Secondary literature sources for the Trans_reg_C domain

For each of the hand selected primary papers associated with the Trans_reg_C domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Crystal structure of Skp, a prefoldin-like chaperone that protects solubleand membrane proteins from aggregation.

Walton TA, Sousa MC
Mol Cell. 2004; 15: 367-74

The Seventeen Kilodalton Protein (Skp) is a trimeric periplasmic chaperonethat assists outer membrane proteins in their folding and insertion intomembranes. Here we report the crystal structure of Skp from E. coli. Thestructure of the Skp trimer resembles a jellyfish with alpha-helicaltentacles protruding from a beta barrel body defining a central cavity.The architecture of Skp is unexpectedly similar to that of Prefoldin/GimC,a cytosolic chaperone present in eukaria and archea, that binds unfoldedsubstrates in its central cavity. The ability of Skp to prevent theaggregation of model substrates in vitro is independent of ATP. Skp caninteract directly with membrane lipids and lipopolysaccharide (LPS). Theseinteractions are needed for efficient Skp-assisted folding of membraneproteins. We have identified a putative LPS binding site on the outersurface of Skp and propose a model for unfolded substrate binding.

Crystal structure of FadR, a fatty acid-responsive transcription factorwith a novel acyl coenzyme A-binding fold.

van Aalten DM, DiRusso CC, Knudsen J, Wierenga RK
EMBO J. 2000; 19: 5167-77

FadR is a dimeric acyl coenzyme A (acyl CoA)-binding protein andtranscription factor that regulates the expression of genes encoding fattyacid biosynthetic and degrading enzymes in Escherichia coli. Here, the 2.0A crystal structure of full-length FadR is described, determined usingmulti-wavelength anomalous dispersion. The structure reveals a dimer and atwo-domain fold, with DNA-binding and acyl-CoA-binding sites located in anN-terminal and C-terminal domain, respectively. The N-terminal domaincontains a winged helix-turn-helix prokaryotic DNA-binding fold.Comparison with known structures and analysis of mutagenesis datadelineated the site of interaction with DNA. The C-terminal domain has anovel fold, consisting of a seven-helical bundle with a crossovertopology. Careful analysis of the structure, together with mutational andbiophysical data, revealed a putative hydrophobic acyl-CoA-binding site,buried in the core of the seven-helical bundle. This structure aids inunderstanding FadR function at a molecular level, provides the firststructural scaffold for the large GntR family of transcription factors,which are keys in the control of metabolism in bacterial pathogens, andcould thus be a possible target for novel chemotherapeutic agents.

A single amino acid substitution in the C terminus of OmpR alters DNArecognition and phosphorylation.

Tran VK, Oropeza R, Kenney LJ
J Mol Biol. 2000; 299: 1257-70

In bacteria and lower eukaryotes, adaptation to changes in the environmentis often mediated by two-component regulatory systems. Such systemsprovide the basis for chemotaxis, nitrogen and phosphate regulation andadaptation to osmotic stress, for example. In Escherichia coli, the sensorkinase EnvZ detects a change in the osmotic environment and phosphorylatesthe response regulator OmpR. Phospho-OmpR binds to the regulatory regionsof the porin genes ompF and ompC, and alters their expression. Recentevidence suggests that OmpR functions as a global regulator, regulatingadditional genes besides the porin genes. In this study, we havecharacterized a previously isolated OmpR2 mutant (V203M) thatconstitutively activates ompF and fails to express ompC. Because thesubstitution was located in the C-terminal DNA-binding domain, it had beenassumed that the substitution would not affect phosphorylation of theN-terminal domain of OmpR. Our results indicate that this substitutioncompletely eliminates phosphorylation by a small phosphate donor, acetylphosphate, but not phosphorylation by the kinase EnvZ. The mutant OmpR hasaltered dephosphorylation kinetics and altered binding affinities to bothompF and ompC sites compared to the wild-type. Thus, a single amino acidsubstitution in the C-terminal DNA-binding domain has dramatic effects onthe N-terminal phosphorylation domain. Most strikingly, we have identifieda single base change in the OmpR binding site of ompC that restoreshigh-affinity binding activity by the mutant. We interpret our results inthe context of a model for porin gene expression.

