Breakdown of Arabidopsis thaliana thioredoxins and glutaredoxins based on electrostatic similarity–Leads to common and unique interaction partners and functions

PLOS ONE RESEARCH ARTICLE Breakdown of Arabidopsis thaliana thioredoxins and glutaredoxins based on electrostatic similarity—Leads to common and unique interaction partners and functions Yana Bodnar ID1,2, Manuela Gellert1, Faruq Mohammed Hossain ID1¤, Christopher Horst Lillig ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Institute for Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald, Germany, 2 Institute for Physics, University of Greifswald, Greifswald, Germany ¤ Current address: Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany * Abstract OPEN ACCESS Citation: Bodnar Y, Gellert M, Hossain FM, Lillig CH (2023) Breakdown of Arabidopsis thaliana thioredoxins and glutaredoxins based on electrostatic similarity—Leads to common and unique interaction partners and functions. PLoS ONE 18(9): e0291272. https://doi.org/10.1371/ journal.pone.0291272 Editor: Timir Tripathi, North-Eastern Hill University, INDIA Received: July 3, 2023 Accepted: August 24, 2023 Published: September 11, 2023 Copyright: © 2023 Bodnar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Deutsche Forschungsgemeinschaft (DFG), grant numbers: Research Training Group 1947 project A1, Li 984/3-2, and Li 984/4-1 to Christopher Horst Lillig, PD Dr. Dr., and Universitätsmedizin Greifswald FOVB-2021-08 and FOVB 2022-16 to Manuel Gellert, Dr. The funders had no role in study design, data collection and The reversible reduction and oxidation of protein thiols was first described as mechanism to control light/dark-dependent metabolic regulation in photosynthetic organisms. Today, it is recognized as an essential mechanism of regulation and signal transduction in all kingdoms of life. Proteins of the thioredoxin (Trx) family, Trxs and glutaredoxins (Grxs) in particular, catalyze thiol-disulfide exchange reactions and are vital players in the operation of thiol switches. Various Trx and Grx isoforms are present in all compartments of the cell. These proteins have a rather broad but at the same time distinct substrate specificity. Understanding the molecular basis of their target specificity is central to the understanding of physiological and pathological redox signaling. Electrostatic complementarity of the redoxins with their target proteins has been proposed as a major reason. Here, we analyzed the electrostatic similarity of all Arabidopsis thaliana Trxs, Grxs, and proteins containing such domains. Clustering of the redoxins based on this comparison suggests overlapping and also distant target specificities and thus functions of the different sub-classes including all Trx isoforms as well as the three classes of Grxs, i.e. CxxC-, CGFS-, and CC-type Grxs. Our analysis also provides a rationale for the tuned substrate specificities of both the ferredoxin- and NADPHdependent Trx reductases. Introduction Redox modifications of cysteinyl side chains are a vital part of numerous signal transduction pathways, in photosynthetic organisms these mechanisms play a vital role, e.g. in light-dark adaptation [1–3]. Redox modifications of protein thiols such as disulfide formation and reduction are catalyzed by members of the Trx family of proteins, i.e. Trxs and Grxs [3–7]. This group of proteins share a common structural motif, the Trx fold. Cysteinyl residues in their active sites are the basis of their redox activity [8]. The proteins of this family catalyze the oxidation and reduction of disulfides in target proteins, including glutathionylation- PLOS ONE | https://doi.org/10.1371/journal.pone.0291272 September 11, 2023 1 / 18 PLOS ONE analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Breakdown of Arabidopsis thaliana thioredoxins and glutaredoxins based on electrostatic similarity deglutathionylation of proteins. Trx family proteins are encoded in essentially all genomes and they were likely already present in the last universal common ancestor of all life forms an earth [9]. Trxs and Grxs are present in all compartments of eukaryotic cells, e.g. the cytosol, ER, mitochondria, nucleus, and plastids–often in multiple isoforms [10]. Most Trxs and Grxs have a broad, but distinct substrate specificity. Understanding the molecular basis of their target specificity is key to their physiological functions and for the understanding of redox regulation in general. This molecular basis is the focus of this work. Trxs are efficient catalysts of thiol-disulfide exchange reactions and the trans-nitrosylation of cysteinyl side chains [11, 12]. During their reaction cycle, Trxs form a disulfide in their CysGly-Pro-Cys active site. This disulfide is reduced by thioredoxin reductases (TrxRs). Photosynthetic organisms contain two types of TrxRs. First, the ferredoxin-dependent FTRs that couple the photosynthetic electron chain directly to redox regulation [13] and, second, the NADPHdependent NTRs that function, among others, in stress defense [14]. Grxs are divided into three classes [4, 15, 16]. The first, also named class-I or CxxC-type Grxs share a Cys-Pro-TyrCys consensus active site motif and catalyze thiol-disulfide exchange reactions, often including glutathione (GSH). Oxidized Grxs are reduced by two molecules of GSH. The resulting GSH disulfide is reduced by GSH reductases at the expense of NADPH. The second subclass of the Grxs, named class-II or CGFS-type Grxs, with a consensus Cys-Gly-Phe-Ser active site, do not catalyze thiol-disulfide exchange reactions. Instead, they function in the regulation of iron metabolism or in the transfer of iron-sulfur centers [17–19]. Two alternative loop structures preceding the active sites and different modes of GSH-binding resulting thereof are the basis for the fundamentally different functions of these two Grx classes [20, 21]. The proteins of the third class are restricted to land plants, they were named ROXYs or CC-type Grxs. These proteins function in the regulation of TGA family transcription factors. So far, neither redox, nor FeS transfer activity of these proteins was described that could be linked to their physiological functions. In most species, and in plants in particular, various Trx family protein isoforms were described and characterized in the same compartment, prompting questions on both overlapping and distinct functions. Proteomic studies screening for interaction partners and target proteins, also summarized in this work, imply a high degree of substrate specificity for the individual isoforms. The basis for this specificity has long been a (...truncated)