Probing the roles of the two different dimers mediated by the receiver domain of the response regulator PhoB.

Structural analysis of the Escherichia coli response regulator transcription factor PhoB indicates that the protein dimerizes in two different orientations that are both mediated by the receiver domain. The two dimers exhibit 2-fold rotational symmetry: one involves the alpha 4-beta 5-alpha 5 surface and the other involves the alpha1/alpha 5 surface. The alpha 4-beta 5-alpha 5 dimer is observed when the protein is crystallized in the presence of the phosphoryl analog BeF(3)(-), while the alpha1/alpha 5 dimer is observed in its absence. From these studies, a model of the inactive and active states of PhoB has been proposed that involves the formation of two distinct dimers. In order to gain further insight into the roles of these dimers, we have engineered a series of mutations in PhoB intended to perturb each of them selectively. Our results indicate that perturbation of the alpha 4-beta 5-alpha 5 surface disrupts phosphorylation-dependent dimerization and DNA binding as well as PhoB-mediated transcriptional activation of phoA, while perturbations to the alpha1/alpha 5 surface do not. Furthermore, experiments with a GCN4 leucine zipper/PhoB chimera protein indicate that PhoB is activated through an intermolecular mechanism. Together, these results support a model of activation of PhoB in which phosphorylation promotes dimerization via the alpha 4-beta 5-alpha 5 face, which enhances DNA binding and thus the ability of PhoB to regulate transcription.