Crystal structure of chloroplastic thioredoxin z defines a type-specific target recognition.

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin-Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX-target recognitions.

Calcium sensing via EF-hand 4 enables thioredoxin activity in the sensor-responder protein calredoxin in the green alga Chlamydomonas reinhardtii.

Calcium (Ca2+) and redox signaling enable cells to quickly adapt to changing environments. The signaling protein calredoxin (CRX) from the green alga Chlamydomonas reinhardtii is a chloroplast-resident thioredoxin having Ca2+-dependent activity and harboring a unique combination of an EF-hand domain connected to a typical thioredoxin-fold. Using small-angle X-ray scattering (SAXS), FRET, and NMR techniques, we found that Ca2+-binding not only induces a conformational change in the EF-hand domain, but also in the thioredoxin domain, translating into the onset of thioredoxin redox activity. Functional analyses of CRX with genetically altered EF-hands revealed that EF-hand 4 is important for mediating the communication between the two domains. Moreover, we crystallized a variant (C174S) of the CRX target protein peroxiredoxin 1 (PRX1) at 2.4 Å resolution, modeled the interaction complex of the two proteins, and analyzed it by cross-linking and MS analyses, revealing that the interaction interface is located close to the active sites of both proteins. Our findings shed light on the Ca2+ binding-induced changes in CRX structure in solution at the level of the overall protein and individual domains and residues.

Impact of key residues within chloroplast thioredoxin-f on recognition for reduction and oxidation of target proteins.

Thioredoxin (Trx) is a redox-responsive protein that modulates the activities of its target proteins mostly by reducing their disulfide bonds. In chloroplasts, five Trx isoforms (Trx-f, Trx-m, Trx-x, Trx-y, and Trx-z) regulate various photosynthesis-related enzymes with distinct target selectivity. To elucidate the determinants of the target selectivity of each Trx isoform, here we investigated the residues responsible for target recognition by Trx-f, the most well-studied chloroplast-resident Trx. As reported previously, we found that positively-charged residues on the Trx-f surface are involved in the interactions with its targets. Moreover, several residues that are specifically conserved in Trx-f (e.g. Cys-126 and Thr-158) were also involved in interactions with target proteins. The validity of these residues was examined by the molecular dynamics simulation. In addition, we validated the impact of these key residues on target protein reduction by studying (i) Trx-m variants into which we introduced the key residues for Trx-f and (ii) Trx-like proteins, named atypical Cys His-rich Trx 1 (ACHT1) and ACHT2a, that also contain these key residues. These artificial or natural protein variants could reduce Trx-f-specific targets, indicating that the key residues for Trx-f are critical for Trx-f-specific target recognition. Furthermore, we demonstrate that ACHT1 and ACHT2a efficiently oxidize some Trx-f-specific targets, suggesting that its target selectivity also contributes to the oxidative regulation process. Our results reveal the key residues for Trx-f-specific target recognition and uncover ACHT1 and ACHT2a as oxidation factors of their target proteins, providing critical insight into redox regulation of photosynthesis.

Distinct electron transfer from ferredoxin-thioredoxin reductase to multiple thioredoxin isoforms in chloroplasts.

Thiol-based redox regulation is considered to support light-responsive control of various chloroplast functions. The redox cascade via ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) has been recognized as a key to transmitting reducing power; however, Arabidopsis thaliana genome sequencing has revealed that as many as five Trx subtypes encoded by a total of 10 nuclear genes are targeted to chloroplasts. Because each Trx isoform seems to have a distinct target selectivity, the electron distribution from FTR to multiple Trxs is thought to be the critical branch point for determining the consequence of chloroplast redox regulation. In the present study, we aimed to comprehensively characterize the kinetics of electron transfer from FTR to 10 Trx isoforms. We prepared the recombinant FTR protein from Arabidopsis in the heterodimeric form containing the Fe-S cluster. By reconstituting the FTR/Trx system in vitro, we showed that FTR prepared here was enzymatically active and suitable for uncovering biochemical features of chloroplast redox regulation. A series of redox state determinations using the thiol-modifying reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate, indicated that all chloroplast Trx isoforms are commonly reduced by FTR; however, significantly different efficiencies were evident. These differences were apparently correlated with the distinct midpoint redox potentials among Trxs. Even when the experiments were performed under conditions of hypothetical in vivo stoichiometry of FTR and Trxs, a similar trend in distinguishable electron transfers was observed. These data highlight an aspect of highly organized circuits in the chloroplast redox regulation network.

Expression of spinach ferredoxin-thioredoxin reductase using tandem T7 promoters and application of the purified protein for in vitro light-dependent thioredoxin-reduction system.

