Cysteine-scanning analysis of the dimerization domain of EnvZ, an osmosensing histidine kinase.
EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in Escherichia coli. The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B. Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A. The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized. From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity. Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A. The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation. Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities. The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity.
Qin
Ling
L
Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Cai
Shengjian
S
Zhu
Yan
Y
Inouye
Masayori
M
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D003470:Culture Media; D003545:Cysteine / Q000235:genetics*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D009154:Mutation; D009994:Osmolar Concentration; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D011494:Protein Kinases; D000071677:Histidine Kinase; C057843:envZ protein, E coli; D010744:Phosphoric Monoester Hydrolases; D003545:Cysteine