Crystal structure of the apo‐PerR‐Zn protein from <i>Bacillus subtilis</i>

Traore, Daouda A K; Ghazouani, Abdelnasser El; Ilango, Sougandi; Dupuy, Jérôme; Jacquamet, Lilian; Ferrer, Jean‐Luc; Caux‐Thang, Christelle; Duarte, Victor; Latour, Jean‐Marc

doi:10.1111/j.1365-2958.2006.05313.x

Cited by 99 publications

(158 citation statements)

References 44 publications

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“…3A). Metal binding to the sensor site promotes a change in PerR conformation from an extended to a more compact form that binds promoter DNA (5,6). The metal-binding-promoted change of PerR to the compact conformation is explained by the fact that H37 and H91, both of which participate directly as metal binding, are located in the two different domains of PerR; thus, these residues can help bring the two PerR domains in each monomer in close proximity and stabilize the compact form.…”

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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“…The other resides in the Ddomain, constructed by either four conserved cysteines near the C terminus (C-site) or residues at the surface of the D-domain pointing toward the hinge region (D-site). Structural comparison between inactive apo-and metallated active forms of BsPerR suggested that regulatory metal binding causes DB-domains to swing around the hinge region with respect to the D-domain, from an open (swung-out) to closed (converged) conformations (22)(23)(24). This process leads to a model that the rearrangement of DBdomains by binding a regulatory metal in the hinge region is responsible for the allosteric transition of all Fur family regulators, from the inactive open conformation to the active closed one (23).…”

mentioning

confidence: 99%

Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur

Shin

Jung

et al. 2011

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

110

189

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Zinc is one of the essential transition metals in cells. Excess or lack of zinc is detrimental, and cells exploit highly sensitive zinc-binding regulators to achieve homeostasis. In this article, we present a crystal structure of active Zur from Streptomyces coelicolor with three zinc-binding sites (C-, M-, and D-sites). Mutations of the three sites differentially affected sporulation and transcription of target genes, such that C- and M-site mutations inhibited sporulation and derepressed all target genes examined, whereas D-site mutations did not affect sporulation and derepressed only a sensitive gene. Biochemical and spectroscopic analyses of representative metal site mutants revealed that the C-site serves a structural role, whereas the M- and D-sites regulate DNA-binding activity as an on-off switch and a fine-tuner, respectively. Consistent with differential effect of mutations on target genes, zinc chelation by TPEN derepressed some genes ( znuA, rpmF2 ) more sensitively than others ( rpmG2 , SCO7682) in vivo. Similar pattern of TPEN-sensitivity was observed for Zur-DNA complexes formed on different promoters in vitro. The sensitive promoters bound Zur with lower affinity than the less sensitive ones. EDTA-treated apo-Zur gained its DNA binding activity at different concentrations of added zinc for the two promoter groups, corresponding to free zinc concentrations of 4.5 × 10 −16 M and 7.9 × 10 −16 M for the less sensitive and sensitive promoters, respectively. The graded expression of target genes is a clever outcome of subtly modulating Zur-DNA binding affinities in response to zinc availability. It enables bacteria to detect metal depletion with improved sensitivity and optimize gene-expression pattern.

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“…1). 10,[12][13]15 To investigate the role of structural Zn 2+ site in peroxide sensing by His oxidation, mutant proteins altered in any of the four cysteine residues were analyzed by MALDI-TOF MS as described above. All the cysteine to serine substitution mutant proteins (C96S, C99S, C136S, C139S) exhibited no detectable oxidation of either T5 containing H37 or T11 containing H91 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1A). 15 In the absence of regulatory metal ion, the apo-PerR (PerR:Zn) adopts a rather flat conformation, where the two DNA-binding domains project opposite directions from the dimerization domain (Fig. 1B).…”

Section: Introductionmentioning

confidence: 99%

Mutational Analysis of the Metal-binding Sites of Peroxide Sensor PerR

Won¹,

Ji²,

Cho³

et al. 2010

Bulletin of the Korean Chemical Society

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Bacillus subtilis PerR is a metal-dependent peroxide-sensing transcription factor which uses metal-catalyzed histidine oxidation for peroxide-sensing. PerR contains two metal binding sites, one for structural Zn 2+ and the other for the regulatory/peroxide-sensing metal. Here we investigated the effect of mutations at both the structural and regulatory metal binding sites on the oxidation of either H37 or H91, two of the peroxide-sensing ligands. All four serine substitution mutants at the structural Zn 2+ site (C96S, C99S, C136S and C139S) exhibited no detectable oxidation at histidine residues. Two of the alanine substitution mutants at regulatory metal site (H37A and D85A) exhibited selective oxidation preferentially at the H91-containing tryptic peptide, whereas no oxidation was detected in the other mutants (H91A, H93A and D104A). Our results suggest that the cysteine residues coordinating structural Zn 2+ are essential for peroxide sensing by PerR, and that the C-terminal regulatory metal binding site composed of H91, H93 and D104 can bind Fe 2+ , providing a possible explanation for the peroxide sensing mechanisms by PerR.

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Crystal structure of the apo‐PerR‐Zn protein from Bacillus subtilis

Cited by 99 publications

References 44 publications

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

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

Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur

Mutational Analysis of the Metal-binding Sites of Peroxide Sensor PerR

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