Crystal structure of PhoU fromPseudomonas aeruginosa, a negative regulator of the Pho regulon

ABSTRACT Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (Pi). Under these conditions, the high-affinity PstSCAB2 protein (i.e., with two PstB proteins) is the primary Pi transporter. Expression from the pstSCAB-phoU operon is regulated by the PhoB/PhoR two-component regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies of phoU have proven to be difficult because deletion of the phoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability of phoU deletions, we employed a promoter-swapping technique that places expression of the phoBR two-component system under control of the Ptac promoter and the lacO ID regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized PphoB ::Ptac and PpstS ::Ptac strains to characterize phenotypes resulting from various ΔphoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB2 transporter, as well as its abundance within the cell. In addition, we used the PphoB ::Ptac ΔphoU strain as a platform to begin characterizing new phoU mutations in plasmids.

Download Full-text

Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure

Journal of Bacteriology ◽

10.1128/jb.185.1.262-273.2003 ◽

2003 ◽

Vol 185 (1) ◽

pp. 262-273 ◽

Cited By ~ 27

Author(s):

Yinghua Chen ◽

Catherine Birck ◽

Jean-Pierre Samama ◽

F. Marion Hulett

Keyword(s):

Crystal Structure ◽

Dna Binding ◽

Transcriptional Activation ◽

Salt Bridge ◽

Pho Regulon ◽

Dimer Interface ◽

And Function ◽

Negative Phenotype

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3′ of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E∼P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

Download Full-text

Purification of the phoU protein, a negative regulator of the pho regulon of Escherichia coli K-12.

Journal of Bacteriology ◽

10.1128/jb.168.2.631-635.1986 ◽

1986 ◽

Vol 168 (2) ◽

pp. 631-635 ◽

Cited By ~ 15

Author(s):

B P Surin ◽

N E Dixon ◽

H Rosenberg

Keyword(s):

Escherichia Coli ◽

Protein A ◽

Negative Regulator ◽

Pho Regulon ◽

K 12

Download Full-text

Crystal structure of Npun_R1517, a putative negative regulator of heterocyst differentiation fromNostoc punctiformePCC 73102

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.22308 ◽

2009 ◽

Vol 74 (3) ◽

pp. 794-798 ◽

Cited By ~ 4

Author(s):

Shuisong Ni ◽

Matthew M. Benning ◽

Matthew J. Smola ◽

Erik A. Feldmann ◽

Michael A. Kennedy

Keyword(s):

Crystal Structure ◽

Negative Regulator ◽

Heterocyst Differentiation

Download Full-text

X-ray Crystal Structure of the Bacterial Conjugation Factor PsiB, a Negative Regulator of RecA

Journal of Biological Chemistry ◽

10.1074/jbc.m110.152298 ◽

2010 ◽

Vol 285 (40) ◽

pp. 30615-30621 ◽

Cited By ~ 3

Author(s):

Vessela Petrova ◽

Kenneth A. Satyshur ◽

Nicholas P. George ◽

Darrell McCaslin ◽

Michael M. Cox ◽

...

Keyword(s):

Crystal Structure ◽

Negative Regulator ◽

Bacterial Conjugation ◽

X Ray

Download Full-text

A differential effect of σ S on the expression of the PHO regulon genes of Escherichia coli

Microbiology ◽

10.1099/mic.0.27124-0 ◽

2004 ◽

Vol 150 (9) ◽

pp. 2985-2992 ◽

Cited By ~ 31

Author(s):

Natalia Pasternak Taschner ◽

Ezra Yagil ◽

Beny Spira

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Phosphate Starvation ◽

Negative Regulator ◽

Transcriptional Level ◽

Pho Regulon ◽

Phosphate Binding ◽

The Core ◽

Phosphate Binding Protein ◽

Positive Effect

The RNA polymerase core associated with σ S transcribes many genes related to stress or to the stationary phase. When cells enter a phase of phosphate starvation, the transcription of several genes and operons, collectively known as the PHO regulon, is strongly induced. The promoters of the PHO genes hitherto analysed are recognized by σ D-associated RNA polymerase. A mutation in the gene that encodes σ S, rpoS, significantly increases the level of alkaline phosphatase activity and the overproduction of σ S inhibits it. Other PHO genes such as phoE and ugpB are likewise affected by σ S. In contrast, pstS, which encodes a periplasmic phosphate-binding protein and is a negative regulator of PHO, is stimulated by σ S. The effect of σ S on the PHO genes is at the transcriptional level. It is shown that a cytosine residue at position −13 is important for the positive effect of σ S on pst. The interpretation of these observations is based on the competition between σ S and σ D for the binding to the core RNA polymerase.

