Protocol for using organic persulfides to measure the chemical reactivity of persulfide sensors

Hydrogen sulfide (H2S) and downstream reactive sulfur species (RSS), including organic persulfides, protect bacterial cells against diverse oxidative stressors. Specialized dithiol-based transcriptional repressors sense persulfides directly to control cellular H2S/RSS and avoid toxicity. Here, we present a protocol to quantify the kinetics of chemical reactivity of cysteines in two bacterial persulfide sensors toward cysteine persulfide and glutathione persulfide, with a LC-ESI-MS analysis that results in a kinetic model. This protocol has potential applications to other cysteine-containing proteins and oxidants. For complete details on the use and execution of this protocol, please refer to Fakhoury et al. (2021) and Capdevila et al. (2021).Fil: Fakhoury, Joseph N.. Indiana University; Estados UnidosFil: Capdevila, Daiana Andrea. Indiana University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Giedroc, David Peter. Indiana University; Estados Unido

Prediction and statistics of pseudoknots in RNA structures using exactly clustered stochastic simulations

Ab initio RNA secondary structure predictions have long dismissed helices interior to loops, so-called pseudoknots, despite their structural importance. Here, we report that many pseudoknots can be predicted through long time scales RNA folding simulations, which follow the stochastic closing and opening of individual RNA helices. The numerical efficacy of these stochastic simulations relies on an O(n^2) clustering algorithm which computes time averages over a continously updated set of n reference structures. Applying this exact stochastic clustering approach, we typically obtain a 5- to 100-fold simulation speed-up for RNA sequences up to 400 bases, while the effective acceleration can be as high as 100,000-fold for short multistable molecules (<150 bases). We performed extensive folding statistics on random and natural RNA sequences, and found that pseudoknots are unevenly distributed amongst RNAstructures and account for up to 30% of base pairs in G+C rich RNA sequences (Online RNA folding kinetics server including pseudoknots : http://kinefold.u-strasbg.fr/ ).Comment: 6 pages, 5 figure

Major Subject: Computer ScienceTECHNIQUES FOR MODELING AND ANALYZING RNA AND PROTEIN FOLDING ENERGY LANDSCAPES

Major Subject: Computer Scienceiii Techniques for Modeling and Analyzing RNA and Protein Folding Energ

Thioredoxin Profiling of Multiple Thioredoxin-Like Proteins in Staphylococcus aureus

Hydrogen sulfide (H2S) is thought to signal through protein S-sulfuration (persulfidation; S-sulfhydration) in both mammalian systems and bacteria. We previously profiled proteome S-sulfuration in Staphylococcus aureus (S. aureus) and identified two thioredoxin-like proteins, designated TrxP and TrxQ, that were capable of reducing protein persulfides as a potential regulatory mechanism. In this study, we further characterize TrxP, TrxQ and the canonical thioredoxin, TrxA, by identifying candidate protein substrates in S. aureus cells using a mechanism-based profiling assay where we trap mixed disulfides that exist between the attacking cysteine of a FLAG-tagged Trx and a persulfidated cysteine on the candidate substrate protein in cells. Largely non-overlapping sets of four, 32 and three candidate cellular substrates were detected for TrxA, TrxP, and TrxQ, respectively, many of which were previously identified as global proteome S-sulfuration targets including for example, pyruvate kinase, PykA. Both TrxA (kcat = 0.13 s-1) and TrxP (kcat = 0.088 s-1) are capable of reducing protein persulfides on PykA, a model substrate detected as a candidate substrate of TrxP; in contrast, TrxQ shows lower activity (kcat = 0.015 s-1). This work reveals that protein S-sulfuration, central to H2S and reactive sulfur species (RSS) signaling, may impact cellular activities and appears to be regulated in S. aureus largely by TrxP under conditions of sulfide stress

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster

Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence 547CNCTNCKCDQTKSCHGGDC565 are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1Δ strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu4-S6] cage structure, characterized by a core of trigonally S3 coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu4-L6 (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster

Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence 547CNCTNCKCDQTKSCHGGDC565 are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1Δ strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu4-S6] cage structure, characterized by a core of trigonally S3 coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu4-L6 (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I)

