Structural and mechanistic basis for redox sensing by the cyanobacterial transcription regulator RexT

Organisms have a myriad of strategies for sensing, responding to, and combating reactive oxygen species, which are unavoidable consequences of aerobic life. In the heterocystous cyanobacterium Nostoc sp. PCC 7120, one such strategy is the use of an ArsR-SmtB transcriptional regulator RexT that senses H2O2 and upregulates expression of thioredoxin to maintain cellular redox homeostasis. Different from many other members of the ArsR-SmtB family which bind metal ions, RexT has been proposed to use disulfide bond formation as a trigger to bind and release DNA. Here, we present high-resolution crystal structures of RexT in the reduced and H2O2-treated states. These structures reveal that RexT showcases the ArsR-SmtB winged-helix-turn-helix fold and forms a vicinal disulfide bond to orchestrate a response to H2O2. The importance of the disulfide-forming Cys residues was corroborated using site-directed mutagenesis, mass spectrometry, and H2O2-consumption assays. Furthermore, an entrance channel for H2O2 was identified and key residues implicated in H2O2 activation were pinpointed. Finally, bioinformatics analysis of the ArsR-SmtB family indicates that the vicinal disulfide “redox switch” is a unique feature of cyanobacteria in the Nostocales order, presenting an interesting case where an ArsR-SmtB protein scaffold has been evolved to showcase peroxidatic activity and facilitate redox-based regulation.

. Structure comparison of RexT with ArsR-SmtB superfamily members. (a) Sequence alignment of four ArsR-SmtB transcription factors identified by the Dali server 2 with RexT shows a well conserved wHTH architecture. Secondary structure labeling is based on the consensus. Colored boxes highlight functional residues that could bind an environmental stimulator, such as a metal ion (green and blue), an arsenite ion (pink), form a disulfide bond in RexT (orange), or an unknown species (purple). (b) RexT (gray) is overlaid with VcHlyU 3 (PDB ID: 4K2E, purple). In VcHlyU Cys38 on the α2 helix is in the sulfenic acid form and is pointing towards Cys104, suggesting that disulfide bond formation may be important to regulation. The root mean squared deviation (rmsd) is 1.82 Å over 396 atoms. The rmsd is 1.45 Å over 404 atoms. (d) RexT (gray) is overlaid with AfArsR 5 (PDB ID: 6J05, pink). AfArsR uses three Cys residues on the C-terminus to bind As 3+ (purple sphere). The rmsd is 1.33 Å over 365 atoms. (e) RexT (gray) is overlaid with SeSmtB 6 (PDB ID: 1R22, light purple). In this protein, two Zn 2+ (gray sphere) ions at the dimer interface are found coordinated to Asp and His residues from the α5 helix of one subunit, and His and Glu residues from the other subunit (only a monomer is shown here). The rmsd is 2.05 Å over 426 atoms. In all panels, the overlay of RexT was performed in COOT 7 using the SSM superimpose feature to align the models based on Cα positions and visualized by PyMOL. Af, Acidithiobacillus ferrooxidans; Se, Synechococcus elongatus PCC 7942; Sa, Staphylococcus aureus; Vc, Vibrio cholerae.
