Mycobacterium tuberculosis PhoP recognizes two adjacent direct-repeat sequences to form head-to-head dimers.

Mycobacterium tuberculosis PhoP of the PhoP-PhoR two-component signaling system orchestrates a complex transcription program and is essential for the growth and virulence of the tubercle bacillus. PhoP comprises a phosphorylation domain at the amino-terminal half and a DNA-binding domain in the carboxy-terminal half of the protein. We show here that the protein recognizes a 23-bp sequence of the phoP upstream region comprising two adjacent direct repeat motifs believed to promote transcription regulation. DNA binding, which involves the recruitment of two monomeric PhoP molecules, was dependent on conserved adenines of the repeat sequences and the orientation of the repeat motifs relative to each other. Although response regulators such as PhoB and FixJ dimerize upon phosphorylation, we demonstrate here that PhoP dimerization can also be stimulated by DNA binding. Using the established asymmetric tandem binding model by members of the OmpR/PhoB protein family as a guide, we set out to examine intermolecular interactions between PhoP dimers by protein cross-linking. Our results are consistent with a model in which two PhoP protomers bind the duplex DNA with a symmetric head-to-head orientation to project their N termini toward one another, arguing against previously proposed head-to-tail tandem dimer formation for members of the OmpR/PhoB protein subfamily.