Functional asymmetry and chemical reactivity of CsoR family persulfide sensors

CstR is a persulfide-sensing member of the functionally diverse copper-sensitive operon repressor (CsoR) superfamily. While CstR regulates the bacterial response to hydrogen sulfide (H2S) and more oxidized reactive sulfur species (RSS) in Gram-positive pathogens, other dithiol-containing CsoR proteins respond to host derived Cu(I) toxicity, sometimes in the same bacterial cytoplasm, but without regulatory crosstalk in cells. It is not clear what prevents this crosstalk, nor the extent to which RSS sensors exhibit specificity over other oxidants. Here, we report a sequence similarity network (SSN) analysis of the entire CsoR superfamily, which together with the first crystallographic structure of a CstR and comprehensive mass spectrometry-based kinetic profiling experiments, reveal new insights into the molecular basis of RSS specificity in CstRs. We find that the more N-terminal cysteine is the attacking Cys in CstR and is far more nucleophilic than in a CsoR. Moreover, our CstR crystal structure is markedly asymmetric and chemical reactivity experiments reveal the functional impact of this asymmetry. Substitution of the Asn wedge between the resolving and the attacking thiol with Ala significantly decreases asymmetry in the crystal structure and markedly impacts the distribution of species, despite adopting the same global structure as the parent repressor. Companion NMR, SAXS and molecular dynamics simulations reveal that the structural and functional asymmetry can be traced to fast internal dynamics of the tetramer. Furthermore, this asymmetry is preserved in all CstRs and with all oxidants tested, giving rise to markedly distinct distributions of crosslinked products. Our exploration of the sequence, structural, and kinetic features that determine oxidant-specificity suggest that the product distribution upon RSS exposure is determined by internal flexibility.Fil: Fakhoury, Joseph N. Indiana University; Estados UnidosFil: Zhang, Yifan. Indiana University; Estados UnidosFil: Edmonds, Katherine A. Indiana University; Estados UnidosFil: Bringas, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Luebke, Justin L. Indiana University; Estados UnidosFil: Gonzalez Gutierrez, Giovanni. Indiana University; Estados UnidosFil: Capdevila, Daiana Andrea. Indiana University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Giedroc, David Peter. Indiana University; Estados Unido

Functional analysis of the SRV-1 RNA frameshifting pseudoknot

Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109–1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base–base and base–sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for −1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in −1 frameshifting

Structural elements of metal selectivity in metal sensor proteins

Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR are homologous zinc/cobalt-responsive and nickel/cobalt-responsive transcriptional repressors in vivo, respectively, and members of the ArsR/SmtB superfamily of prokaryotic metal sensor proteins. We show here that Zn(II) is the most potent negative allosteric regulator of czr operator/promoter binding in vitro with the trend Zn(II)>Co(II)≫Ni(II), whereas the opposite holds for the binding of NmtR to the nmt operator/promoter, Ni(II)>Co(II)>Zn(II). Characterization of the metal coordination complexes of CzrA and NmtR by UV/visible and x-ray absorption spectroscopies reveals that metals that form four-coordinate tetrahedral complexes with CzrA [Zn(II) and Co(II)] are potent regulators of DNA binding, whereas metals that form five- or six-coordinate complexes with NmtR [Ni(II) and Co(II)] are the strongest allosteric regulators in this system. Strikingly, the Zn(II) coordination complexes of CzrA and NmtR cannot be distinguished from one another by x-ray absorption spectroscopy, with the best fit a His-3-carboxylate complex in both cases. Inspection of the primary structures of CzrA and NmtR, coupled with previous functional data, suggests that three conserved His and one Asp from the C-terminal α5 helix donate ligands to create a four-coordinate complex in both CzrA and NmtR, with NmtR uniquely capable of expanding its coordination number in the Ni(II) and Co(II) complexes by recruiting additional His ligands from a C-terminal extension of the α5 helix

Effects of various metal ions on the gene expression of iron exporter ferroportin-1 in J774 macrophages

Macrophages play a key role in iron metabolism by recycling iron through erythrophagocytosis. Ferroportin-1 (FPN1) is a transporter protein that is known to mediate iron export from macrophages. Since divalent metals often interact with iron metabolism, we examined if divalent metals could regulate the expression of FPN1 in macrophages. J774 macrophage cells were treated with copper, manganese, zinc, or cobalt at 10, 50, or 100 µM for 16 to 24 h. Then, FPN1 mRNA and protein levels were determined by quantitative real-time PCR and Western blot analyses, respectively. In addition, effects of divalent metals on FPN1 promoter activity were examined by luciferase reporter assays. Results showed that copper significantly increased FPN1 mRNA levels in a dose-dependent manner. The copper-induced expression of FPN1 mRNA was associated with a corresponding increase in FPN1 protein levels. Also, copper directly stimulated the activity of FPN1 promoter-driven reporter construct. In contrast, manganese and zinc had no effect on the FPN1 gene expression in J774 cells. Interestingly, cobalt treatment in J774 cells decreased FPN1 protein levels without affecting FPN1 mRNA levels. In conclusion, our study results demonstrate that divalent metals differentially regulate FPN1 expression in macrophages and indicate a potential interaction of divalent metals with the FPN1-mediated iron export in macrophages