title: A monomeric histidine kinase derived from EnvZ, an Escherichia coli osmosensor.
abstract: Histidine kinases function as dimers. The kinase domain of the osmosensing histidine kinase EnvZ of Escherichia coli consists of two domains: domain A (67 residues) responsible for histidine phosphotransfer and dimerization, and domain B (161 residues) responsible for the catalytic and ATP-binding function. The individual structures of these two domains have been recently solved by NMR spectroscopy. Here, we demonstrate that an enzymatically functional monomeric histidine kinase can be constructed by fusing in tandem two domains A and one domain B to produce a single polypeptide (A-A-B). We show that this protein, EnvZc[AAB], is soluble and exists as a stable monomer. The autophosphorylation and OmpR kinase activities of the monomeric EnvZc[AAB] are similar to that of the wild-type EnvZ, while OmpR-binding and phosphatase functions are reduced. V8 protease digestion and mutational analyses indicate that His-243 of only the amino proximal domain A is phosphorylated. Based on these results, molecular models are proposed for the structures of EnvZc[AAB] and the kinase domain of EnvZ. The present results demonstrate for the first time the construction of a functional, monomeric histidine kinase, further structural studies of which may provide important insights into the structure-function relationships of histidine kinases.
authors.affiliation: Department of Biochemistry, UMDNJ, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
authors.forename: L
authors.initials: L
authors.lastname: Qin
authors.forename: R
authors.initials: R
authors.lastname: Dutta
authors.forename: H
authors.initials: H
authors.lastname: Kurokawa
authors.forename: M
authors.initials: M
authors.lastname: Ikura
authors.forename: M
authors.initials: M
authors.lastname: Inouye
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D020134:Catalytic Domain; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D006358:Hot Temperature; D008958:Models, Molecular; D009097:Multienzyme Complexes; D009997:Osmotic Pressure; D011487:Protein Conformation; D015202:Protein Engineering; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D011494:Protein Kinases; D000071677:Histidine Kinase; C057843:envZ protein, E coli