Supplementary Fig. 3. The ability of RexT to bind DNA is not impacted by the presence of metal ions. (a) A DNA probe (20 nM) was incubated with RexT (100 nM) and 1, 2.5, or 5 equivalents of As(III), Cd 2+ , and Zn 2+ . This electrophoretic mobility shift assay (EMSA) showed that RexT binds to DNA similarly in the absence (lane 2) and presence of the different tested metal ions (lanes 3-11). (b) A labeled DNA probe shows changes in fluorescence anisotropy following the addition of RexT. These differences allowed for calculation of the Kd for RexT in the presence (orange) and absence (gray) of Cd 2+ (see Supplementary  Table 2). As shown in panel A, adding a metal ion to RexT in a 2.5-fold excess does not markedly change its ability to bind DNA. Each data point for the experiments is shown as an open shape. In panel b, data was measured using n=3 independent experiments and is presented with the individual measurements (open shapes) and as the mean value of these measurements ± SD (closed shapes). Source data are provided as a Source Data file. Red dotted lines show subunit-specific interactions. In chain A, the chloride interacts with a nearby water and the side chain oxygen of Asn74. In chain B, the chloride may have a weak interaction with the sulfur atom of Cys40. (b) The 2Fo-Fc (gray) and Fo-Fc (green/red) electron density map calculated for the refined structure of RexT around the modeled chloride ion. These maps are shown contoured at 1.0σ and ±3.0σ, respectively. (c) The 2Fo-Fc (gray) and Fo-Fc (green/red) electron density map calculated after chloride was omitted from the structure of RexT and water was modeled in its place. These maps are shown contoured at 1.0σ and ±3.0σ, respectively. (d) The 2Fo-Fc (gray) and Fo-Fc (green/red) electron density map calculated after the chloride ion was omitted from the structure of RexT. These maps are shown contoured at 1.0σ and ±3.0σ, respectively.
Supplementary Fig. 7. Mass spectrometry reveals key insights into disulfide bond formation. (a) To probe the identity of the disulfide bond forming Cys residues, mass spectrometry experiments were performed on wild-type (WT) RexT and each of its Cys variants in the presence of H2O2. The mass of RexT with a disulfide bond should be 2 Da less than when a disulfide bond is not formed. (b) As disulfide bond formation proceeds through formation of a sulfenic acid moiety, mass spectrometry was also used to look for incorporation of the small molecule dimedone into RexT following the addition of H2O2. This experiment was performed to identify the peroxidatic Cys residue. Incorporation of one molecule of dimedone into RexT results in a 138 Da increase in the mass of the protein. In chain B, there is space for a disulfide bond to be formed between Cys40 and Cys41. In chain A, a symmetry molecule is packed up against where the bond would be formed. (b) Chain B of RexT is overlaid with chain A to show the clash that would occur upon disulfide bond formation.
Supplementary Fig. 10. A significant conformational change occurs in the RexT structure following oxidation. (a) The reduced structure of RexT (gray) has a different conformation than the oxidized in the α3 helical region. Shown in this panel are the 2Fo-Fc (gray) and Fo-Fc (green/red) electron density maps contoured at 1.0σ and ±2.0σ, respectively, when the structure of the reduced RexT is used as a model for the oxidized RexT data. (b) Modeling the oxidized structure (cyan) with a disulfide bond is a better fit of the data. Again, the 2Fo-Fc (gray) and Fo-Fc (green/red) electron density maps contoured at 1.0σ and ±2.0σ, respectively. (c) The refined 2Fo-Fc (gray) and Fo-Fc (green/red) electron density maps contoured at 1.0σ and ±2.0σ, respectively, around the oxidized structure in the region of the modeled disulfide bond are shown.
(d) A Fo-Fc omit (green) electron density map contoured at ±2.0σ is shown around the α3 helical region after it was omitted from the refined structure of oxidized RexT. (e) Alignment of the oxidized RexT structure with the DNA-bound NolR structure (PDB: 4ON0) 8 shows that the wHTH regions of RexT that interact with DNA are highly flexible. The bottom of the calculated cavity that surrounds Cys41 contains residues arranged in a way that suggests they are important players in activation of H2O2. (c) The electron density maps for the residues and glycerol molecule shown in panel B are displayed. The refined 2Fo-Fc (gray) and Fo-Fc (green/red) electron density maps contoured at 0.9σ and ±3.0σ, respectively. (d) The 2Fo-Fc and Fo-Fc electron density map calculated after glycerol was omitted from the structure of reduced RexT. These maps are shown contoured at 0.9σ and ±3.0σ, respectively. (e) The 2Fo-Fc (gray) and Fo-Fc (green/red) electron density maps for the residues and H2O2 molecule from Fig. 5B are displayed contoured at 0.9σ and ±3.0σ, respectively. (f) The 2Fo-Fc and Fo-Fc electron density map calculated after H2O2 was omitted from chain A of the oxidized RexT structure are displayed contoured at 0.9σ and ±3.0σ, respectively.
