K+ Stimulates Specifically the Autokinase Activity of Purified and Reconstituted EnvZ of Escherichia coli

Jung, Kirsten; Hamann, Knut; Revermann, Anne

doi:10.1074/jbc.m107871200

Cited by 37 publications

(38 citation statements)

References 49 publications

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“…They are most consistent with a model in which the autophosphorylation of EnvZ or the phosphotransfer to OmpR (or both) increases with increasing osmolarity. This view is further supported by a recent study that reported elevated EnvZ autokinase activity at high potassium concentrations, with no change in the phosphotransfer or phosphatase activities (30). Spontaneous dephosphorylation of OmpR-P would then lead to OmpR-P decay.…”

Section: Phosphorylation Decreases the Affinity Of Ompr Forsupporting

confidence: 68%

Phosphorylation Alters the Interaction of the Response Regulator OmpR with Its Sensor Kinase EnvZ

Mattison¹,

Kenney

2002

Journal of Biological Chemistry

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OmpR and EnvZ comprise a two-component system that regulates the porin genes ompF and ompC in response to changes in osmolarity. EnvZ is autophosphorylated by intracellular ATP on a histidine residue, and it transfers the phosphoryl group to an aspartic acid residue of OmpR. EnvZ can also dephosphorylate phospho-OmpR (OmpR-P) to control the cellular level of OmpR-P. At low osmolarity, OmpR-P levels are low because of either low EnvZ kinase or high EnvZ phosphatase activities. At high osmolarity, OmpR-P is elevated. It has been proposed that EnvZ phosphatase is the activity that is regulated by osmolarity. OmpR is a twodomain response regulator; phosphorylation of OmpR increases its affinity for DNA, and DNA binding stimulates phosphorylation. The step that is affected by DNA depends upon the phosphodonor employed. In the present work, we have used fluorescence anisotropy and phosphotransfer assays to examine OmpR interactions with EnvZ. Our results indicate that phosphorylation greatly reduces the affinity of OmpR for the kinase, whereas DNA does not affect their interaction. The results presented cast serious doubts on the role of the EnvZ phosphatase in response to signaling in vivo.The predominant paradigm for signal transduction in prokaryotes is the two-component regulatory system (see Ref. 1 for reviews and references). The first component is a sensor kinase, often a membrane protein, which senses the appropriate environmental signal and is phosphorylated from intracellular ATP on a histidine residue. The sensor phosphokinase then transfers the phosphoryl group to an aspartic acid residue of the second component, the response regulator. Phosphorylation of the response regulator generally leads to its activation, often by increasing its affinity for DNA with a subsequent effect on transcription. In a postulated second level of regulation, it has been suggested that the sensor kinase can also stimulate dephosphorylation of the phosphorylated response regulator via a phosphatase activity, thus limiting the level of the activated regulator and resetting the system.The two-component regulatory system that governs expression of the outer membrane porins OmpF and OmpC consists of the sensor kinase EnvZ and the response regulator OmpR. Activation of EnvZ by an unknown signal, related to the osmolarity of the growth medium, leads to phosphorylation of OmpR at aspartate 55 (2-4). Phosphorylation of OmpR results in an increased affinity for the regulatory regions upstream from the ompF and ompC genes (5-8). Recent in vitro studies demonstrated that the corollary is also true, i.e. the presence of DNA increases the level of OmpR phosphorylation (9, 10). As a result of these studies, we proposed that OmpR exists as an equilibrium mixture of four distinct states (see Fig. 1): unphosphorylated OmpR (A), phosphorylated OmpR (B), unphosphorylated OmpR bound to DNA with low affinity (C), and phosphorylated OmpR bound to DNA with high affinity (D). A similar model has been proposed for the single-domain response regulator Ch...

