Characterization of the DNA‐binding site in the ferric uptake regulator protein from <i>Escherichia coli</i> by UV crosslinking and mass spectrometry

Tiss, Ali; Barré, Olivier; Michaud‐Soret, Isabelle; Forest, Éric

doi:10.1016/j.febslet.2005.08.067

Cited by 34 publications

(41 citation statements)

References 28 publications

(36 reference statements)

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“…4D). The residues that showed larger decreases in LonA binding on both the alanine and aspartate substitution blots were all located on helix 4 of PerR, a region highly conserved among PerR homologs (see Fur, a Paralog of PerR in B. subtilis, Is Not a LonA Substrate) and known to be involved in DNA binding (11,12).…”

Section: Resultsmentioning

confidence: 99%

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

Ahn

Baker

2015

Proc. Natl. Acad. Sci. U.S.A.

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Peroxide operon regulator (PerR) is a broadly conserved hydrogen peroxide sensor in bacteria, and oxidation of PerR at its regulatory metal-binding site is considered irreversible. Here, we tested whether this oxidation specifically targets PerR for proteolysis. We find that oxidizing conditions stimulate PerR degradation in vivo, and LonA is the principal AAA+ (ATPases associated with diverse cellular activities) protease that degrades PerR. Degradation of PerR by LonA is recapitulated in vitro, and biochemical dissection of this degradation reveals that the presence of regulatory metal and PerR-binding DNA dramatically extends the half-life of the protein. We identified a LonA-recognition site critical for oxidation-controlled PerR turnover. Key residues for LonA-interaction are exposed to solvent in PerR lacking metal, but are buried in the metal-bound form. Furthermore, one residue critical for Lon recognition is also essential for specific DNAbinding by PerR, thus explaining how both the metal and DNA ligands prevent PerR degradation. This ligand-controlled allosteric mechanism for protease recognition provides a compelling explanation for how the oxidation-induced conformational change in PerR triggers degradation. Interestingly, the critical residues recognized by LonA and exposed by oxidation do not function as a degron, because they are not sufficient to convert a nonsubstrate protein into a LonA substrate. Rather, these residues are a conformation-discriminator sequence, which must work together with other residues in PerR to evoke efficient degradation. This mechanism provides a useful example of how other proteins with only mild or localized oxidative damage can be targeted for degradation without the need for extensive oxidation-dependent protein denaturation.oxidative damage | proteolysis | degradation signals | Lon

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Section: Resultsmentioning

confidence: 99%

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

Ahn

Baker

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…VOL. 192, 2010 GONOCOCCAL Fur REGULON 81 (50) attribute the binding to the putative Fur DNA helix recognition region. The intragenic FB sequence logo, in contrast to the promoter-proximal FB sequence logo, showed a reduced frequency of thymidine residues able to bind Fur (Fig.…”

Section: Resultsmentioning

confidence: 99%

Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon

et al. 2010

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To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies-temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)-to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.The acquisition of iron is essential for bacterial pathogenesis. Because iron is insoluble in aqueous environments at neutral pH and has the potential to produce damaging free radicals, the mammalian host tightly sequesters iron (1, 51), lowering the level of free iron available to invading microorganisms to well below what is needed to survive (34). Since survival in the host and hence virulence is dependent on the acquisition of iron, pathogenic bacteria must be able to sense iron availability and globally regulate the transcription of iron acquisition genes. For many gram-positive and gram-negative organisms, including the sexually transmitted disease pathogen Neisseria gonorrhoeae, the crucial balance between acquiring enough iron to grow and avoiding the toxic effects of excess iron is controlled by the ferric uptake regulator (Fur) protein (12, 27, 49). Typically, Fur acts as a transcriptional repressor by binding as a dimer, along with ferrous iron as a corepressor, to regulatory Fur box (FB) sequences in the promoters of iron-regulated genes under iron-rich growth conditions. As intracellular iron stores are depleted, the Fur-Fe 2ϩ dimer dissociates from the promoter, allowing the entry of RNA polymerase and subsequent transcription (22). Fur acts as a global regulator controlling not only the expression of iron acquisition...

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“…The optional C-terminal tail has been associated with metal binding, folding (Pohl et al, 2003) are shown. BS, potential binding site residues for iron; Y56, tyrosine 56 which is in direct contact with DNA target (Tiss et al, 2005); C92-C95 and C132-C135, cysteine residues involved in disulfide bridge formation in E. coli (D'Autréaux et al, 2007). At each position an asterisk indicates identical residues, a colon denotes conserved substitutions, and a dot represents semi-conserved substitutions.…”

Section: Basic Properties Of Fur From Members Of the Vibrionaceae Familymentioning

confidence: 99%

“…It is unclear to us if the strandspecific contribution of T13 is due to real differences in interactions between the DNA and the Fur homodimer, or if it is due to an artifact in the MD simulations and binding free energy calculations. Using a mass spectrometry-based method, EC-Fur and the E. coli "classical" Fur box, Tiss and coworkers (Tiss et al, 2005) showed that T12 and T13 (correspond to T15 and T16 in E. coli) directly interact with the protein. In summary, A14 and C16, which are conserved in B. subtilis and Y. pestis, represent new potential interaction sites between Fur and DNA.…”

Section: Vibrio-specific Fur Box Residues Involved In Interaction Witmentioning

confidence: 99%

“…In a PA-Fur-DNA structure model helix 4 was implicated in DNA binding, and in EC-Fur a tyrosine residue (Tyr 56) in helix 4 is in direct contact with the DNA target, and is in close proximity with two thymines (pos. 15 and 16) in the Fur binding site (Tiss et al, 2005). By using computerbased methods we recently showed that several other Fur residues (e.g., K13, R18, R56, R69, Q60, and H76) might also significantly contribute to Fur-DNA interactions (Ahmad et al, 2009a).…”

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confidence: 98%

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Experimental and computational characterization of the ferric uptake regulator from Aliivibrio salmonicida (Vibrio salmonicida)

et al. 2010

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The Ferric uptake regulator (Fur) is a global transcription factor that affects expression of bacterial genes in an iron-dependent fashion. Although the Fur protein and its iron-responsive regulon are well studied, there are still important questions that remain to be answered. For example, the consensus Fur binding site also known as the "Fur box" is under debate, and it is still unclear which Fur residues directly interact with the DNA. Our long-term goal is to dissect the biological roles of Fur in the development of the disease cold-water vibriosis, which is caused by the psychrophilic bacteria Aliivibrio salmonicida (also known as Vibrio salmonicida). Here, we have used experimental and computational methods to characterise the Fur protein from A. salmonicida (AS-Fur). Electrophoretic mobility shift assays show that AS-Fur binds to the recently proposed vibrio Fur box consensus in addition to nine promoter regions that contain Fur boxes. Binding appears to be dependent on the number of Fur boxes, and the predicted "strength" of Fur boxes. Finally, structure modeling and molecular dynamics simulations provide new insights into potential AS-Fur-DNA interactions.

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Characterization of the DNA‐binding site in the ferric uptake regulator protein from Escherichia coli by UV crosslinking and mass spectrometry

Cited by 34 publications

References 28 publications

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon

Experimental and computational characterization of the ferric uptake regulator from Aliivibrio salmonicida (Vibrio salmonicida)

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