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

Abstract

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 NADPH-dependent Trx reductases.

Fig 1. Clustering of Arabidopsis thaliana redoxins.

(A) Phylogram based on primary structure comparison, computed by Clustal Omega and CLC sequence viewer. (B) Similarity tree based on the similarity of the 3D structures extracted from the pdb and generated by homology modeling; the tree was computed using PDBeFold server (https://www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver) and the iTOL (https://itol.embl.de/) (C) The electrostatic similarity of the whole proteins was computed as outlined in the methods section; the tree was generated using ‘R’, the tree representation was done with iTOL (https://itol.embl.de/). The protein abbreviations highlighted in green correspond to the ‘ROXY’ classes in A and B.

Fig 2. Electrostatic features of the active site contact areas of exemplary redoxins from the seven electrostatic groups of Arabidopsis thaliana redoxins.

(A) Representative redoxins for each group. Depicted are the cartoon representations of the redoxins with helices in purple, sheets in yellow and the N-terminal CxxC cysteinyl residues in ball-and-stick model. and the electrostatic potential isosurfaces at + and—1 k·T·e^-1, blue: positive, red negative potential. (B) Interaction between barley (Hordeum vulgare, H.v.) TrxH2 and barley alpha-amylase/subtilisin inhibitor protein (BASI), pdb entry: 2iwt. The protein complex was opened (middle) to expose the interaction surfaces highlighted in cyan. The electrostatic potential isosurfaces of the interaction surfaces are shown at + and—1 k·T·e^-1. The orientation of TrxH2 in (B) is the same as one for the other Trxs in (A).

Fig 3. Venn diagram of the potential overlapping interaction partners of the indicated redoxins.

The potential or suggested interaction partners are listed in the S1 Table in S1 File and were received from the literature and various interaction data bases.

Fig 4. Hierarchical clustering of all redoxins with experimentally determined structures in the pdb.

The electrostatic similarity of the whole proteins was computed as outlined in the methods section. All redoxins from photosynthetic organisms were highlighted. The color codes were included in the figure. Modified from reference [27].

Fig 5. Electrostatic features of the active site contact areas of the thioredoxin reductases from Arabidopsis thaliana.

(A-C) the contact surface of FTR with TrxF2 (pdb 7c2b) computed with UCSF Chimera. (D-F) the electrostatic features of FTR from 7c2b. The image of the electrostatic isosurface (E) was scaled down by a factor of 5 for better visibility, lines represent the scale. (G-I) electrostatic features of NTR2 domain containing the active site cysteinyl residues analyzed based on pdb entry 1vdc (the FAD domain was removed as explained in the maintext).(J-K): electrostatic features of NTR1. (M-O) electrostatic features of NTRC. NTR1 and NTRC were modeled and based in this structure and analyzed accordingly. The electrostatic potential isosurfaces are shown at +/- 1 k·T·e-1. The electrostatic potential mapped to the water-accessible surface of the proteins at +/- 4 k·T·e-1. Blue: positive, red negative potential.