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Structural basis for persulfide-sensing specificity in a transcriptional regulator


Cysteine thiol-based transcriptional regulators orchestrate the coordinated regulation of redox homeostasis and other cellular processes by ‘sensing’ or detecting a specific redox-active molecule, which in turn activates the transcription of a specific detoxification pathway. The extent to which these sensors are truly specific in cells for a singular class of reactive small-molecule stressors, for example, reactive oxygen or sulfur species, is largely unknown. Here, we report structural and mechanistic insights into the thiol-based transcriptional repressor SqrR, which reacts exclusively with oxidized sulfur species such as persulfides, to yield a tetrasulfide bridge that inhibits DNA operator–promoter binding. Evaluation of crystallographic structures of SqrR in various derivatized states, coupled with the results of a mass spectrometry-based kinetic profiling strategy, suggest that persulfide selectivity is determined by structural frustration of the disulfide form. These findings led to the identification of an uncharacterized repressor from the bacterial pathogen Acinetobacter baumannii as a persulfide sensor.

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Fig. 1: Specificity of C9S SqrR oxidation.
Fig. 2: Thiol reactivity is determined by steric hindrance.
Fig. 3: The disulfide crosslink introduces high local structural frustration relative to the tetrasulfide form.
Fig. 4: The kinetics of tetrasulfide bond formation in SqrR.
Fig. 5: Identification of an uncharacterized RSS-sensing repressor from A. baumannii.

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Data availability

The coordinates and structural factors have been submitted to the Protein Data Bank with accession codes 6O8L (C9S SqrR, reduced), 6O8K (wild-type SqrR, reduced), 6O8N (C9S SqrR, tetrasulfide form), 6O8O (SeMet C9S SqrR, disulfide form) and 6O8M (C9S SqrR, derivatized with TMAD). The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary information files. All raw data are available from the corresponding author upon request.


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We thank the National Institutes of Health (grant no. R35 GM118157 to D.P.G.) and the Pew Foundation Latin American Fellows and Williams foundation (to D.A.C.) for financial support of this work. B.J.C.W. was supported in part by a Quantitative and Chemical Biology Training Fellowship provided by the NIH (grant no. T32 GM109825; grant no. T32 GM131994) and the Kratz Fellowship provided by the Indiana University Department of Chemistry. We thank H. Wu for his help with NMR data acquisition. We thank Y.-C. Huang for her help in protein purification and M. Villarruel for his help with figure editing. We thank S. Xu and M. Xian from Washington State University, who kindly provided the SNAP persulfide donor. We gratefully acknowledge use of the Macromolecular Crystallography Facility at the Molecular and Cellular Biochemistry Department, Indiana University, Bloomington. We also thank J. Nix for his assistance during X-ray data collection at beamline 4.2.2, ALS.

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Authors and Affiliations



D.A.C., B.J.C.W., Y.Z. and C.D. conducted biochemical and chemical studies and G.G.-G. solved the crystal structures. D.A.C. and D.P.G. conceptualized the study and supervised the research. D.P.G. was responsible for direction and resource acquisition. D.A.C. and D.P.G. wrote the manuscript, and all authors edited and reviewed it.

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Correspondence to David P. Giedroc.

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Extended data

Extended Data Fig. 1 Illustration of quantitative capping of reduced C9S SqrR with IAM under native conditions and the reaction of C9S SqrR with TMAD, RNS and RCS.

LC-ESI-MS spectra of a, reduced and untreated C9S SqrR (yellow trace) and IAM treated C9S SqrR (black trace); b, Reactive nitrogen species (RNS)-treated where MAHMA NONOate spontaneously generates nitric oxide at physiological pH. nitrate, sodium nitrite; c, TMAD-treated, and d, reactive chlorine species (RCS)-treated C9S SqrR followed addition of an excess of IAM (900x). Solution conditions: 150 mM sodium phosphate, pH 7.4, 1 mM EDTA. Cl-T, chloramine-T. The observed addition of 1 or 2 oxygen atoms upon mixing with RCS suggests that the dithiol pair in SqrR may be protected by sacrificial methionine oxidation by both chloramine-T (Cl-T) and bleach58. Data are representative of three independent experiments.

