scholarly journals Genetic Evidence that the α5 Helix of the Receiver Domain of PhoB Is Involved in Interdomain Interactions

2001 ◽  
Vol 183 (7) ◽  
pp. 2204-2211 ◽  
Author(s):  
Mindy P. Allen ◽  
Kimberly B. Zumbrennen ◽  
William R. McCleary
Keyword(s):  
Response Regulator ◽  
Response Regulators ◽  
Receiver Domain ◽  
Intracellular Signals ◽  
Regulator Protein ◽  
Phosphorylation Cascade ◽  
Α Helix ◽  
Interdomain Interactions ◽  
Response Regulator Protein ◽  
Output Domain

ABSTRACT Two-component signaling proteins are involved in transducing environmental stimuli into intracellular signals. Information is transmitted through a phosphorylation cascade that consists of a histidine protein kinase and a response regulator protein. Generally, response regulators are made up of a receiver domain and an output domain. Phosphorylation of the receiver domain modulates the activity of the output domain. The mechanisms by which receiver domains control the activities of their respective output domains are unknown. To address this question for the PhoB protein from Escherichia coli, we have employed two separate genetic approaches, deletion analysis and domain swapping. In-frame deletions were generated within the phoB gene, and the phenotypes of the mutants were analyzed. The output domain, by itself, retained significant ability to activate transcription of the phoA gene. However, another deletion mutant that contained the C-terminal α-helix of the receiver domain (α5) in addition to the entire output domain was unable to activate transcription of phoA. This result suggests that the α5 helix of the receiver domain interacts with and inhibits the output domain. We also constructed two chimeric proteins that join various parts of the chemotaxis response regulator, CheY, to PhoB. A chimera that joins the N-terminal ∼85% of CheY's receiver domain to the β5-α5 loop of PhoB's receiver domain displayed phosphorylation-dependent activity. The results from both sets of experiments suggest that the regulation of PhoB involves the phosphorylation-mediated modulation of inhibitory contacts between the α5 helix of its unphosphorylated receiver domain and its output domain.

scholarly journals Phosphate flow in the chemotactic response system of Helicobacter pylori

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3299-3311 ◽  
Author(s):  
María-Antonieta Jiménez-Pearson ◽  
Isabel Delany ◽  
Vincenzo Scarlato ◽  
Dagmar Beier
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Chemotactic Response ◽  
Receiver Domain ◽  
Regulator Protein ◽  
Infection Models ◽  
Animal Infection ◽  
H Pylori ◽  
Response Regulator Protein

It is well established that motility is an essential virulence trait of the human gastric pathogen Helicobacter pylori. Accordingly, chemotaxis contributes to the ability of H. pylori to colonize animal infection models. Chemotactic signal transduction in H. pylori differs from the enterobacterial paradigm in several respects. In addition to a separate CheY response regulator protein (CheY1), H. pylori contains a CheY-like receiver domain (CheY2) which is C-terminally fused to the histidine kinase CheA. Furthermore, the genome of H. pylori encodes three CheV proteins consisting of an N-terminal CheW-like domain and a C-terminal receiver domain, while there are no orthologues of the chemotaxis genes cheB, cheR and cheZ. To obtain insight into the mechanisms controlling the chemotactic response of H. pylori, we investigated the phosphotransfer reactions between the purified two-component signalling modules in vitro. We demonstrate that both CheY1 and CheY2 are phosphorylated by CheA∼P and that the three CheV proteins mediate the dephosphorylation of CheA∼P, but with a clearly reduced efficiency as compared to CheY1 and CheY2. Furthermore, our data indicate retrophosphorylation of CheAY2 by CheY1∼P, suggesting a role of CheY2 as a phosphate sink to modulate the half-life of CheY1∼P.

scholarly journals Phosphorylation-Induced Signal Propagation in the Response Regulator NtrC

2000 ◽  
Vol 182 (18) ◽  
pp. 5188-5195 ◽  
Author(s):  
Jonghui Lee ◽  
Jeffrey T. Owens ◽  
Ingyu Hwang ◽  
Claude Meares ◽  
Sydney Kustu
Keyword(s):  
Transcriptional Activation ◽  
Atp Hydrolysis ◽  
Response Regulator ◽  
Regulatory System ◽  
Signal Propagation ◽  
Protein Cleavage ◽  
Iron Chelate ◽  
Receiver Domain ◽  
Α Helix ◽  
Output Domain

