognate sites. Together, our observations raise the possibility that PhoP, in the unphosphorylated and phosphorylated forms, may be capable of adopting different orientations as it binds to a vast array of genes to activate or repress transcription.