A complex role for the gamma-butyrolactone SCB1 in regulating antibioticproduction in Streptomyces coelicolor A3(2).

Takano E, Chakraburtty R, Nihira T, Yamada Y, Bibb MJ
Mol Microbiol. 2001; 41: 1015-28

Many streptomycetes produce extracellular gamma-butyrolactones. In severalcases, these have been shown to act as signals for the onset of antibioticproduction. Synthesis of these molecules appears to require a member ofthe AfsA family of proteins (AfsA is required for A-factor synthesis ofthe gamma-butyrolactone A-factor and consequently for streptomycinproduction in Streptomyces griseus). An afsA homologue, scbA, wasidentified in Streptomyces coelicolor A3(2) and was found to lie adjacentto a divergently transcribed gene, scbR, which encodes agamma-butyrolactone binding protein. Gel retardation assays and DNase Ifootprinting studies revealed DNA binding sites for ScbR at - 4 to - 33 ntwith respect to the scbA transcriptional start site, and at - 42 to - 68nt with respect to the scbR transcriptional start site. Addition of thegamma-butyrolactone SCB1 of S. coelicolor resulted in loss of theDNA-binding ability of ScbR. A scbA mutant produced nogamma-butyrolactones, yet overproduced two antibiotics, actinorhodin (Act)and undecylprodigiosin (Red), whereas a deletion mutant of scbR alsofailed to make gamma-butyrolactones and showed delayed Red production.These phenotypes differ markedly from those expected by analogy with theS. griseus A-factor system. Furthermore, transcription of scbR increased,and that of scbA was abolished, in an scbR mutant, indicating that ScbRrepresses its own expression while activating that of scbA. In the scbAmutant, expression of both genes was greatly reduced. Addition of SCB1 tothe scbA mutant induced transcription of scbR, but did not restore scbAexpression, indicating that the deficiency in scbA transcription in thescbA mutant is not solely due to the inability to produce SCB1, and thatScbA is a positive autoregulator in addition to being required forgamma-butyrolactone production. Overall, these results indicate a complexmechanism for gamma-butyrolactone-mediated regulation of antibioticbiosynthesis in S. coelicolor.

Structural comparison of the PhoB and OmpR DNA-binding/transactivationdomains and the arrangement of PhoB molecules on the phosphate box.

Okamura H, Hanaoka S, Nagadoi A, Makino K, Nishimura Y
J Mol Biol. 2000; 295: 1225-36

PhoB is a transcriptional activator that binds to the phosphate box in thepromoters of the phosphate genes of Escherichia coli. PhoB contains twofunctional domains, an N-terminal phosphorylation domain and a C-terminalDNA-binding/transactivation domain. Here, the three-dimensional structureof the DNA-binding/transactivation domain has been determined by NMR. Itconsists of an N-terminal four-stranded beta-sheet, a central threehelical bundle and a C-terminal beta-hairpin. The second and third helicesform a helix-turn-helix (HTH) variant containing a longer turn than thecorresponding turn of the classical HTH motif. The overall architecture isvery close to that of the OmpR DNA-binding/transactivation domain,however, the conformation of the long turn region of PhoB, a putativeinteraction site for the RNA polymerase sigma subunit, is entirelydifferent from that of the corresponding turn of OmpR, which interactswith the alpha subunit. In addition, the third helix of PhoB is threeamino acid residues longer than the corresponding helix of OmpR. Thebinding site of PhoB is a TGTCA sequence and the phospahte box containsthe two binding sites. NMR studies of the complexes of the PhoBDNA-binding/transactivation domain bound to several different DNAmolecules have revealed that two PhoB molecules bind in a tandem array onthe phosphate box. In each complex of PhoB the third helix of theDNA-binding/transactivation domain is likely to recognize the TGTCAsequence from the major groove of DNA and the C-terminal beta-hairpincontacts on the minor groove of the 3' site out of the TGTCA sequence in anon-specific manner. The long turn region facing outward is likely tointeract with the sigma subunit.

