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

2004 ◽  
Vol 385 (1) ◽  
pp. 255-264 ◽  
Author(s):  
Ahmad KHORCHID ◽  
Masayori INOUYE ◽  
Mitsuhiko IKURA
Keyword(s):  
Escherichia Coli ◽  
Histidine Kinase ◽  
Analytical Ultracentrifugation ◽  
Hydrophobic Interactions ◽  
Kinase Domain ◽  
Regulatory Function ◽  
Core Domain ◽  
Sensor Histidine Kinase ◽  
Helical Domain

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 (Ala38–Arg162). 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 (Glu83–Arg162) 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.

Structure of the mRNA splicing complex component Cwc2: insights into RNA recognition

2011 ◽  
Vol 441 (2) ◽  
pp. 591-597 ◽  
Author(s):  
Peilong Lu ◽  
Guifeng Lu ◽  
Chuangye Yan ◽  
Li Wang ◽  
Wenqi Li ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Hydrophobic Interactions ◽  
Mrna Splicing ◽  
Regulatory Function ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Core Domain ◽  
Intron Removal

The Prp19-associated complex [NTC (nineteen complex)] plays a crucial role in intron removal during premature mRNA splicing in eukaryotes. Only one component of the NTC, Cwc2, is capable of binding RNA. In the present study we report the 1.9 Å (1 Å=0.1 nm) X-ray structure of the Cwc2 core domain, which is both necessary and sufficient for RNA binding. The Cwc2 core domain contains two sub-domains, a CCCH-type ZnF (zinc finger) and a RRM (RNA recognition motif). Unexpectedly, the ZnF domain and the RRM form a single folding unit, glued together by extensive hydrophobic interactions and hydrogen bonds. Structure-guided mutational analysis revealed that the intervening loop [known as the RB loop (RNA-binding loop)] between ZnF and RRM plays an essential role in RNA binding. In addition, a number of highly conserved positively charged residues on the β-strands of RRM make an important contribution to RNA binding. Intriguingly, these residues and a portion of the RB loop constitute an extended basic surface strip that encircles Cwc2 halfway. The present study serves as a framework for understanding the regulatory function of the NTC in RNA splicing.

scholarly journals The Histidine Kinase Domain of UhpB Inhibits UhpA Action at the Escherichia coli uhpT Promoter

2000 ◽  
Vol 182 (22) ◽  
pp. 6279-6286 ◽  
Author(s):  
Jesse S. Wright ◽  
Igor N. Olekhnovich ◽  
Gail Touchie ◽  
Robert J. Kadner
Keyword(s):  
Escherichia Coli ◽  
Histidine Kinase ◽  
Response Regulator ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Signaling Complex ◽  
Regulatory Systems ◽  
Negative Effect ◽  
Polytopic Membrane Protein

ABSTRACT The histidine kinase (HK) component of many two-component regulatory systems exhibits regulated ability to phosphorylate itself and to participate in transfer of phosphate to and from its cognate response regulator. The signaling system that controls expression of the UhpT sugar phosphate transporter in Escherichia coli in response to external glucose 6-phosphate includes the HK protein UhpB and the polytopic membrane protein UhpC, a UhpT homolog which is required for responsiveness to an inducer and activation of UhpB. The existence of a UhpBC signaling complex is suggested by the requirement for UhpC for the activity of certain constitutively active variants of UhpB, the dominance and epistasis relationships of uhpalleles, and the finding that expression of UhpB in excess of UhpC has a strong dominant-negative effect. Expression of a hybrid protein containing the cytoplasmic C-terminal half of UhpB fused to glutathioneS-transferase (GST) also interfered with Uhp signaling. This interference phenotype could not result solely from the phosphatase activity of UhpB, because interference affected both overexpressed UhpA and UhpA variants which are active in the absence of phosphorylation. Variant forms of UhpB which were active in the absence of UhpC carried amino acid substitutions near motifs conserved in HK proteins. The GST fusion protein inhibited the ability of UhpA to bind and activate transcription at the uhpT promoter. Unlike the wild-type situation, a GST fusion variant carrying one of the UhpB-activating substitutions, R324C, displayed autokinase activity and phosphate transfer to UhpA but retained the ability to sequester UhpA when it was altered in the conserved residues important for phosphate transfer. Thus, the default state of UhpB is kinase off, and activation of its phosphate transfer activity requires either the action of UhpC or the occurrence of certain mutations in UhpB. The interference phenotype shown by UhpB in excess of UhpC appears to include the binding and sequestration of UhpA.

scholarly journals Characterization of GlnK1 from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1

2002 ◽  
Vol 184 (4) ◽  
pp. 1028-1040 ◽  
Author(s):  
Claudia Ehlers ◽  
Roman Grabbe ◽  
Katharina Veit ◽  
Ruth A. Schmitz
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Ammonium Assimilation ◽  
Regulatory Function ◽  
Native Form ◽  
E Coli ◽  
Methanosarcina Mazei ◽  
Apparent Homogeneity

ABSTRACT Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK1 is encoded in the glnK1 -amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK1 was cloned and overexpressed in E. coli, and GlnK1 was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E. coli GlnK. First, GlnK1 from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.

scholarly journals Characterization of the RcsC Sensor Kinase from Erwinia amylovora and Other Enterobacteria

