Molecular Basis for the Interactions of Human Thioredoxins with Their Respective Reductases.

Md Faruq Hossain,Manuela Gellert,Elias S J Arnér,Calvin Klein,Yana Bodnar,Christopher Horst Lillig,Clara Ortegón Salas,Jesús Tejero

doi:10.1155/2021/6621292

Abstract

The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in kinetic assays. For human Trx1 and TrxR1, complementary electrostatic surfaces within the area covered in the encounter complex appear to control the affinity of the reductase for its substrate Trx. Electrostatic compatibility was even observed in areas that do not form direct molecular interactions in the encounter complex, and our results suggest that the electrostatic complementarity in these areas influences the catalytic efficiency of the reduction. The human genome encodes ten cytosolic Trx-like or Trx domain-containing proteins. In agreement with our hypothesis, the proteins that have been characterised as TrxR1 substrates also show the highest similarity in their electrostatic properties.

Highlights

The thioredoxin (Trx) family of proteins comprises many key enzymes in redox signalling that catalyse specific reversible redox reactions, e.g., dithiol-disulphide exchange,glutathionylation, transnitrosylation, or peroxide reduction
E. coli thioredoxin reductases (TrxR) has a much narrower substrate specificity compared to the mammalian enzyme and cannot reduce human Trx1, while the mammalian-type TrxR1 can reduce both bacterial and mammalian Trxs
The results presented here confirm that the binding of Trx1 to TrxR1 is independent of a subsequent redox reaction between the proteins

Summary

Introduction

The thioredoxin (Trx) family of proteins comprises many key enzymes in redox signalling that catalyse specific reversible redox reactions, e.g., dithiol-disulphide exchange, (de)glutathionylation, transnitrosylation, or peroxide reduction. The Trx family of proteins is defined by a common structural motif that, in its most basic form, consists of a central four-stranded β-sheet surrounded by three alpha helices—the thioredoxin fold [8, 9]. The reduction of protein disulphides, for instance, is initiated by a nucleophilic attack of the more N-terminal active site cysteinyl residue, characterised by a low pKa value, on a sulphur atom of the disulphide in the target protein. This results in an intermediate mixed disulphide that, in the second reaction step, is reduced by the C-terminal

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