Structure of the DNA-binding domain of the OmpR family of responseregulators.

Mizuno T, Tanaka I
Mol Microbiol. 1997; 24: 665-7

A phosphorylation site mutant of OmpR reveals different bindingconformations at ompF and ompC.

Mattison K, Oropeza R, Byers N, Kenney LJ
J Mol Biol. 2002; 315: 497-511

In Escherichia coli, the two-component regulatory system that controls theexpression of outer membrane porins in response to environmentalosmolarity consists of the sensor kinase EnvZ and the response regulatorOmpR. Phosphorylated OmpR activates expression of the OmpF porin at lowosmolarity, and at high osmolarity represses ompF transcription andactivates expression of OmpC. We have characterized a substitution in theamino-terminal phosphorylation domain of OmpR, T83I, its phenotype isOmpF(-) OmpC(-). The mutant protein is not phosphorylated by smallmolecule phosphodonors such as acetyl phosphate and phosphoramidate, butit is phosphorylated by the cognate kinase EnvZ. Interestingly, the activesite T83I substitution alters the DNA binding properties of thecarboxyl-terminal effector domain. DNase I protection assays indicate thatDNA binding by the mutant protein is similar to wild-type OmpR at the ompFpromoter, but at ompC, the pattern of protection is different from OmpR.Our results indicate that all three of the OmpR binding sites at the ompCpromoter must be filled in order to activate gene expression. Furthermore,it appears that OmpR-phosphate must adopt different conformations whenbound at ompF and ompC. A model is presented to account for the reciprocalregulation of OmpF and OmpC porin expression.

Structural relationships in the OmpR family of winged-helix transcriptionfactors.

Martinez-Hackert E, Stock AM
J Mol Biol. 1997; 269: 301-12

OmpR, a protein that regulates expression of outer membrane porin proteinsin enteric bacteria, belongs to a large family of transcription factors.These transcription factors bind DNA and interact productively with RNApolymerase to activate transcription. The two functions, DNA-binding andtranscriptional activation, have been localized within the 100 amino acidDNA-binding domain that characterizes members of the OmpR family. Both DNAbinding and transcriptional activation by OmpR related proteins haveremained poorly understood for lack of structural information or lack ofsequence homology with transcription factors of known three-dimensionalstructure. The recently determined crystal structures of the Escherichiacoli OmpR DNA-binding domain (OmpRc) have defined a new subfamily of"winged-helix-turn-helix" DNA-binding proteins. Structural elements ofOmpRc can be assigned functional roles by analogy to other winged-helixDNA-binding proteins. A structure based sequence analysis of the OmpRfamily of transcription factors indicates specific roles for all conservedamino acid residues. Mutagenesis studies performed on several members ofthis family, OmpR, PhoB, ToxR and VirG, can now be interpreted withrespect to the structure.

Mutational analysis of the highly conserved C-terminal residues of theXylS protein, a member of the AraC family of transcriptional regulators.

Manzanera M, Marques S, Ramos JL
FEBS Lett. 2000; 476: 312-7

The XylS protein of the TOL plasmid of Pseudomonas putida belongs to theso-called AraC/XylS family of regulators, that includes more than 100different bacterial proteins. A conserved stretch of about 100 amino acidsis present at the C-terminal end. This conserved region is believed tocontain seven alpha-helices, including two helix-turn-helix (HTH) DNAbinding motifs (alpha(2)-T-alpha(3) and alpha(5)-Talpha-(6)), connected bya linker alpha-helix (alpha(4)), and two flanking alpha-helices (alpha(1)and alpha(7)). The second HTH motif is the region with the highesthomology in the proteins of the family, with certain residues showingalmost 90% identity. We have constructed XylS single mutants in the mostconserved residues and have analysed their ability to stimulatetranscription from its cognate promoter, Pm, fused to 'lacZ. The analysisrevealed that mutations in the alpha(5)-helix conserved residues hadlittle effect on the XylS transcriptional activity, whereas thedistribution of polarity in the alpha(6)-helix was important for theactivity. The strongest effect of the mutations was observed in conservedresidues located outside the DNA binding domain, namely, Gly-290 in theturn between the two helices, Pro-309 located downstream of alpha(6), andLeu-313, in the small last helix alpha(7), that seems to play an importantrole in the activation of RNA-polymerase. Our analysis shows thatconservation of amino acids in the family reflects structural requirementsrather than functionality in specific DNA interactions.

