Signal detection by the PhoQ sensor-transmitter. Characterization of the sensor domain and a response-impaired mutant that identifies ligand-binding determinants.

The PhoP-PhoQ two-component system is required for virulence and/or regulatory stress responses in enteric bacteria. The PhoQ protein responds to low concentrations of extracellular divalent cations by activating PhoP-mediated transcription of a set of genes. PhoQ is a member of a family of transmembrane proteins that contain a periplasmic sensor domain coupled to a cytoplasmic transmitter domain. Here, we describe the cloning, purification, and properties of a fragment of Escherichia coli PhoQ corresponding to the sensor domain. This fragment is monomeric in solution and has a circular dichroism spectrum indicative of a mixture of alphahelix and beta-sheet. Divalent cations do not affect the oligomeric state, circular dichroism spectrum, or fluorescence spectrum of the sensor domain but do stabilize this domain to denaturation in a fashion expected for a direct binding model. We have also constructed a mutant in which a cluster of acidic amino acids (EDDDDAE) in the sensor domain is replaced with conservative, uncharged residues (QNNNNAQ). The mutant sensor domain is indistinguishable from wild type in terms of oligomeric form and spectral properties but differs in being substantially more stable to urea denaturation, showing no additional stabilization in the presence of divalent cations, and showing little activation of PhoP-mediated transcription in response to divalent-cation starvation in vivo. These data are consistent with a model in which divalent cations bind to the acidic cluster of the wild-type sensor domain and stabilize a conformation that is inactive in signaling. Substituting uncharged residues for the acidic cluster appears to mimic the effect of divalent-cation binding by stabilizing the inactive conformation.