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

Autor(en): Klare, Johann P.
Lucana, Dario Ortiz de Orue
Stichwörter: Biochemistry & Molecular Biology; BIOLOGY; DISTANCES; DNA-BINDING CHARACTERISTICS; Endocrinology & Metabolism; ISONIAZID RESISTANCE; MYCOBACTERIUM-TUBERCULOSIS; REGULATOR FURS; RESIDUES; RESONANCE; RHODOPSIN; STREPTOMYCES-RETICULI
Erscheinungsdatum: 2012
Herausgeber: MARY ANN LIEBERT INC
Journal: ANTIOXIDANTS & REDOX SIGNALING
Volumen: 16
Ausgabe: 7
Startseite: 639
Seitenende: 648
Zusammenfassung: 
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 (H2O2)-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 a-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. Antioxid. Redox Signal. 16, 639-648.
ISSN: 15230864
DOI: 10.1089/ars.2011.4080

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