Functional and structural characterization of EnvZ, an osmosensing histidine kinase of E. coli.
EnvZ is an osmosensing histidine kinase located in the inner membrane, and one of the most extensively studied Escherichia coli histidine kinases. Because of its structural complexity, functional and structural studies have been quite challenging. It is a multidomain transmembrane protein consisting of 450 amino acid residues. In addition, it must form a dimer to function as a histidine kinase like all the other histidine kinases. EnvZ consists of the 115-residue periplasmic domain, two transmembrane domains (TM1 and TM2), and the cytoplasmic domain consisting of the 43-residue linker (HAMP) domain and the 228-residue kinase domain. It has been shown that the kinase domain of EnvZ, responsible for its enzymatic activities, contains all of the conserved regions of histidine kinases such as H, F, N, G1, G2, and G3 boxes. Therefore, the 271-residue cytoplasmic domain of EnvZ (termed EnvZc) has been used as a model system to establish fundamental characteristics of histidine kinases. The DNA fragment encoding EnvZc was cloned in pET vector and EnvZc was expressed and purified. It is highly soluble and retains all the enzymatic activities of EnvZ. We demonstrated that it consists of two functional domains, domain A and domain B. NMR spectroscopic studies of these two domains revealed, for the first time, the structure of a histidine kinase. Domain A is responsible for dimerization of EnvZc forming a four-helical bundle containing two alpha-helical hairpin structures, while domain B is a monomer and has an ATP-binding pocket formed by regions conserved among the histidine kinases. In this chapter, we describe functional and structural studies of EnvZc, which can be applied to characterize other histidine kinases.
Yoshida
Takeshi
T
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ, USA.
Phadtare
Sangita
S
Inouye
Masayori
M
D000255:Adenosine Triphosphate / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D001426:Bacterial Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D006639:Histidine / Q000031:analogs & derivatives / Q000737:chemistry; D000071677:Histidine Kinase; D006867:Hydrolases / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; C482905:OmpX protein, E coli; D006639:Histidine; D000255:Adenosine Triphosphate; D004247:DNA; D011494:Protein Kinases; D000071677:Histidine Kinase; C057843:envZ protein, E coli; D006867:Hydrolases; C005436:phosphohistidine