Download Full-text

Discovery and Structure-Based Design of Macrocyclic Peptides Targeting STUB1

10.26434/chemrxiv-2021-1108b ◽

2021 ◽

Author(s):

Simon Ng ◽

Alexander Brueckner ◽

Soheila Bahmanjah ◽

Qiaolin Deng ◽

Jennifer Johnston ◽

...

Keyword(s):

Crystal Structure ◽

Interferon Gamma ◽

Negative Regulator ◽

Malignant Cells ◽

Target Validation ◽

Exposed Region ◽

Macrocyclic Peptides ◽

Cellular Permeability ◽

Hydrophilic Solvent ◽

Further Development

STIP1 homology and U-Box containing protein 1 (STUB1) plays a key role in maintaining cell health during stress and aging. Recent evidence suggested STUB1 also helps regulate immunity with the potential of clearing malignant cells. Indeed, we and others have shown that STUB1 is a pivotal negative regulator of interferon gamma sensing – a process critical to the immunosurveillance of tumors and pathogens. Thus far, investigation of STUB1’s role relies mostly on genetic approaches as pharmacological inhibitors of this protein are lacking. Identification of a STUB1 tool compound is important as it would allow therapeutically relevant target validation in a broader sense. Accordingly, we leveraged phage display and computational modeling to identify and refine STUB1 binders. Screening of >10E9 macrocyclic peptides resulted in several conserved motifs as well as structurally diverse leads. Co-crystal structure of the peptide hit and STUB1 has enabled us to employ structure-based in silico design for further optimization. Of the modifications employed, replacing the hydrophilic solvent-exposed region of the macrocyclic peptides with a hydrophobic scaffold improved cellular permeability, while the binding conformation was maintained. Further substitution of the permeability-limiting terminal aspartic acid with a tetrazole bioisostere retained the binding to certain extent while improving permeability, suggesting a path forward. The current lead, although not optimal for cellular study, provides a valuable template for further development into selective tool compounds for STUB1 to enable target validation.

Download Full-text

Crystal Structure Determination of Glycerol-Containing Lipids from Electron Diffraction Data. Use of Analog Compounds Based upon Configurational Isomers of Cyclopentane-1, 2, 3-TRIOL

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077761 ◽

1977 ◽

Vol 35 ◽

pp. 24-25

Author(s):

Douglas L. Dorset ◽

Anthony J. Hancock

Keyword(s):

Crystal Structure ◽

Electron Diffraction ◽

Diffraction Data ◽

Structure Determination ◽

Crystal Surface ◽

Coherent Scattering ◽

Crystal Structure Determination ◽

Electron Diffraction Data ◽

Polar Group ◽

Axis Orientation

Lipids containing long polymethylene chains were among the first compounds subjected to electron diffraction structure analysis. It was only recently realized, however, that various distortions of thin lipid microcrystal plates, e.g. bends, polar group and methyl end plane disorders, etc. (1-3), restrict coherent scattering to the methylene subcell alone, particularly if undistorted molecular layers have well-defined end planes. Thus, ab initio crystal structure determination on a given single uncharacterized natural lipid using electron diffraction data can only hope to identify the subcell packing and the chain axis orientation with respect to the crystal surface. In lipids based on glycerol, for example, conformations of long chains and polar groups about the C-C bonds of this moiety still would remain unknown.One possible means of surmounting this difficulty is to investigate structural analogs of the material of interest in conjunction with the natural compound itself. Suitable analogs to the glycerol lipids are compounds based on the three configurational isomers of cyclopentane-1,2,3-triol shown in Fig. 1, in which three rotameric forms of the natural glycerol derivatives are fixed by the ring structure (4-7).

Download Full-text