The antioxidant function of Sco proteins depends on a critical surface-exposed residue
Graphical abstract
Introduction
The evolutionary conserved Sco (synthesis of cytochrome c oxidase) proteins exist in all kingdoms of life. The number of SCO genes and their function may vary across organisms [1], but most eukaryotic Sco proteins share the following features: an N-terminal mitochondrial targeting sequence, a single transmembrane domain, a highly conserved CxxxC motif and histidine residue – crucial for copper binding – and a thioredoxin fold [2].
The first Sco protein (Sco1) was identified in the yeast Saccharomyces (S.) cerevisiae as an essential component for the assembly of the cytochrome c oxidase (COX) [3]. Subsequent studies in different organisms have shown divergent roles of Sco proteins including copper homeostasis [[4], [5], [6], [7]] and redox signaling [[8], [9], [10]]. Most recently, a role of Sco proteins in the defense against oxidative stress was reported [[11], [12], [13], [14]]. The presence of the thioredoxin fold in Sco proteins has long been pointed out as a structural homology to antioxidant enzymes [10,[15], [16], [17]]. However, the investigation of the antioxidant activity of Sco proteins is hampered by their concomitant role in the assembly of the respiratory chain. This difficulty can be overcome by using the facultative aerobic organism S. cerevisiae that can gain energy exclusively by glycolysis. Complementation studies in this model organism nicely figured out the conservation of the antioxidant role of eukaryotic Sco homologs among distant species independent of the extent of their sequence homology [14].
Structural characterization of human Sco proteins and the analysis of pathogenic mutations of human Sco2 led to the identification of several amino acid(s) (aa) with special importance for conformational stability as well as for physical and binding properties [10,18]. Subsequent analyses revealed the negative impact of the respective mutations on copper homeostasis [6], oxidoreductase activity [19] and antioxidant function [14].
In addition, several studies have emphasized the role of charged residues on both structural and functional features of Sco proteins and pointed out the contribution of salt bridges to the structure of human Sco proteins [15,16]. For instance, S. cerevisiae Sco1 interacts with the copper chaperone Cox17 and the Cox2 subunit of mitochondrial cytochrome c oxidase through complementary electrostatic surfaces [20].
In the present work, we aimed at investigating functionally important residues for the antioxidant role of eukaryotic Sco proteins. In the light of previous findings [14], we included the Sco homologs from Arabidopsis (A.) thaliana, human and Kluyveromyces (K.) lactis and analyzed the conserved site(s) by putting special emphasis on charged aa. Our results point out a novel functional site – 15 aa downstream of the CxxxC domain – which is crucial for oxidative stress defense. Mutagenesis data demonstrate charge-driven changes in the antioxidant function and propose a role to this conserved site in mediating electrostatic interactions.
Section snippets
Bioinformatic analysis
The protein sequences were retrieved from the UniProt database [21] and alignments were done by Clustal Omega [22]. The MitoFates tool [23] was used to predict mitochondrial targeting sequences. The transmembrane domain was predicted with TMpred [24] for SpSco and K07152; for the other Sco homologs this information was retrieved from the UniProt database.
The protein structures were retrieved from Protein Data Bank (PDB) and model structures were built by SWISS-MODEL [25] using the top-ranked
Identification of a critical aa site for antioxidant function by multiple sequence alignment of Sco proteins
In our previous study, we described the oxidative stress sensitivity of a double mutant yeast strain lacking SCO2 and SOD1 – encoding superoxide dismutase 1. We used this strain, ∆sco2∆sod1, to investigate the ability of different eukaryotic Sco proteins to rescue the oxidative stress sensitivity [14]. Based on these results, the analyzed Sco homologs were grouped into functional (ySco2, SpSco, hSco1, hSco2, Scox, HCC2) and non-functional (HCC1, K07152) homologs. The finding that the K. lactis
Discussion
The defensive role of eukaryotic Sco proteins against oxidative stress has been shown previously in a model system with a yeast strain harboring concomitantly a deletion of SCO2 and SOD1, Δsco2Δsod1 [14]. The similar phenotype of this mutant and of the Δtrx3Δsod1 mutant strain under oxidative stress has supported the idea of a possible thioredoxin-like activity of Sco proteins as predicted by structural analyses [49] and shown before for both prokaryotic Sco proteins [[50], [51], [52]] and
Author contributions
AEK and UG conceptualized the study; AEK designed and performed experiments; AEK, GR and UG validated and analyzed data; GR and UG supervised the project; AEK wrote the manuscript; GR and UG made manuscript revisions; GR acquired the funding.
Funding
This work was supported by the Dresden International Graduate School for Biomedicine and Bioengineering (DIGS-BB) funded by the German Research Foundation (DFG grant GSC 97) to AEK.
Declaration of Competing Interest
The authors state no conflict of interest.
Acknowledgements
We are grateful to Prof. Michael Schroeder and Dr. Sebastian Salentin for their assistance in bioinformatic analysis. We are thankful to MS facility of the Max Planck Institute of Molecular Biology and Genetics (MPI-CBG) for mass spectrometry and statistical analyses.
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Current address: Department of Biology, University of Fribourg, Chemin du Musée 10, 1700 Fribourg, Switzerland.