ATP and Mg2+ Promote the Reversible Oligomerization and Aggregation of Chloroplast 2-Cys Peroxiredoxin

Abstract

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous peroxidases with important roles in cellular antioxidant defense and hydrogen peroxide-mediated signaling. Post-translational modifications of conserved cysteines cause the transition from low to high molecular weight oligomers, triggering the functional change from peroxidase to molecular chaperone. However, it remains unclear how non-covalent interactions of 2-Cys Prx with metabolites modulate the quaternary structure. Here, we disclose that ATP and Mg 2؉(ATP/Mg) promote the self-polymerization of chloroplast 2-Cys Prx (polypeptide 23.5 kDa) into soluble higher order assemblies (>2 MDa) that proceed to insoluble aggregates beyond 5 mM ATP. Remarkably, the withdrawal of ATP or Mg 2؉ brings soluble oligomers and insoluble aggregates back to the native conformation without compromising the associated functions. As confirmed by transmission electron microscopy, ATP/Mg drive the toroidlike decamers (diameter 13 nm) to the formation of large spherelike particles (diameter ϳ30 nm). Circular dichroism studies on ATP-labeled 2-Cys Prx reveal that ATP/Mg enhance the proportion of ␤-sheets with the concurrent decrease in the content of ␣-helices. In line with this observation, the formation of insoluble aggregates is strongly prevented by 2,2,2-trifluoroethanol, a cosolvent employed to induce ␣-helical conformations. We further find that the response of self-polymerization to ATP/Mg departs abruptly from that of the associated peroxidase and chaperone activities when two highly conserved residues, Arg 129 and Arg 152 , are mutated. Collectively, our data uncover that non-covalent interactions of ATP/Mg with 2-Cys Prx modulate dynamically the quaternary structure, thereby coupling the non-redox chemistry of cell energy with redox transformations at cysteine residues.

Peroxiredoxins in Parasites

“…The S P O 2 H of such sensitive Prxs can in many organisms be reactivated (Step 5) by ATP-dependent reduction by sulfiredoxin (Srx) or, perhaps, the less-characterized sestrin (46,113). How these Prxs facilitate peroxide signaling is not yet well understood but may involve a floodgate-like mechanism (265) and/or the formation of larger aggregates with molecular chaperone or other activities (12,89,197). Prx1 activity has also been reported to be regulated by phosphorylation (264).…”

The C-Terminal Extension of Chloroplast 2-Cys Peroxiredoxin Is Critical for Interaction with ATP

“…Our previous studies revealed that ATP interacts with Cys R , , but nucleotide binding motives in 2-Cys Prx were not identified in an exhaustive search of protein databases. Therefore, we used QUANTUM version 3.3 that screens the interactions of proteins with drugs and metabolites to explore docking of ATP to different regions of 2-Cys Prx (Protein Data Bank entry ).…”

“…Recently, circular dichroism (CD) spectroscopy revealed that changes in the secondary structure of 2-Cys Prx accompany the self-polymerization mediated by ATP/Mg. Again, if the C-terminal region is critical for the binding of ATP to His 6 -tagged 2-Cys Prx, the length of this extension should condition the response of the secondary structure to the binding of the nucleotide.…”

“…This dynamic self-polymerization (decamers ⇔ soluble oligomers ⇔ insoluble aggregates) indicates that 2-Cys Prx not only acts as a redox regulatory protein but also effectively responds to nonredox processes that occur in the cellular milieu. 13,14 Our previous studies revealed that ATP interacts with Cys R , 15,16 but nucleotide binding motives in 2-Cys Prx were not identified in an exhaustive search of protein databases. Therefore, we used QUANTUM version 3.3 that screens the interactions of proteins with drugs and metabolites to explore docking of ATP to different regions of 2-Cys Prx (Protein Data Bank entry 1qmv).…”

“…These findings complemented a recent study in which Arg129, an amino acid residue close to the peroxidatic site, is necessary for the assembly of large protein complexes (Figure S-3 of the Supporting Information). 16 The investigation of the intrinsic fluorescence of His 6 -tagged 2-Cys Prx further supported the idea that the C-terminal extension plays a causative role in the binding of ATP/Mg to 2-Cys Prx. If ATP/Mg quenches the emission of Trp179 but not that of Trp88, 15 Prx and should be ineffective on Δ183 2-Cys Prx.…”