Thiol redox switches regulate the oligomeric state of cyanobacterial Rre1, RpaA and RpaB response regulators

Abstract

Cyanobacteria employ two-component sensor-response regulator systems to monitor and respond to environmental challenges. The response regulators RpaA, RpaB, Rre1 and RppA are integral to circadian clock function and abiotic stress acclimation in cyanobacteria. RpaA, RpaB and Rre1 are known to interact with ferredoxin or thioredoxin, raising the possibility of their thiol regulation. Here, we report that Synechocystis sp. PCC 6803 Rre1, RpaA and RpaB exist as higher-order oligomers under oxidising conditions and that reduced thioredoxin A converts them to monomers. We further show that these response regulators contain redox-responsive cysteine residues with an E_m7 around -300 mV. These findings suggest a direct thiol modulation of the activity of these response regulators, independent of their cognate sensor kinases.

Keywords: Hik2; RpaA; RpaB; Rre1; TrxA; thiol regulation.

Fig. 1

Sequence alignment of full‐length Synechocystis sp. PCC 6803 Rre1, RpaA, RpaB, and RppA response regulators. Putative redox‐active cysteines are shown in red. The divalent cation Mg²⁺‐binding motif “D2”, phosphorylation site “D1”, and the lysine (K) motifs are shown. The receiver domain is shaded in white, and the DNA binding domain in yellow.

Fig. 2

Effect of redox environment on the oligomeric state of Rre1, RpaA, RpaB, and RppA. (A–D) redox‐treated proteins as resolved by urea‐SDS/PAGE. (E–H) oligomeric state of response regulators as determined by size exclusion chromatography. Panels (E–H) correspond to samples oxidized with 2 mm H₂O₂ or reduced with 2 mm DTT, respectively.

Fig. 3

Redox titration of Rre1, RpaA, and RpaB. (A,C & E) Redox‐titrated proteins separated by nonreducing urea‐SDS/PAGE and stained with Coomassie brilliant blue. (B,D & F) Plots of redox titration. From the intensity of the reduced and oxidized band species, the midpoint potential E_m was computed by fitting the data to the Nernst equation. SHE, standard hydrogen electrode potential. Error bars represent ± SE from two replicate titrations.

Fig. 4

TrxA‐mediated reduction of the redox‐active cysteines of Rre1, RpaA, RpaB, and RppA. TrxA‐treated proteins as separated on a nonreducing urea‐SDS/PAGE gel and stained with Coomassie. + or − sign indicates the presence or absence of 0.1 mm DTT or 5 mm TrxA, respectively.