Thioredoxins (Trxs) regulate the activity of target proteins in the chloroplast redox regulatory system. In vivo, a disulfide bond within Trxs is reduced by photochemically generated electrons via ferredoxin (Fd) and ferredoxin-thioredoxin reductase (FTR: EC 1.8.7.2). FTR is an αß-heterodimer, and the ß-subunit has a 4Fe-4S cluster that is indispensable for the electron transfer from Fd to Trxs. Reconstitution of the light-dependent Fd/Trx system, including FTR, is required for the biochemical characterization of the Trx-dependent reduction pathway in the chloroplasts. In this study, we generated functional FTR by simultaneously expressing FTR-α and -ß subunits under the control of tandem T7 promoters in Escherichia coli, and purifying the resulting FTR complex protein. The purified FTR complex exhibited spectroscopic absorption at 410 nm, indicating that it contained the Fe-S cluster. Modification of the expression system and simplification of the purification steps resulted in improved FTR complex yields compared to those obtained in previous studies. Furthermore, the light-dependent Trx-reduction system was reconstituted by using Fd, the purified FTR, and intact thylakoids.

Arabidopsis mitochondrial voltage-dependent anion channel 3 (AtVDAC3) protein interacts with thioredoxin m2.

Voltage-dependent anion channels (VDACs) are conserved mitochondrial outer membrane proteins. A yeast two-hybrid screen identified interaction between Arabidopsis VDAC3 and the chloroplast protein thioredoxin m2 (AtTrx m2). This was confirmed via pull-down assay. A bimolecular fluorescence complementation assay located the interaction in mitochondria. AtVDAC3 and AtTrx m2 transcripts were expressed in multiple tissues and up-regulated by abiotic stress. Under NaCl stress, AtVDAC3 overexpression inhibited growth and increased H2O2 accumulation, while AtTrx m2 overexpression conferred resistance to NaCl and reduced H2O2. Results indicate that both AtVDAC3 and AtTrx m2 are involved in ROS signaling and play opposite roles in NaCl stress response.

Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins.

Plant redox-related proteins were overexpressed using a genetic codon substitution downstream of the translation initiation codon. This method significantly improved recombinant protein expression levels of Arabidopsis chloroplastic thioredoxins and cytosolic nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (E.C. 1.8.1.9) in Escherichia coli. Using these proteins, the in vitro chloroplastic thioredoxins-reduction system was reconstituted in an NADPH-dependent manner. This system could convert the five classes of chloroplastic Arabidopsis thioredoxins and two chloroplastic Spinach thioredoxins to their reduced forms, independent of dithiothreitol and the photosynthetic electron transport system.

Plastid thioredoxins f and m are related to the developing and salinity response of post-germinating seeds of Pisum sativum.

Plastid thioredoxins (TRXs) f and m have long been considered to regulate almost exclusively photosynthesis-related processes. Nonetheless, some years ago, we found that type-f and m TRXs were also present in non-photosynthetic organs such as roots and flowers of adult pea plants. In the present work, using pea seedlings 2-5 days old, we have determined the mRNA expression profile of the plastid PsTRX f, m1, and m2, together with the ferredoxin NADP reductase (FNR). Our results show that these TRX isoforms are expressed in cotyledons, underlying similar expression levels in roots for PsTRX m2. We have also noted plastid TRX expression in cotyledons of etiolated seedlings of Arabidopsis thaliana lines carrying constructs corresponding to PsTRX f and m1 promoters fused to the reporter gene GUS, pointing to a role in reserve mobilization. Furthermore, the response of plastid TRXs to NaCl and their capacity in restoring the growth of a TRX-deficient yeast under saline conditions suggest a role in the tolerance to salinity. We propose that these redox enzymes take part of the reserve mobilization in seedling cotyledons and we suggest additional physiological functions of PsTRX m2 in roots and PsTRX m1 in the salinity-stress response during germination.

A small family of chloroplast atypical thioredoxins.

The reduction and the formation of regulatory disulfide bonds serve as a key signaling element in chloroplasts. Members of the thioredoxin (Trx) superfamily of oxidoreductases play a major role in these processes. We have characterized a small family of plant-specific Trxs in Arabidopsis (Arabidopsis thaliana) that are rich in cysteine and histidine residues and are typified by a variable noncanonical redox active site. We found that the redox midpoint potential of three selected family members is significantly less reducing than that of the classic Trxs. Assays of subcellular localization demonstrated that all proteins are localized to the chloroplast. Selected members showed high activity, contingent on a dithiol electron donor, toward the chloroplast 2-cysteine peroxiredoxin A and poor activity toward the chloroplast NADP-malate dehydrogenase. The expression profile of the family members suggests that they have distinct roles. The intermediate redox midpoint potential value of the atypical Trxs might imply adaptability to function in modulating the redox state of chloroplast proteins with regulatory disulfides.