Supplementary Fig. 12. The HTH ArsR-SmtB-type DNA-binding domain and related protein families were used to generate a sequence similarity network (SSN). Proteins and their structures that have an annotated "HTH ArsR-type DNA-binding domain" (IPR001845, large oval shape). This classification encompasses a few protein families, predominantly PF01022 (HTH_5, shaded in pink) and PF12840 (HTH_20, shaded in blue). Notably a few proteins that belong to PF12840 do not have the annotated IPR001845 domain (shaded in blue, outside of the large oval). Proteins and PDB IDs shown in italics do not have an accompanying publication.
Supplementary Fig. 13. Sequence alignment of previously characterized members of the ArsR-SmtB family. Conserved secondary structures are labeled as cylinders (α-helices) and arrows (β-strands) based on the consensus. Key amino acid residues that are implicated in regulatory process are colored: blue, His/Asp/Glu-rich motif for binding of "hard" metal ions such as Ni 2+ and Zn 2+ ; green, Cys-rich motif for binding "soft" metal ions such as Pb 2+ and Cd 2+ ; magenta, Cys-rich motif for binding of arsenite or methylarsenite; purple, key Cys residues that are involved in redox processes to bind RSS or an unknown species; yellow, key Cys residues that are involved in the ROS response; gray, residues that resembles abovementioned conserved motifs but do not exhibit any function and are likely the remnant of evolution. The residues responsible for regulation in NolR, Rv0081, Rv2034 and BaPagR are unknown.  Fig. 6 were submitted to the server). It is analyzed in Cytoscape at the cut-off value of e -32 . The network is further annotated by the EFI-genome neighborhood tool 9,10 by checking the ten or three genes upstream and downstream of the RexT homologs. The nodes are color-coded based on the numbering of the multi-node clusters in order of decreasing number of sequences. The Pfam for thioredoxin (PF00085) was used as a query to identify its co-occurrence with the RexT homologs. Of interest to this work, in 121 and 108 instances, a thioredoxin gene was found to be within the ten or three-gene neighborhood of RexT homologs, respectively. However, in only 105 of the identified instances is a thioredoxin gene adjacent to a RexT homolog (large teal nodes). 102 of the 105 RexT homologs are from Cyanobacteria (circled with a solid line), two are from unclassified bacteria and one is from Abditibacteria (circled with a dashed line). RexT from Nostoc sp. PCC 7120 (NoRexT) is highlighted in dark red. CgCyeR is found in a different cluster in this network.  Fig. 16. A rooted phylogentic tree for RexT homologs from three orders of Cyanobacteria. This analysis was conducted in MEGA X 12,13 and involved 96 RexT homologs. The RexT sequence from Abditibacterium utsteinense (UniProt ID: A0A2S8SU85) was used as the outgroup. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model 14 .
The tree with the highest log likelihood (-4801.45) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.3614)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The 11 RexT homologs from Synechococcales and Oscillatoriales are colored in green and the 84 RexT homologs from Nostocales are colored in blue. The RexT homolog from Tolypothrix sp. NIES-4075 that does not contain Cys40 is highlighted by a pink dot, and RexT homologs from Nostocales that contain an additional Cys105 are highlighted by a blue dot. RexT from Nostoc sp. PCC 7120 studied in this work is indicated by an arrow.   Note: Regulatory residues are colored. Cyan: residues in the Asp/Glu and His-rich metal binding motifs; Green: residues in the Cys-rich metalbinding motifs; Pink: Cys residues that bind arsenite or methyl-arsenite; Purple: Cys residues that bind an RSS or an unknown species; Yellow: Cys residues that bind an ROS; Gray, residues that resembles above-mentioned conserved motifs but do not exhibit any function and are likely the remnant of evolution.