Role of the cysteinyl residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis-trans isomerase and redox-related functions
Résumé
Cyclophilins (Cyps) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved Cys-residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast located CYP20-3, site-directed mutagenised Cys->Ser-variants were generated and analyzed for enzymatic and conformational properties under reducing and oxidising conditions. Compared to the wild type form elimination of three out of the four cysteinyl residues decreased the catalytic efficiency of peptidyl-prolyl cis-trans isomerase (PPI) activity of the reduced CYP20-3 indicating a regulatory role of dithiol-disulfide transitions in protein function. Oxidation was accompanied by conformational changes with a predominant role in the structural rearrangement of the disulfide bridge formed between Cys 54} and Cys 171}. The rather negative midpoint redox potential of -319 mV places CYP20-3 into the redox hierarchy of the chloroplast suggesting the activation of CYP20-3 in the light under condition of limited acceptor availability for photosynthesis as realised under environmental stress. Chloroplast peroxiredoxins (Prx) were identified as interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in the reduction of 2-Cys PrxA and B was assigned to Cys 129} and Cys 171}. In addition it was shown that the isomerisation and disulfide reduction activities are two independent functions of CYP20-3 that both are regulated by the redox state of its active centre.
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