2011 ◽  
Vol 101 (6) ◽  
pp. 710-717 ◽  
Author(s):  
Dongping Wang ◽  
Schuyler S. Korban ◽  
P. Lawrence Pusey ◽  
Youfu Zhao
Keyword(s):  
Histidine Kinase ◽  
Erwinia Amylovora ◽  
Kinase Domain ◽  
Sensor Kinase ◽  
Pear Fruit ◽  
E Coli ◽  
Pantoea Stewartii ◽  
Mutant Strains

RcsC is a hybrid sensor kinase which contains a sensor domain, a histidine kinase domain, and a receiver domain. We have previously demonstrated that, although the Erwinia amylovora rcsC mutant produces more amylovoran than the wild-type (WT) strain in vitro, the mutant remains nonpathogenic on both immature pear fruit and apple plants. In this study, we have comparatively characterized the Erwinia RcsC and its homologs from various enterobacteria. Results demonstrate that expression of the Erwinia rcsC gene suppresses amylovoran production in various amylovoran overproducing WT and mutant strains, thus suggesting the presence of a net phosphatase activity of Erwinia RcsC. Findings have also demonstrated that rcsC homologs from other enterobacteria could not rescue amylovoran production of the Erwinia rcsC mutant in vitro. However, virulence of the Erwinia rcsC mutant is partially restored by rcsC homologs from Pantoea stewartii, Yersinia pestis, and Salmonella enterica but not from Escherichia coli on apple shoots. Domain-swapping experiments have indicated that replacement of the E. coli RcsC sensor domain by those of Erwinia and Yersinia spp. partially restores virulence of the Erwinia rcsC mutant, whereas chimeric constructs containing the sensor domain of E. coli RcsC could not rescue virulence of the Erwinia rcsC mutant on apple. Interestingly, only chimeric constructs containing the histidine kinase and receiver domains of Erwinia RcsC are fully capable of rescuing amylovoran production. These results suggest that the sensor domain of RcsC may be important in regulating bacterial virulence, whereas the activity of the histidine kinase and receiver domains of Erwinia RcsC may be essential for amylovoran production in vitro.

Characterization of the sensor domain of QseE histidine kinase from Escherichia coli

2016 ◽  
Vol 126 ◽  
pp. 122-126 ◽  
Author(s):  
Kwon Joo Yeo ◽  
Jin-Wan Park ◽  
Eun-Hee Kim ◽  
Young Ho Jeon ◽  
Kwang Yeon Hwang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Histidine Kinase

scholarly journals In vivocross-linking and transmembrane helix dynamics support a bidirectional non-piston model of signaling withinE. coliEnvZ

2017 ◽  
Author(s):  
Rahmi Yusuf ◽  
Tuyết Linh Nguyễn ◽  
Annika Heininger ◽  
Robert J. Lawrence ◽  
Benjamin A. Hall ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Histidine Kinase ◽  
In Silico ◽  
Transmembrane Domain ◽  
Coarse Grained ◽  
Sensor Histidine Kinase ◽  
Rotational Components ◽  
Stimulus Perception ◽  
Standing Question

AbstractIn Gram-negative bacteria, porins span the outer membrane and control the influx of several prominent groups of antibiotics. Thus, it should not be surprising that expression of these porins is often altered in clinical isolates exhibiting multidrug resistance (MDR). The major regulator of porin expression inEscherichia coliis EnvZ, a canonical sensor histidine kinase (SHK). It allosterically processes periplasmic interactions with MzrA and cytoplasmic osmosensing into a single unified change in the ratio of its kinase and phosphatase activities. Unfortunately, the role of the EnvZ transmembrane domain (TMD) in bidirectional communication of these signals remains not well understood. Here, we employedin vivosulfhydryl-reactivity to probe the dynamics of the TM2 helices and demonstrate that upon stimulus perception, only the region proximal to the periplasm undergoes conformational rearrangement. Furthermore,in silicocoarse-grained molecular dynamics (CG-MD) simulations with aromatically tuned variants of EnvZ TM2 demonstrate the existence of both tilting and azimuthal rotational components to transmembrane communication while ruling out piston-type repositioning of TM2. Finally, in contrast to a similar analysis of TM1, we identified position-specific mutants possessing a “flipped” phenotype by dual-color fluorescent reporter analysis suggesting that both the periplasmic and cytoplasmic ends of TM2 are critical for maintenance of EnvZ signal output. Taken together, these data strongly support that EnvZ employs a non-piston-type mechanism during transmembrane communication. We conclude by discussing these results within the context of allosteric processing by EnvZ and propose that these results can be used to predict and classify transmembrane communication by various SHKs.ImportanceThe EnvZ sensor histidine kinase serves as the major regulator of porin expression withinEscherichia coli. A long-standing question is how stimulus perception by a bacterial receptor on one side of a biological membrane is transmitted to the opposite side of the membrane. To address this question, we monitored the dynamics of the transmembrane domain of EnvZin vivoand coupled these results within silicosimulations of membrane-embedded EnvZ transmembrane domains. Taken together, these results demonstrate that detection of osmotic stress by the cytoplasmic domain of EnvZ results in non-piston communication across the inner membrane ofE. coli.Thus, in addition to understanding how EnvZ regulates porin balance and antibiotic influx, these results contribute to answering the long-standing question of how transmembrane communication is performed by bacterial receptors. Our work concludes with a framework that correlates receptor domain composition and signal transduction mechanisms that could be employed by other research groups on their particular receptors of interest.

Sign in / Sign up

Export Citation Format

Share Document