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Conformational changes in the novel redox sensor protein HbpS studied by site-directed spin labeling and its turnover in dependence on the catalase-peroxidase CpeB.

ABSTRACT: AIMS:To establish conditions to study the oligomeric assembly of heme-binding protein (HbpS) in solution by applying the tools of site-directed spin labeling combined with pulse electron paramagnetic resonance (SDSL EPR) spectroscopy, as well as to analyze redox stress-based conformational changes in HbpS subunits within the oligomer in solution. In vivo elucidation of molecular mechanisms that control the downregulation of the novel redox-system HbpS-SenS-SenR. RESULTS:Using a set of specifically generated HbpS mutants, and SDSL EPR spectroscopy, we show the octomeric assembly of HbpS in solution, and demonstrate that iron-mediated stress induces conformational changes in HbpS subunits within the octamer. We further demonstrate that the catalase-peroxidase CpeB protects HbpS from hydrogen peroxide (H(2)O(2))-mediated oxidative attack in vivo. Moreover, chromosomal inactivation of cpeB results in an enhanced sensitivity of the mutant to redox-cycling compounds. INNOVATION:SDSL EPR has been used in this work for the first time to monitor redox-mediated conformational changes in a redox-sensing protein in solution. This work substantially explains redox-dependent dynamics in HbpS at the atomic level, and presents novel molecular mechanisms supporting downregulation of a signaling cascade. CONCLUSION:Iron-mediated stress induces movements of subunits within the HbpS octomeric assembly. We suggest a motion of the C-terminal ?-helix toward the preceding helical segment. These events upregulate the activity of the HbpS-SenS-SenR system, in which HbpS acts as an accessory element. The mycelia-associated CpeB, under the control of HbpS-SenS-SenR, protects the extracellular HbpS from oxidation in vivo. Thus, de novo synthesized HbpS proteins downregulate the HbpS-SenS-SenR signaling cascade.

SUBMITTER: Klare JP

PROVIDER: S-EPMC3277929 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Conformational changes in the novel redox sensor protein HbpS studied by site-directed spin labeling and its turnover in dependence on the catalase-peroxidase CpeB.

Klare Johann P JP Ortiz de Orué Lucana Darío D

Antioxidants & redox signaling 20111019 7

<h4>Aims</h4>To establish conditions to study the oligomeric assembly of heme-binding protein (HbpS) in solution by applying the tools of site-directed spin labeling combined with pulse electron paramagnetic resonance (SDSL EPR) spectroscopy, as well as to analyze redox stress-based conformational changes in HbpS subunits within the oligomer in solution. In vivo elucidation of molecular mechanisms that control the downregulation of the novel redox-system HbpS-SenS-SenR.<h4>Results</h4>Using a se ...[more]

PMID: 21846213

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Project description:The secreted protein HbpS, the membrane-embedded sensor kinase SenS and the cytoplasmic response regulator SenR from streptomycetes have been shown to form a novel type of signaling pathway. Based on structural biology as well as different biochemical and biophysical approaches, redox stress-based post-translational modifications in the three proteins were shown to modulate the activity of this signaling pathway. In this study, we show that the homologous system, named here HbpSc-SenSc-SenRc, from the model species Streptomyces coelicolor A3(2) provides this bacterium with an efficient defense mechanism under conditions of oxidative stress. Comparative analyses of the transcriptomes of the Streptomyces coelicolor A3(2) wild-type and the generated hbpSc-senSc-senRc mutant under native and oxidative-stressing conditions allowed to identify differentially expressed genes, whose products may enhance the anti-oxidative defense of the bacterium. Amongst others, the results show an up-regulated transcription of genes for biosynthesis of cysteine and vitamin B12, transport of methionine and vitamin B12, and DNA synthesis and repair. Simultaneously, transcription of genes for degradation of an anti-oxidant compound is down-regulated in a HbpSc-SenSc-SenRc-dependent manner. It appears that HbpSc-SenSc-SenRc controls the non-enzymatic response of Streptomyces coelicolor A3(2) to counteract the hazardous effects of oxidative stress. Binding of the response regulator SenRc to regulatory regions of some of the studied genes indicates that the regulation is direct. The results additionally suggest that HbpSc-SenSc-SenRc may act in concert with other regulatory modules such as a transcriptional regulator, a two-component system and the Streptomyces B12 riboswitch. The transcriptomics data, together with our previous in vitro results, enable a profound characterization of the HbpS-SenS-SenR system from streptomycetes. Since homologues to HbpS-SenS-SenR are widespread in different actinobacteria with ecological and medical relevance, the data presented here will serve as a basis to elucidate the biological role of these homologues.

Dataset Information

Conformational changes in the novel redox sensor protein HbpS studied by site-directed spin labeling and its turnover in dependence on the catalase-peroxidase CpeB.

Publications

Conformational changes in the novel redox sensor protein HbpS studied by site-directed spin labeling and its turnover in dependence on the catalase-peroxidase CpeB.

Similar Datasets

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