Extended Data Fig. 2 Cysteine trisulfide synthesis and reaction with C9S SqrR.

a, Reaction scheme for synthesis of cysteine trisulfide (CSSSC, red)29,59 proceeding through a cystine-S-monoxide intermediate (blue) from cystine (green) starting material. Table includes the expected m/z for these species. b, ESI-MS of impure (top) and pure (bottom) synthetic preparations of cysteine trisulfide. Peaks are highlighted for each molecule based on the color scheme in panel (a); masses given are the observed m/z. c, LC-ESI-MS spectra of CSSSC-reacted C9S SqrR using 20-fold excess of impure (blue), pure (red) CSSSC and cystine (CSSC, green), followed by addition of excess IAM. Solution conditions: 30 μM protomer C9S SqrR,150 mM phosphate buffer pH 7.4, 1 mM EDTA.

Extended Data Fig. 3 rPA-MS single time point assays used to compare the relative reactivities of C9, C41 and C107 in SqrR variants.

a, Reaction scheme illustrated for wild-type SqrR. Heavy d5 (grey shaded) and light H5 (red shaded) C9- b, C107- c, and C41- d, containing peptides for the different protein variants (indicated) at 30s pulse time with a 3-fold protomer mol excess of reagent. C9 is more reactive than C107 (increased relative abundance of the “heavy” d5-NEM used for the alkylation pulse), which is significantly higher than C41 in all the variants tested. Substitution of the non-conserved C9 decreases the reactivity of the other Cys residues consistent with a slight protective role of the N-terminal unstructured (Extended Data Fig. 5) region in SqrR. Serine substitution of C41 or C107 increases the apparent nucleophilicity on the remaining Cys with little impact on C9. Solution conditions: 25 mM HEPES, pH 7.0, 200 mM NaCl, 5 mM EDTA. Data are representative of three independent experiments.

Extended Data Fig. 4 Cα ribbon diagram superpositions and pair-wise Cα root-mean square deviations (RMSD) in pairs of SqrR structures presented here.

Superposition of the structures, depicted as a Cα trace, of reduced C9S SqrR with a, disulfide-crosslinked C9S SqrR; b, tetrasulfide-crosslinked C9S SqrR; c, the C9S SqrR diamide adduct, and d, wild-type reduced SqrR. The RMSD for Cα atoms for the monomer (calculated from the coordinates of chain A when there is more than one protomer in the unit cell) is shown at the bottom of each panel. The positions of the secondary structure elements are depicted as cartoons in each panel.

Extended Data Fig. 5 1H, 15N HSQC spectra and 1H–{15N} heteronuclear nuclear Overhauser enhancement (hNOE) of reduced C9S SqrR.

a, Resonance assigned 1H,15N HSQC spectrum of C9S SqrR in the reduced state. Backbone assignments are shown in the one-letter code (assignments to be reported elsewhere). b, Backbone hNOE as a function of residue number, indicating relative backbone flexibility of the N-terminal region (the shaded area indicates the expected values for a rigid backbone on the sub-ns timescale). The position of the secondary structure motifs is shown above. c, 1H,15N HSQC spectra of C9S SqrR in the tetrasulfide state compared to the reduced state (green contour; inly a single contour line is shown). The solution conditions for all the experiments were the same: 25 mM MES, 50 mM NaCl, pH 5.1, 40 °C).

Extended Data Fig. 6 Frustration patterns in C9S SqrR in various allosteric states.

Frustration patterns in C9S SqrR in the tetrasulfide a, d, reduced b, e, and disulfide states c, f. The backbones of the proteins are shown as gray cartoons (the N-terminal region in the disulfide state is represented with a line), minimally frustrated contacts are depicted with green lines, and highly frustrated interactions with red lines. Neutral interactions were omitted for clarity. A higher density of highly frustrated contacts is observed in the vicinity of the disulfide bond (c), whereas another region of high local frustration for all three states of the protein is the N-terminal region of α4 (d-f).

Extended Data Fig. 7 Electron density maps of the dithiol pocket in C9S SqrR in the reduced and tetrasulfide states.

Electron density maps of the dithiol pocket in C9S SqrR in the reduced a, and tetrasulfide-oxidized b, states. Interpreted electron density (grey) and uninterpreted electron density (positive, green and negative, red) were obtained from the deposited maps for 6O8L (a) and 6O8N (b, with the water in line representation near the arrow not included in the refinement for this figure). The arrows indicate uninterpreted electron density that was not modelled the deposited structures and may derive from residual electron density from methionine oxidation to a sulfone (black) and the backbone (blue). The red arrow indicate uninterpreted electron density modelled the deposited structure as a water, that instead may derive from an alternative tetrasulfide geometry or the presence of small amount of pentasulfide linkage (see Extended Data Fig. 10). c, Validation that the oxidation-sensitive methionines (M17, M38) are not necessary for the formation of the tetrasulfide linkage in the presence of GSSH. Data (c) are representative of three independent experiments.