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Section: Phosphorylation Decreases the Affinity Of Ompr Forsupporting

confidence: 68%

Phosphorylation Alters the Interaction of the Response Regulator OmpR with Its Sensor Kinase EnvZ

Mattison¹,

Kenney

2002

Journal of Biological Chemistry

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“…Studies on these chimaeric proteins [13,14], combined with mutagenesis analysis [15,16], have shown that the transmembrane, as well as the linker helices, plays a role in signal propagation. While in vitro studies on reconstituted E. coli EnvZ have shown a response to changes in K + concentration [17], the mechanisms underlying the sensing function have not been elucidated. Replacement of up to 91 amino acids (Met 56 -Arg 146 ) from the C-terminal region of EnvZ per with the non-homologous periplasmic domain of PhoR had no effect on the regulation of OmpF and OmpC expression [18], thus putting in question the role of EnvZ per in sensing osmolarity signal(s).…”

Section: Introductionmentioning

confidence: 99%

Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization

Khorchid

Inouye

Ikura

2004

Biochemical Journal

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Escherichia coli EnvZ is a membrane sensor histidine kinase that plays a pivotal role in cell adaptation to changes in extracellular osmolarity. Although the cytoplasmic histidine kinase domain of EnvZ has been extensively studied, both biochemically and structurally, little is known about the structure of its periplasmic domain, which has been implicated in the mechanism underlying its osmosensing function. In the present study, we report the biochemical and biophysical characterization of the periplasmic region of EnvZ (Ala 38 -Arg 162 ). This region was found to form a dimer in solution, and to consist of two well-defined domains: an N-terminal α-helical domain and a C-terminal core domain (Glu 83 -Arg 162 ) containing both α-helical and β-sheet secondary structures. Our pull-down assays and analytical ultracentrifugation analysis revealed that dimerization of the periplasmic region is highly sensitive to the presence of CHAPS, but relatively insensitive to salt concentration, thus suggesting the significance of hydrophobic interactions between the homodimeric subunits. Periplasmic homodimerization is mediated predominantly by the C-terminal core domain, while a regulatory function may be attributed mainly to the N-terminal α-helical domain, whose mutations have been shown previously to produce a high-osmolarity phenotype.

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“…the transporters ProP of E. coli (Culham et al, 2003), BetP of C. glutamicum (Rübenhagen et al, 2001) and OpuA of L. lactis (van der Heide et al, 2001), and the sensor kinases KdpD and EnvZ of E. coli (Jung et al, 2001). We observed that BetT mutants carrying C-terminal deletions of 6, 12 or 18 amino acid residues displayed a gradual loss in osmotic activation of choline transport.…”

Section: Discussionmentioning

confidence: 67%

Membrane topology and mutational analysis of the osmotically activated BetT choline transporter of Escherichia coli

Tøndervik

Strøm

2007

Microbiology

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For osmoprotection, Escherichia coli can synthesize glycine betaine from externally supplied choline by the Bet system (betTIBA products). The major carrier of choline is the high-affinity, proton-driven, secondary transporter BetT, which belongs to the BCCT family of transporters. Fusion proteins consisting of N-terminal fragments of BetT linked to b-galactosidase (LacZ) or alkaline phosphatase (PhoA) were constructed. By analysis of 51 fusion proteins with 37 unique fusion-points, the predictions that BetT comprised 12 membrane-spanning regions and that its N-and C-terminal extensions of about 12 and 180 amino acid residues, respectively, were situated in the cytoplasm were confirmed. This is believed to represent the first experimental examination of the membrane topology of a BCCT family protein. Osmotic upshock experiments were performed with spectinomycin-treated E. coli cells that had expressed the wild-type or a mutant BetT protein during growth at low osmolality (160 mosmol kg "1 ). The choline transport activity of wild-typeBetT increased tenfold when the cells were stressed with 0.4 M NaCl (total osmolality 780 mosmol kg

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K+ Stimulates Specifically the Autokinase Activity of Purified and Reconstituted EnvZ of Escherichia coli

Cited by 37 publications

References 49 publications

Phosphorylation Alters the Interaction of the Response Regulator OmpR with Its Sensor Kinase EnvZ

Phosphorylation Alters the Interaction of the Response Regulator OmpR with Its Sensor Kinase EnvZ

Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization

Membrane topology and mutational analysis of the osmotically activated BetT choline transporter of Escherichia coli

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