Crystal structure of the Escherichia coli Rob transcription factor incomplex with DNA.

Kwon HJ, Bennik MH, Demple B, Ellenberger T
Nat Struct Biol. 2000; 7: 424-30

The Escherichia coli Rob protein is a transcription factor belonging tothe AraC/XylS protein family that regulates genes involved in resistanceto antibiotics, organic solvents and heavy metals. The genes encodingthese proteins are activated by the homologous proteins MarA and SoxS,although the level of activation can vary for the different transcriptionfactors. Here we report a 2.7 A crystal structure of Rob in complex withthe micF promoter that reveals an unusual mode of binding to DNA. TheRob-DNA complex differs from the previously reported structure of MarAbound to the mar promoter, in that only one of Rob's dual helix-turn-helix(HTH) motifs engages the major groove of the binding site. Biochemicalstudies show that sequence specific interactions involving only one ofRob's HTH motifs are sufficient for high affinity binding to DNA. The twodifferent modes of DNA binding seen in crystal structures of Rob and MarAalso match the distinctive patterns of DNA protection by AraC at severalsites within the pBAD promoter. These and other findings suggest that geneactivation by AraC/XylS transcription factors might involve twoalternative modes of binding to DNA in different promoter contexts.

Escherichia coli positive regulator OmpR has a large loop structure at theputative RNA polymerase interaction site.

Kondo H, Nakagawa A, Nishihira J, Nishimura Y, Mizuno T, Tanaka I
Nat Struct Biol. 1997; 4: 28-31

The C-terminal DNA-binding domain of OmpR, a positive regulator involvedin osmoregulation expression of the ompF and ompC genes in Escherichiacoli, has a helix-turn-helix variant motif. The 'turn' region, consistingof 11 residues, forms an RNA polymerase contact site.

Membrane protein folding on the example of outer membrane protein A ofEscherichia coli.

Kleinschmidt JH
Cell Mol Life Sci. 2003; 60: 1547-58

The biophysical principles and mechanisms by which membrane proteinsinsert and fold into a biomembrane have mostly been studied withbacteriorhodopsin and outer membrane protein A (OmpA). This reviewde-scribes the assembly process of the monomeric outer membrane proteinsof Gram-negative bacteria, for which OmpA has served as an example. OmpAis a two-domain outer membrane protein composed of a 171-residueeight-stranded beta-barrel transmembrane domain and a 154-residueperiplasmic domain. OmpA is translocated in an unstructured form acrossthe cytoplasmic membrane into the periplasm. In the periplasm, unfoldedOmpA is kept in solution in complex with the molecular chaperone Skp.After binding of periplasmic lipopolysaccharide, OmpA insertion andfolding occur spontaneously upon interaction of the complex with thephospholipid bilayer. Insertion and folding of the beta-barreltransmembrane domain into the lipid bilayer are highly synchronized, i.e.the formation of large amounts of beta-sheet secondary structure andbeta-barrel tertiary structure take place in parallel with the same rateconstants, while OmpA inserts into the hydrophobic core of the membrane.In vitro, OmpA can successfully fold into a range of model membranes ofvery different phospholipid compositions, i. e. into bilayers of lipids ofdifferent headgroup structures and hydrophobic chain lengths. Threemembrane-bound folding intermediates of OmpA were discovered in foldingstudies with dioleoylphosphatidylcholine bilayers. Their formation wasmonitored by time-resolved distance determinations by fluorescencequenching, and they were structurally distinguished by the relativepositions of the five tryptophan residues of OmpA in projection to themembrane normal. Recent studies indicate a chaperone-assisted, highlysynchronized mechanism of secondary and tertiary structure formation uponmembrane insertion of beta-barrel membrane proteins such as OmpA thatinvolves at least three structurally distinct folding intermediates.