Extended Data Fig. 8 SqrO–DNA binding isotherms for C9S and wild-type SqrRs in the reduced, disulfide- and tetrasulfide-crosslinked states.

DNA binding isotherms for a, C9S SqrR in different oxidation states (at 200 mM NaCl), b, C9S SqrR in the reduced and tetrasulfide states (300 mM NaCl), the arrow indicates addition of TCEP. Inset, raw anisotropy as a function of upon addition of 5 mM TCEP revealing rapid SqrR association after reduction. c, C9S SqrR in the reduced state (2 mM TCEP added) at different [NaCl]. Inset, [NaCl] (M)-dependence of Ka. d, C9S SqrR in the reduced state, no TCEP added (in 300 mM NaCl) (black continuous line) compared to C9S SqrR (reduced, black dashed lines; tetrasulfide, red dashed lines). The arrow indicates addition of IAM. Inset, raw anisotropy as a function of time following addition of SqrR first (up-arrow) and then 1 mM IAM (down-arrow) to free DNA. These data suggest a relatively slow kinetics of dissociation of IAM-derivatized SqrR. e, C9S SqrR binding to the SqrR operator (SqrO) present in the promoter regions of the rcc01451 (black) and rcc0785 (brown) genes (200 mM NaCl), and as a control, no binding to a canonical CstR operator (green) (in 300 mM NaCl) f, Reduced wild-type SqrR (black) compared to reduced and oxidized C9S SqrRs (reduced, black dashed lines; tetrasulfide, red dashed lines) (in 300 mM NaCl). Conditions: 10 mM Hepes, pH 7.0, 25.0 °C, [SqrO]=5 nM rcc01451 unless indicated otherwise with the indicated [NaCl]. Data are derived from three independent experiments presented as mean values ± SD.

Extended Data Fig. 9 Evidence for a mixed disulfide intermediate in GSSH-treated single Cys-containing SqrR variants and kinetic trapping of the disulfide.

a, LC-ESI-MS spectra of C9S SqrR in the disulfide state obtained following a 1 h incubation with 1 mM TMAD and reacted with a 20-fold molar excess of GSSH, followed by a large excess of IAM (900x) to cap. b, LC-ESI-MS spectra of reduced C9S/C41S SqrR (30 µM protomer) treated for 1 h with a 20-fold molar excess of GSSG or GSSH. c, LC-ESI-MS spectra of reduced C9S/C107S SqrR (30 µM protomer) treated for 1 h with a 20-fold molar excess of GSSG or GSSH. Solution conditions: 150 mM sodium phosphate, pH 7.4, 1 mM EDTA. Data are representative of three independent experiments.

Extended Data Fig. 10 Comparison of the structures and polysulfide product distributions of C9S RcSqrR and a BigR-like RSS sensor.

a, (left) The cysteine pocket of C9S RcSqrR illustrating the distance between the thiols compared to that of XfBigR in the reduced form (PDB 3PQJ)51. The XfBigR shows a folded N-terminal α-helix (labeled α1’) that packs against the Cys cavity preventing the two Cys from getting closer to one another. (right) AbBigR polysulfide length comparison with C9S SqrR showing that the pentasulfide is a predominant species in AbBigR while the tetrasulfide is the dominant product for C9S SqrR. This suggests that the polysulfide length distribution may be dictated at least in part by the distance between the two reduced thiolate Sγ atoms, supporting the idea that this product distribution is largely dictated by the crosslinked product(s) that give rise to a minimal structural perturbation. Data are derived from five independent experiments presented as mean values ± SD. b, Multiple sequence alignment of SqrR/BigR-like proteins that are functionally characterized to varying degrees as follows: purple: in vivo evidence for persulfide specificity24,43; black: persulfide reactivity can be inferred on the basis of the function of the regulated genes44,45; green: no information of the nature of the inducer in cells is currently available.

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Capdevila, D.A., Walsh, B.J.C., Zhang, Y. et al. Structural basis for persulfide-sensing specificity in a transcriptional regulator. Nat Chem Biol 17, 65–70 (2021).

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