Quantitative assessment of the determinant structural differences between redox-active and inactive glutaredoxins
| Autor: | Jannik Zimmermann, Linda Liedgens, Hugo Laporte, Marcel Deponte, Bruce A. Morgan, Lucas Wäschenbach, Holger Gohlke, Fabian Geissel |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Models
Molecular Protein Conformation alpha-Helical 0301 basic medicine Saccharomyces cerevisiae Proteins Science Mutant General Physics and Astronomy Saccharomyces cerevisiae Molecular Dynamics Simulation medicine.disease_cause 01 natural sciences Article Catalysis General Biochemistry Genetics and Molecular Biology 03 medical and health sciences chemistry.chemical_compound Enzyme activator Thioredoxins Protein structure Catalytic Domain Glutaredoxin 0103 physical sciences medicine Moiety Amino Acid Sequence Disulfides lcsh:Science Glutaredoxins Enzyme Assays Mutation Multidisciplinary 010304 chemical physics General Chemistry Glutathione Yeast Enzymes Enzyme Activation Kinetics 030104 developmental biology chemistry Biochemistry Biocatalysis lcsh:Q ddc:500 Oxidation-Reduction |
| Zdroj: | Nature Communications 11(1), 1725 (2020). doi:10.1038/s41467-020-15441-3 Nature Communications Nature Communications, Vol 11, Iss 1, Pp 1-18 (2020) |
| Popis: | 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. Glutaredoxins play a central role in numerous biological processes including cellular redox homeostasis and Fe-S cluster biogenesis. Here the authors establish the molecular basis for glutaredoxin redox catalysis through comprehensive biochemical and structural analyses. |
| Databáze: | OpenAIRE |
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