Comparative molecular modeling study of Arabidopsis NADPH-dependent thioredoxin reductase and its hybrid protein.

Yuno Lee,Sang Yeol Lee,Prettina Lazar,Songmi Kim,Kyun Oh Lee,Swan Hwang,Keun Woo Lee,Jeong Chan Moon,Sundarapandian Thangapandian,Youngsik Shon,Peter Csermely

doi:10.1371/journal.pone.0046279

Abstract

2-Cys peroxiredoxins (Prxs) play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC) was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C) of thioredoxin reductase (TrxR) in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx) domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D)), which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD) simulations on AtNTRC and AtNTRA-(Trx-D) proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD) for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D) cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D) because of following reasons: i) unstable and unfavorable interaction of the linker region, ii) shifted Trx domain, and iii) different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.

Highlights

Redox regulation plays an important role in a variety of biological processes
AtNTR isotype A (AtNTRA) was connected with Trx domain of AtNTRC to confirm the role of Trx domain with AtNTRA
Functional domains like flavin adenine dinucleotide (FAD), the linker region between FAD and Trx, pyridine nucleotide domain (PD) and Trx domain were well aligned in both the enzymes and along with the important cysteine residues (Figure 1D)

Summary

Introduction

Redox regulation plays an important role in a variety of biological processes. in order to maintain redox homeostasis, cells have developed many compartmentalized enzymatic and non-enzymatic antioxidative systems. The cysteine residue is oxidized by peroxides during the catalytic cycle, and it is regenerated through intra- or intermolecular disulfide bond formation [6] via numerous reducing systems, such as thioredoxin (Trx), glutaredoxin (Grx), cyclophilin, or AhpF and AhpD [7,8,9,10]. It is already a proven fact that 2-Cys Prxs play important roles in the antioxidative defense systems of plant chloroplasts. AtNTRC contained N-terminal thioredoxin reductase (TrxR) and C-terminal Trx domains. It exhibited both TrxR and Trx activities and co-localized with 2-Cys Prx-A in chloroplasts [13]. According to the previous experimental results it was suggested that AtNTRC functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TrxR/Trx system in chloroplasts [14]

Methods

Results

Discussion

Conclusion