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The target specificity of thioredoxin family proteins is determined by electrostatic compatibility
(2021)
The thioredoxin (Trx) family of proteins comprises many key enzymes in redox signaling, that catalyzes specific reversible redox reactions, e.g. dithiol-disulfide exchange reactions, (de-)glutathionylation, trans-nitrosylation, or peroxide reduction. With the analysis of a large number of proteins, as well as a certain redox couple in [article 1] and [article 4], we demonstrated that electrostatic complementarity is the major distinguishing feature that controls the specific interactions of Trxs with their target proteins. The primary aim of this work was to determine the importance of this specific interaction and the prediction, modulation, and engineering of functional redox interactions of Trx family proteins. To understand the role of electrostatic complementarity for the mammalian Trx1-TrxR complex, we generated more than 20 hTrx1 mutants and systematically engineered the electrostatic potential within and outside the contact area with TrxR [article 1]. The effects of these specific alterations distributed all over the protein surface were analyzed by enzyme kinetics, differential scanning fluorimetry (DSF), circular dichroism (CD) spectroscopy, and MD simulations. Trx family proteins have a broad and very distinct substrate specificity, which is a prerequisite for redox switching. In [article 4], we comprehensively compared the classification of various redoxins from all kingdoms of life based on their similarity in amino acid sequence, tertiary structure, and electrostatic properties. These similarities were then correlated to the existence of common interaction partners. Our analyses confirmed that the primary and tertiary structure similarities do not correlate to the target specificity of the proteins as thiol-disulfide oxidoreductases. However, we demonstrated that the electrostatic properties of the protein from both Trx or Grx subfamilies is the major determinant for their target specificity.
Although structurally very similar, CxxC/S-type or class I Grxs act as oxidoreductases and CGFS-type or class II Grxs act as FeS cluster transferases. In [article 3], we re-investigated the structural differences between the two main classes of Grxs to solve the mystery of the missing FeS transferase activity of the CxxC/S-type and the lack of oxidoreductase activity of the CGFS-type Grxs. The presence of a distinct loop structure adjacent to the active site is the major determinant of the Grx function. We confirmed that the function of Grxs can be switched from oxidoreductase to FeS cluster transferase by construction of a CxxC/S-type Grx with a CGFS-type Grx loop and vice versa. Results of several in vitro and in vivo assays together with the detailed structural analyses indicate that not a radically different substrate specificity accounts for the lack of activity, but rather slightly different modes of GSH binding, which is an essential nucleophile required in redox and iron homeostasis.
Various processes within the cell depend on GSH, including redox reactions, reversible posttranslational modifications, and iron metabolim. GSH is not only important in the export of FeS precursors from mitochondria, but it is also an essential cofactor for cluster binding in iron sulfur Grxs. In [article 2], we discussed the role of GSH and iron sulfur Grxs in iron metabolism, the physiological role of CGFS-type Grx interactions with BolA- like proteins, and the cluster transfer between Grxs and recipient proteins. The first well characterized physiological function of a Grx-BolA hetero complex is presented with the Grx3/4-Fra2-mediated regulation of iron homeostasis in yeast.
In synopsis, the results presented and discussed in these articles and the manuscript support the concept of electrostatic properties as the main determinant in substrate specificity towards functional predictions in Trx family proteins. The mathematical model presented here showed significantly accuracy and precision in function prediction. We are aware that our findings are focused on Trx family proteins as a particular family of proteins, but by using a machine learning strategy this mathematical model is being trained with numerous different protein models for better efficacy and accuracy, that may lead to new insights also in the specific interactions of other protein families. The new concept for the substrate specificity determinant doesn’t eliminate previously described aspects for molecular recognition, instead it reveals a deeper understanding of the protein-protein interaction. The 3D structural elements of a protein play a significant role in the specificity and function. We have been able to activate an inactive protein by replacing defined structural elements. Elimination of the loop structure from CGFS-type Grx5 transformed it from an FeS transferase into an oxidoreductase and the activity was further increased by modification of the active site. We believe that the present findings may be useful to investigate proteins in great detail regarding their function based on structure and electrostatic properties. Understanding the nature of the specific interactions may enable us to specifically modify the signal transduction pathways.
