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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.