Quantitative assessment of the determinant structural differences between redox-active and inactive glutaredoxins

Linda Liedgens, Jannik Zimmermann, Lucas Wäschenbach, Fabian Geissel, Hugo Laporte, Holger Gohlke (), Bruce Morgan () and Marcel Deponte ()
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Linda Liedgens: Fachbereich Chemie, Abteilung Biochemie, Technische Universität Kaiserslautern
Jannik Zimmermann: Institut für Biochemie, Zentrum für Human- und Molekularbiologie (ZHMB), Universität des Saarlandes
Lucas Wäschenbach: Mathematisch-Naturwissenschaftliche Fakultät, Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf
Fabian Geissel: Fachbereich Chemie, Abteilung Biochemie, Technische Universität Kaiserslautern
Hugo Laporte: Institut für Biochemie, Zentrum für Human- und Molekularbiologie (ZHMB), Universität des Saarlandes
Holger Gohlke: Mathematisch-Naturwissenschaftliche Fakultät, Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf
Bruce Morgan: Institut für Biochemie, Zentrum für Human- und Molekularbiologie (ZHMB), Universität des Saarlandes
Marcel Deponte: Fachbereich Chemie, Abteilung Biochemie, Technische Universität Kaiserslautern

Nature Communications, 2020, vol. 11, issue 1, 1-18

Abstract: Abstract Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.

Date: 2020
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DOI: 10.1038/s41467-020-15441-3

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