Structure-function relationships of EnvZ, an osmosensory histidine kinase/phosphatase in Escherichia col

EnvZ is a prototypical histidine kinase/phosphatase involved in the osmoregulatory His-Asp phosphorelay pathway of Escherichia coli. Investigations conducted toward establishing the structure-function relationship of the cytoplasmic domain of this protein constitute the subject of this dissertation.;Collaborative work with Dr. M. Ikura and his group led to the resolution of NMR-solution structures of two domains A (residues 223--289) and B (residues 290--450) that comprise the kinase/phosphatase domain of EnvZ. A review was undertaken to evaluate previous knowledge in the light of the new structural information.;An interesting homology was observed between the ATP-binding domains of histidine kinases, DNA gyrase, heat-shock protein Hsp90 and DNA-mismatch-repair protein MutL. A review of the structure-function relationship of these four families of proteins was undertaken with an emphasis on the common ATP-binding fold. The features of this novel Bergerat ATP-binding fold have been identified.;The dimeric histidine kinase, presents difficulties for structural analysis. A monomeric histidine kinase was derived from EnvZ by fusing another domain A to an AB domain. The biochemical and biophysical characterization of this protein, EnvZc[AAB], reveals that it functions as a histidine kinase. A molecular model of the EnvZ C-terminal domain has been proposed.;Asn347 in the B domain is a highly conserved residue in histidine kinases. Biochemical characterization of the C-terminal domain of this mutant protein revealed an interesting reverse phospho-transfer phenomenon from Asp55 on OmpR to His243 on EnvZ, implicating the latter reidue in the phosphatase reaction of EnvZ. UV-crosslinking studies demonstrated that this mutant protein had lost its ability to bind ATP.;Mutational analysis was conducted on the conserved residue, Thr247, on domain A of EnvZ. Biochemical analysis of substitution mutants of this residue revealed that Thr247 is a critical residue in the active site of EnvZ. Besides influencing the kinase activity it might play an active role in the phosphatase activity of EnvZ.;The relative contributions of the DHp and the CA domains in EnvZ enzymatic activities were examined through the biochemical characterization of an EnvZc[T247R/N347D] double mutant protein. On the basis of these results models are proposed for the autokinase, phosphotransferase and phosphatase activities of EnvZ.