ABSTRACT The bacterial enhancer-binding protein NtrC is a well-studied response regulator in a two-component regulatory system. The amino (N)-terminal receiver domain of NtrC modulates the function of its adjacent output domain, which activates transcription by the ς54 holoenzyme. When a specific aspartate residue in the receiver domain of NtrC is phosphorylated, the dimeric protein forms an oligomer that is capable of ATP hydrolysis and transcriptional activation. A chemical protein cleavage method was used to investigate signal propagation from the phosphorylated receiver domain of NtrC, which acts positively, to its central output domain. The iron chelate reagent Fe-BABE was conjugated onto unique cysteines introduced into the N-terminal domain of NtrC, and the conjugated proteins were subjected to Fe-dependent cleavage with or without prior phosphorylation. Phosphorylation-dependent cleavage, which requires proximity and an appropriate orientation of the peptide backbone to the tethered Fe-EDTA, was particularly prominent with conjugated NtrCD86C, in which the unique cysteine lies near the top of α-helix 4. Cleavage occurred outside the receiver domain itself and on the partner subunit of the derivatized monomer in an NtrC dimer. The results are commensurate with the hypothesis that α-helix 4 of the phosphorylated receiver domain of NtrC interacts with the beginning of the central domain for signal propagation. They imply that the phosphorylation-dependent interdomain and intermolecular interactions between the receiver domain of one subunit and the output domain of its partner subunit in an NtrC dimer precede—and may give rise to—the oligomerization needed for transcriptional activation.

scholarly journals Candida albicans Response Regulator Gene SSK1 Regulates a Subset of Genes Whose Functions Are Associated with Cell Wall Biosynthesis and Adaptation to Oxidative Stress

2003 ◽  
Vol 2 (5) ◽  
pp. 1018-1024 ◽  
Author(s):  
Neeraj Chauhan ◽  
Diane Inglis ◽  
Elvira Roman ◽  
Jesus Pla ◽  
Dongmei Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Signal Transduction ◽  
Hydrogen Peroxide ◽  
Cell Wall ◽  
Response Regulator ◽  
Cell Wall Biosynthesis ◽  
Regulator Protein ◽  
Two Component ◽  
Response Regulator Protein ◽  
Mutant Cells

ABSTRACT Ssk1p of Candida albicans is a putative response regulator protein of the Hog1 two-component signal transduction system. In Saccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that C. albicans SSK1 does not complement the ssk1 mutant of S. cerevisiae and that the ssk1 mutant of C. albicans is not sensitive to sorbitol. In this study, we show that the C. albicans ssk1 mutant is sensitive to several oxidants, including hydrogen peroxide, t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by the ssk1 mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30°C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). In S. cerevisiae, three (AHP1, HSP12, and PYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis in C. albicans. Of the down-regulated genes, ALS1 is a known cell adhesin in C. albicans. Verification of the microarray data was obtained by reverse transcription-PCR for HSP12, AHP1, CHK1, PYC2, GPH1, ALS1, MNN4-4, and FLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in C. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in the ssk1 mutant of C. albicans when grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in the ssk1 mutant of C. albicans when grown in a hyperosmotic medium but was not phosphorylated in the ssk1 mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate that C. albicans utilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further, SSK1 appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions of C. albicans SSK1 differ from those of S. cerevisiae SSK1.

scholarly journals Switched or Not?: the Structure of Unphosphorylated CheY Bound to the N Terminus of FliM

2006 ◽  
Vol 188 (21) ◽  
pp. 7354-7363 ◽  
Author(s):  
Collin M. Dyer ◽  
Frederick W. Dahlquist
Keyword(s):  
Binding Site ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Active Role ◽  
Crystallographic Structure ◽  
Coupling Mechanism ◽  
Allosteric Communication ◽  
Regulator Protein ◽  
Target Binding ◽  
Response Regulator Protein

ABSTRACT Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF3 −-CheY, a phosphorylated CheY (CheY∼P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY∼P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM16]). While the peptide conformation and binding site are similar to those of the BeF3 −-CheY-FliM16 complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg2+-CheY-FliM16 complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF3 −-activated CheY. This structure makes clear the active role that the β4-α4 loop plays in the Tyr87-Tyr106 coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.

scholarly journals Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Varisa Huangyutitham ◽  
Zehra Tüzün Güvener ◽  
Caroline S. Harwood
Keyword(s):  
Signal Transduction ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Cluster Formation ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Regulator Protein ◽  
Response Regulator Protein

ABSTRACT WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. IMPORTANCE Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

scholarly journals Phospho-dependent Signaling during the General Stress Response by the Atypical Response Regulator and ClpXP Adaptor RssB

2021 ◽  
Author(s):  
Jacob Schwartz ◽  
Jonghyeon Son ◽  
Christiane Brugger ◽  
Alexandra M. Deaconescu
Keyword(s):  
Stress Response ◽  
Response Regulator ◽  
Model Organism ◽  
Coupling Scheme ◽  
Response Regulators ◽  
General Stress Response ◽  
Signaling Model ◽  
Receiver Domain ◽  
General Stress ◽  
Promoter Specificity

ABSTRACTIn the model organism Escherichia coli and related species, the general stress response relies on tight regulation of the intracellular levels of the promoter specificity subunit RpoS. RpoS turnover is exclusively dependent on RssB, a two-domain response regulator that functions as an adaptor that delivers RpoS to ClpXP for proteolysis. Here we report crystal structures of the receiver domain of RssB both in its unphosphorylated form and bound to the phosphomimic BeF3−. Surprisingly, we find only modest differences between these two structures, suggesting that truncating RssB may partially activate the receiver domain to a “meta-active” state. Our structural and sequence analysis points to RssB proteins not conforming to either the Y-T coupling scheme for signaling seen in prototypical response regulators, such as CheY, or to the signaling model of the less understood FATGUY proteins.

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