Structural and mutational analysis of the PhoQ histidine kinase catalytic domain. Insight into the reaction mechanism.

PhoQ is a transmembrane histidine kinase belonging to the family of two-com ponent signal transducing systems common in prokaryotes and lower eukaryote s. In response to changes in environmental Mg2+ concentration, PhoQ regulat es the level of phosphorylated PhoP, its cognate transcriptional response-r egulator. The PhoQ cytoplasmic region comprises two independently folding d omains: the histidine-containing phosphotransfer domain and the ATP-binding kinase domain. We have determined the structure of the kinase domain of Es cherichia coli PhoQ complexed with the non-hydrolyzable ATP analog adenosin e 5'-(beta,gamma -imino)triphosphate and Mg2+. Nucleotide binding appears t o be accompanied by conformational changes in the loop that surrounds the A TP analog (ATP-lid) and has implications for interactions with the substrat e phosphotransfer domain. The high resolution (1.6 Angstrom) structure reve als a detailed view of the nucleotide-binding site, allowing us to identify potential catalytic residues. Mutagenic analyses of these residues provide new insights into the catalytic mechanism of histidine phosphorylation in the histidine kinase family. Comparison with the active site of the related GHL ATPase family reveals differences that are proposed to account for the distinct functions of these proteins.