PMID:10760160
| Citation |
Qin, L, Dutta, R, Kurokawa, H, Ikura, M and Inouye, M (2000) A monomeric histidine kinase derived from EnvZ, an Escherichia coli osmosensor. Mol. Microbiol. 36:24-32 |
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| 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. |
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| Keywords |
Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Bacterial Proteins; Catalytic Domain; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli Proteins; Hot Temperature; Models, Molecular; Multienzyme Complexes; Osmotic Pressure; Protein Conformation; Protein Engineering; Protein Kinases/chemistry; Protein Kinases/genetics; Protein Kinases/metabolism; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/metabolism; Signal Transduction; Solubility; Trans-Activators/metabolism |
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