Relative binding affinities of OmpR and OmpR-phosphate at the ompF andompC regulatory sites.

Head CG, Tardy A, Kenney LJ
J Mol Biol. 1998; 281: 857-70

In Escherichia coli, porin gene expression is regulated, in part, by thetwo-component regulatory system consisting of the two proteins EnvZ andOmpR. EnvZ is an integral inner membrane protein that is phosphorylated bycytoplasmic ATP on a histidine residue. EnvZ modulates the activity ofOmpR by phosphorylation and dephosphorylation. Phospho-OmpR (OmpR-P) bindsto the porin genes ompF and ompC to regulate their expression. The simpleaffinity model predicts that as the concentration of OmpR-P increases,initially high-affinity binding sites on ompF are filled. Then bindingsites of lower affinity on ompF and ompC are occupied and this orderedbinding accounts for the differential expression of the porin genes. Wedemonstrate that acetyl phosphate phosphorylates OmpR at aspartate 55, thesame residue phosphorylated by the kinase EnvZ. Quantification of thelevel of OmpR-P by HPLC and direct measurement of the binding affinitiesenabled us to test the affinity model. Our results indicate thatphosphorylation dramatically increases the affinity of OmpR for itsbinding sites (greater than tenfold). We also show that the affinities ofOmpR-P for F1 and C1 binding sites are not sufficiently different toprovide a strong basis for discrimination. The consequences of theseobservations for the simple affinity model are considered.

Structural characterization of GntR/HutC family signaling domain.

Gorelik M, Lunin VV, Skarina T, Savchenko A
Protein Sci. 2006; 15: 1506-11

The crystal structure of Escherichia coli PhnF C-terminal domain (C-PhnF) was solved at 1.7 A resolution by the single wavelength anomalous dispersion (SAD) method. The PhnF protein belongs to the HutC subfamily of the large GntR transcriptional regulator family. Members of this family share similar N-terminal DNA-binding domains, but are divided into four subfamilies according to their heterogenic C-terminal domains, which are involved in effector binding and oligomerization. The C-PhnF structure provides for the first time the scaffold of this domain for the HutC subfamily, which covers about 31% of GntR-like regulators. The structure represents a mixture of alpha-helices and beta-strands, with a six-stranded antiparallel beta-sheet at the core. C-PhnF monomers form a dimer by establishing interdomain eight-strand beta-sheets that include core antiparallel and N-terminal two-strand parallel beta-sheets from each monomer. C-PhnF shares strong structural similarity with the chorismate lyase fold, which features a buried active site locked behind two helix-turn-helix loops. The structural comparison of the C-PhnF and UbiC proteins allows us to propose that a similar site in the PhnF structure is adapted for effector binding.

Solution structure of the IRF-2 DNA-binding domain: a novel subgroup ofthe winged helix-turn-helix family.

Furui J, Uegaki K, Yamazaki T, Shirakawa M, Swindells MB, Harada H, Taniguchi T, Kyogoku Y
Structure. 1998; 6: 491-500

BACKGROUND: The transcription of interferon (IFN) and IFN-inducible genesis mainly regulated by the interferon regulatory factor (IRF) family ofproteins, which recognize a unique AAGTGA hexamer repeat motif in theregulatory region of IFN genes. A DNA-binding domain of approximately 100amino acids has been commonly found in the IRF family of proteins, but ithas no sequence homology to known DNA-binding motifs. Elucidation of thestructures of members of the IRF family is therefore useful to theunderstanding of the regulation and evolution of the immune system at thestructural level. RESULTS: The solution structure of the DNA-bindingdomain of interferon regulatory factor-2 (IRF-2) has been determined byNMR spectroscopy. It is composed of a four-stranded antiparallel betasheet and three alpha helices, and its global fold is similar to those ofthe winged helix-turn-helix (wHTH) family of proteins. A long loop(Pro37-Asp51) is found immediately before the HTH motif, which is notfound in other wHTH proteins. The NMR signals of residues in this longloop, as well as the second helix of the HTH motif, are strongly affectedupon the addition of the hexamer repeat DNA, suggesting that thesestructural elements participate in DNA recognition and binding.CONCLUSIONS: The structural similarity of the DNA-binding domain of IRF-2with those of proteins in the wHTH family shows that the IRF proteinsbelong to the wHTH family, even though there is no apparent sequencehomology among proteins of the two families. The sequential structurealignment program (SSAP) shows that IRF-2 has a slightly differentstructure from typical wHTH proteins, mainly in the orientation of helix2. The IRF family of proteins should therefore be categorized into asubfamily of the wHTH family. The evidence here implies that theevolutional pathway of the IRF family is distinct from that of the otherwHTH proteins, in other words, the immune system diverged from anevolutional stem at an early stage.

