Purification and Characterization of the Periplasmic Domain of EnvZ Osmosensor inEscherichia coli

Egger, Linda A.; Inouye, Masayori

doi:10.1006/bbrc.1996.6007

Cited by 16 publications

(14 citation statements)

References 31 publications

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“…Analysis of the CD spectra using the K2D software indicates that EnvZ per consists of 41 % α-helical and 20 % β-sheet structures. This is in close agreement with data reported previously [22], as well as the PHD prediction of 45 % α-helices and 22 % β-sheets in the corresponding sequence of EnvZ. To determine that EnvZ per is properly folded into tertiary structure, we measured the near UV (250-330 nm) CD spectrum, which is indicative of the chemical environment of aromatic residues, such as phenylalanine (250-270 nm), tyrosine (270-290 nm) and tryptophan (280-300 nm).…”

Section: Structural Characterizationsupporting

confidence: 94%

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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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Section: Structural Characterizationsupporting

confidence: 94%

“…15 [22]. The protein expression and yield reported were too low for detailed biophysical analysis, and the construct used lacked seven highly conserved N-terminal amino acids from the periplasmic region (Pro 41 -Asn 47 ) ( Figure 1B).…”

Section: Nmr Spectroscopymentioning

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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“…This result implies that the activation signal is in excess or that additional domains (i.e. transmembrane) are required for titrating the activation signal (Egger & Inouye 1997a). Transmembrane mutations result in various phenotypes which indicates their importance in transmembrane signalling.…”

Section: Genetic Analysismentioning

confidence: 99%

Signal transduction via the histidyl‐aspartyl phosphorelay

1997

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The histidyl-aspartyl phosphorelay, formerly described as the two-component system, is the predominant mode of signal transduction in bacteria. Adaptation to environmental changes occurs through a sensor histidine protein kinase and a response regulator. The histidine protein kinase is usually a transmembrane receptor and the response regulator is a cytoplasmic protein.Together the histidyl-aspartyl phosphorelay proteins mediate reversible phosphorylation events that control downstream effectors. Following autophosphorylation at a conserved histidine residue, the histidine kinase serves as a phospho-donor for the response regulator. Once phosphorylated, the response regulator mediates changes in gene expression or cellular locomotion. The EnvZ-OmpR phosphorelay system in Escherichia coli, which monitors external osmolarity and responds by differentially modulating the expression of the OmpF and OmpC major outer membrane porins, will be described as a model system. While histidine kinases were thought to be present only in prokaryotes, they have recently been identified in eukaryotic systems. Here, we review the unique and conserved features of this growing family of signal transducers.

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“…In order to respond to changes in environmental conditions, E. coli adjusts OmpF and OmpC expression through a complex regulatory network utilizing both transcriptional and translational regulation (for review, see Forst and Inouye, 1988; Pratt et al, 1996; Vogel and Papenfort, 2006). For instance, the osmolarity-dependent transcriptional control of ompF and ompC is exerted via the EnvZ/OmpR two-component signal transduction system in which EnvZ, an inner membrane histidine protein kinase, senses osmotic signals and transmits them to the transcription factor OmpR (Igo et al, 1989; Egger and Inouye, 1997; Yoshida et al, 2006). High osmolarity leads to lower OmpF levels, while relative expression of OmpC is increased (Forst and Inouye, 1988; Mizuno and Mizushima, 1990; Pratt et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

A Novel Regulatory Cascade Involving BluR, YcgZ, and Lon Controls the Expression of Escherichia coli OmpF Porin

et al. 2017

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In Escherichia coli, OmpF is an important outer membrane protein, which serves as a passive diffusion pore for small compounds including nutrients, antibiotics, and toxic compounds. OmpF expression responds to environmental changes such as temperature, osmolarity, nutrients availability, and toxic compounds via complex regulatory pathways involving transcriptional and post-transcriptional regulation. Our study identified a new regulatory cascade that controls the expression of OmpF porin. This pathway involves BluR, a transcriptional regulator repressing the expression of the ycgZ-ymgABC operon. We showed that BluR was responsible for the temperaturedependent regulation of the ycgZ-ymgABC operon. Furthermore, our results showed that independent expression of YcgZ led to a decreased activity of the ompF promoter, while YmgA, YmgB, and YmgC expression had no effect. We also determined that YcgZ accumulates in the absence of the Lon protease. Thus, mutation in bluR leads to derepression of ycgZ-ymgABC transcription. With a second mutation in lon, YcgZ protein accumulates to reach levels that do not allow increased expression of OmpF under growth conditions that usually would, i.e., low temperature. With BluR responding to blue-light and temperature, this study sheds a new light on novel signals able to regulate OmpF porin.

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Purification and Characterization of the Periplasmic Domain of EnvZ Osmosensor inEscherichia coli

Cited by 16 publications

References 31 publications

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

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

Signal transduction via the histidyl‐aspartyl phosphorelay

A Novel Regulatory Cascade Involving BluR, YcgZ, and Lon Controls the Expression of Escherichia coli OmpF Porin

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