Bacteria are exposed to oxidative stress as an unavoidable consequence of their aerobic lifestyle. Reactive oxygen species (ROS) are generated in the stepwise one-electron reduction of molecular oxygen during the respiration. Pathogens encounter ROS during the oxidative burst of macrophages as part of the host immune defense. Besides ROS, bacteria also have to cope with reactive chlorine, electrophilic and nitrogen species (RCS, RES, RNS). To cope with these reactive species, bacteria have evolved different defense and repair mechanisms. To maintain the reduced state of the cytoplasm, they utilize low molecular weight (LMW) thiols. LMW thiols are small thiol-containing compounds that can undergo post-translational thiolmodifications with protein thiols, termed as S-thiolations. S-thiolations function as major redox regulatory and thiol-protection mechanism under oxidative stress conditions. In eukaryotes and Gram-negative bacteria, the tripeptide glutathione (GSH) functions as major LMW thiol, which is present in millimolar concentrations. The Actinomycetes, such as Mycobacterium and Corynebacterium species do not produce GSH and utilize instead mycothiol (MSH) as their alternative LMW thiol. In Firmicutes, including Bacillus and Staphylococcus species, bacillithiol (BSH) functions as the major LMW thiol. LMW thiols protect protein thiols against the irreversible overoxidation of cystein residues to sulfinic and sulfonic acids. In addition, LMW thiols contribute to the virulence and survival of pathogens, function in metal homeostasis and serve as enzyme cofactors for detoxification of xenobiotics and antibiotics. In this doctoral thesis, we aimed to investigate the roles of MSH and BSH in redox regulation of main metabolic enzymes under oxidative stress in the pathogens Corynebacterium diphtheriae and Staphylococcus aureus. Previous redox proteomics studies identified the glyceraldehyde-3-phosphate dehydrogenase GapDH and the aldehyde dehydrogenase AldA as S-thiolated in S. aureus and C. diphtheriae. Thus, we aimed to study the redox regulation of the metabolic enzyme GapDH in C. diphtheriae in response to NaOCl and H2O2 stress by S-mycothiolation, which is described in chapter 1. Moreover, we studied the involvement of the mycoredoxin-1 (Mrx1) and thioredoxin (Trx) pathways in reactivation of S-mycothiolated GapDH in vitro. Using shotgun proteomics, 26 S-mycothiolated proteins were identified under NaOCl stress in C. diphtheriae. These are involved in energy metabolism (Ndh, GlpD) and in the biosynthesis of amino acids (ThrA, LeuB), purines (PurA) and cell wall metabolites (GlmS). The glycolytic GapDH was identified as conserved target for S-thiolation across Gram-positive bacteria. GapDH was the most abundant protein, contributing with 0.75 % to the total cystein proteome. Moreover, GapDH is a conserved target for redox regulation and S-glutathionylation in response to oxidative stress in several prokaryotic and eukaryotic organisms. Treatment of GapDH with NaOCl and H2O2 in the absence of MSH resulted in irreversible enzyme inactivation due to overoxidation. Pretreatment of GapDH with MSH prior to H2O2 or NaOCl exposure resulted in reversible inactivation due to S-mycothiolation of the active site Cys153. Since S-mycothiolation is faster compared to overoxidation, S-mycothiolation efficiently protects the GapDH active site against overoxidation. The activity of S-mycothiolated GapDH could be restored by both, the Mrx1 and Trx pathway in vitro. Interestingly, the recovery of Smycothiolated GapDH by Mrx1 was faster compared to its reduction by the Trx pathway. In previous studies, the reactivation of S-mycothiolated Mpx and MrsA by the mycoredoxin pathway occurred also faster compared to the Trx pathway, which is consistent with our results. We were further interested to analyze the redox regulation of the glyceraldehyde-3phosphate dehydrogenase Gap of S. aureus under NaOCl and H2O2 stress, which is described in chapter 2. Using the quantitative redox proteomic approach OxICAT, 58 NaOCl-sensitive cystein residues with >10% thiol oxidation under NaOCl stress were identified. Gap and AldA showed the highest oxidation increase of 29% under NaOCl stress at their active site cystein residues. Using shotgun proteomics, five S-bacillithiolated proteins were identified, including Gap, AldA, GuaB, RpmJ and PpaC. Gap contributed with 4 % as most abundant cystein protein to the total cystein proteome. Our activity assays demonstrated that Gap of S. aureus is highly sensitive to overoxidation by H2O2 and NaOCl in vitro in the absence of BSH. The active site Cys151 of Gap was oxidized to the BSH mixed disulfide under H2O2 and NaOCl stress in the presence of BSH in vitro, which resulted in the reversible Gap inactivation. Moreover, inactivation of Gap by NaOCl and H2O2 due to S-bacillithiolation was faster compared to overoxidation, indicating that S-bacillithiolation protects the Gap active site against overoxidation in vitro. We further showed that the bacilliredoxin Brx catalyzes the reduction of S-bacillithiolated Gap in vitro. Molecular docking of BSH into the Gap active site revealed that S-bacillithiolation does not require major structural changes. Apart from Gap, the aldehyde dehydrogenase AldA was identified as S-bacillithiolated at its active site Cys279 under NaOCl stress in S. aureus previously. Thus, the expression, function, redox regulation and structural changes of AldA were analysed under NaOCl and aldehyde stress in S. aureus as summarized in chapter 3. AldA was S-bacillithiolated in the presence of H2O2 and BSH as demonstrated in BSH-specific Western blots in vitro. The expression of aldA was previously shown to be regulated by the alternative sigma factor SigmaB in S. aureus. Transcription of aldA was strongly increased in a SigmaB-independent manner under formaldehyde, NaOCl and diamide stress in S. aureus. Using an aldA deletion mutant, we demonstrated that aldA is required for growth and survival under NaOCl stress in S. aureus. The purified AldA enzyme was shown to catalyze the oxidation of various aldehyde substrates, including formaldehyde, methylglyoxal, glycolaldehyde and acetaldehyde in vitro. In addition, the function of the conserved Cys279 for AldA activity was investigated in vivo and in vitro. The purified AldAC279S mutant was shown to be inactive for aldehyde oxidation in vitro. Moreover, the aldAC279S mutant was very sensitive under NaOCl stress in vivo, and this phenotype could be reversed using the aldA complemented strain. These experiments demonstrate the function of Cys279 for AldA activity both in vitro and in vivo. AldA activity assays showed that AldA is sensitive to overoxidation and irreversible inactivation by H2O2 alone in vitro. In the presence of BSH, AldA is protected against overoxidation by reversible Sbacillithiolation in vitro. Molecular docking and molecular dynamics simulations revealed that BSH occupies two different positions in the Cys279 active site, which depend on the NAD+ cofactor. In the apoenzyme, BSH forms the disulfide with Cys279 in the “resting” state position, while Cys279 is S-bacillithiolated in the “attacking” state position in the holoenzyme in the presence of the NAD+ cofactor.