Signal transduction by the EnvZ-OmpR phosphotransfer system in bacteria.

Forst SA, Roberts DL
Res Microbiol. 1994; 145: 363-73

Disruption of a gene encoding a putative gamma-butyrolactone-bindingprotein in Streptomyces tendae affects nikkomycin production.

Engel P, Scharfenstein LL, Dyer JM, Cary JW
Appl Microbiol Biotechnol. 2001; 56: 414-9

A 2.6-kb BamHI fragment from the genome of the wild-type,nikkomycin-producing strain of Streptomyces tendae ATCC 31160 was clonedand sequenced. This 2.6-kb BamHI fragment corresponds to the DNA sitewhere transposon Tn4560 had inserted to create a nikkomycin-nonproducingmutant. A possible ORF of 660 nucleotides was found in this 2.6-kb BamHIfragment, in which the third base of each codon was either G or C in 92%of the codons. The deduced amino acid sequence coded by this ORF (TarA,tendae autoregulator receptor) shows strong homology with severalGamma-butyrolactone-binding proteins that negatively regulate antibioticproduction in other streptomycetes and have a helix-turn-helix DNA-bindingmotif. A portion (179 nucleotides) of tarA that encodes thehelix-turn-helix motif was replaced with ermE, and wild-type S. tendae wastransformed with this construct borne in pDH5, a gene-disruption vector.Southern hybridization indicated that ermE had inserted in the 2.6-kbBamHI region in one isolate that is erythromycin resistant. Northernhybridization indicated that tarA disruption significantly increased theamount of disrupted-tarA mRNA. This suggests that TarA negativelyregulates its own synthesis. Nikkomycin production by the tarA disruptantwas delayed but reached the wild-type level after longer incubation inproduction medium.

C-terminal DNA binding stimulates N-terminal phosphorylation of the outermembrane protein regulator OmpR from Escherichia coli.

Ames SK, Frankema N, Kenney LJ
Proc Natl Acad Sci U S A. 1999; 96: 11792-7

Expression of the porin genes of Escherichia coli is regulated in part bythe osmolarity of the growth medium. The process is controlled by thehistidine kinase EnvZ and the response regulator OmpR. We have previouslyshown that phosphorylation of OmpR increases its affinity for the upstreamregulatory regions of ompF and ompC. We now report that, in the presenceof DNA, there is a dramatic stimulation in the level of phospho-OmpR. Thiseffect is independent of the source of phosphorylation, i.e., stimulationof phosphorylation is observed with a small phosphorylating agent such asacetyl phosphate or with protein-catalyzed phosphorylation by the kinaseEnvZ. The dephosphorylation rate of phospho-OmpR is affected only slightlyby the presence of DNA; thus, the increased level is largely caused by anincreased rate of phosphorylation. Stimulation of phosphorylation requiresspecific binding of DNA by OmpR. Occupancy of the DNA binding domainexposes a trypsin cleavage site in the linker, which connects thephosphorylation domain with the DNA binding domain. Our results indicatethat when DNA binds in the C terminus, it enhances phosphorylation in theN terminus, and the linker undergoes a conformational change. Ageneralized mechanism involving a four-state model for response regulatorsis proposed.