Site Cys Research Articles

SPEAR: A proteomics approach for simultaneous protein expression and redox analysis

Oxidation and reduction of protein cysteinyl thiols serve as molecular switches, which is considered the most central mechanism for redox regulation of biological processes, altering protein structure, biochemical activity, subcellular localization, and binding affinity. Redox proteomics allows global identification of redox-modified cysteine (Cys) sites and quantification of their reversible oxidation/reduction responses, serving as a hypothesis-generating platform to stimulate redox biology mechanistic research. Here, we developed Simultaneous Protein Expression and Redox (SPEAR) analysis, a new redox-proteomics approach based on differential labeling of reversibly oxidized and reduced cysteines with light and heavy isotopic forms of commercially available isotopically-labeled N-ethylmaleimide (NEM). The presented method does not require enrichment for labeled peptides, thus enabling simultaneous quantification of Cys reversible oxidation state and protein abundance. Using SPEAR, we were able to quantify the in-vivo reversible oxidation state of thousands of cysteines across the Arabidopsis proteome under steady-state and oxidative stress conditions. Functional assignment of the identified redox-sensitive proteins demonstrated the widespread effect of oxidative conditions on various cellular functions and highlighted the enrichment of chloroplastic proteins. SPEAR provides a simple, straightforward, and cost-effective means of studying redox proteome dynamics. The presented data provide a global quantitative view of the reversible oxidation of well-known redox-regulated active sites and many novel redox-sensitive sites whose role in plant acclimation to stress conditions remains to be further explored.

Structural basis for UFM1 transfer from UBA5 to UFC1

Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

Mass spectrometry-based direct detection of multiple types of protein thiol modifications in pancreatic beta cells under endoplasmic reticulum stress

Thiol-based post-translational modifications (PTMs) play a key role in redox-dependent regulation and signaling. Functional cysteine (Cys) sites serve as redox switches, regulated through multiple types of PTMs. Herein, we aim to characterize the complexity of thiol PTMs at the proteome level through the establishment of a direct detection workflow. The LC-MS/MS based workflow allows for simultaneous quantification of protein abundances and multiple types of thiol PTMs. To demonstrate its utility, the workflow was applied to mouse pancreatic β-cells (β-TC-6) treated with thapsigargin to induce endoplasmic reticulum (ER) stress. This resulted in the quantification of >9000 proteins and multiple types of thiol PTMs, including intra-peptide disulfide (S–S), S-glutathionylation (SSG), S-sulfinylation (SO2H), S-sulfonylation (SO3H), S-persulfidation (SSH), and S-trisulfidation (SSSH). Proteins with significant changes in abundance were observed to be involved in canonical pathways such as autophagy, unfolded protein response, protein ubiquitination pathway, and EIF2 signaling. Moreover, ~500 Cys sites were observed with one or multiple types of PTMs with SSH and S–S as the predominant types of modifications. In many cases, significant changes in the levels of different PTMs were observed on various enzymes and their active sites, while their protein abundance exhibited little change. These results provide evidence of independent translational and post-translational regulation of enzyme activity. The observed complexity of thiol modifications on the same Cys residues illustrates the challenge in the characterization and interpretation of protein thiol modifications and their functional regulation.

Discovery of a Fluorogenic Probe for In Situ Pyruvate Kinase M2 Isoform (PKM2) Labeling through Chemoselective SNAr with a Binding Site Lysine Residue.

The key challenge of developing reaction-based turn-on probes is to establish latent electrophilic fluorophores exhibiting high reactivity only upon binding to a specific protein(s). Herein, we identified such a fluorophore, 6-arylthioether-substituted 3-cyano-1-oxo-1H-phenalene-2-carboxylate, which chemoselectively labels binding site Cys or Lys residues. Based on this fluorophore, we developed the first reaction-based turn-on pyruvate kinase M2 isoform (PKM2) fluorescent probe AT-OPC1, which selectively labels PKM2 with the binding site Lys305. The latent electrophilic reactivity of the fluorophore endows the probe with precise detection of the expression of PKM2 in situ by means of both in-gel fluorescence imaging at the proteome level and real-time no-wash cell imaging approaches, which has the potential to be applied in cancer diagnoses.

Disulfide reductase activity of thioredoxin-h2 imparts cold tolerance in Arabidopsis

Many thioredoxin-h (Trx-h) proteins, cytosolic isotypes of Trxs, have been functionally characterized in plants; however, the physiological function of Arabidopsis Trx-h2, which harbors two active site cysteine (Cys) residues and an N-terminal extension peptide containing a fatty acid acylation site, remains unclear. In this study, we investigated the physiological function of Trx-h2 by performing several abiotic stress treatments using trx-h1–3 knockout mutant lines, and found that the reductase function of Trx-h2 is critical for cold resistance in Arabidopsis. Plants overexpressing Trx-h2 in the trx-h2 mutant background (Trx-h2OE/trx-h2) showed strong cold tolerant phenotypes compared with Col-0 (wild type) and trx-h2 mutant plants. By contrast, Trx-h2(C/S)OE/trx-h2 plants expressing a variant Trx-h2 protein, in which both active site Cys residues were substituted by serine (Ser) residues, showed high cold sensitivity, similar to trx-h2 plants. Moreover, cold-responsive (COR) genes were highly up-regulated in Trx-h2OE/trx-h2 plants but not in trx-h2 and Trx-h2(C/S)OE/trx-h2 plants under cold conditions. These results explicitly suggest that the cytosolic Trx-h2 protein relays the external cold stress signal to downstream cold defense signaling cascades through its protein disulfide reductase function.

Stomatal regulation: Role of H2S-induced persulfidation in ABA signaling

Stomatal regulation: Role of H2S-induced persulfidation in ABA signaling

Structural and Kinetic Characterization of Hyperthermophilic NADH-Dependent Persulfide Reductase from Archaeoglobus fulgidus.

NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H2S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S0 and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting kcat/Km on the order of 104 M−1 s−1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2′-phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S0 or serve in another metabolic role that has yet to be identified.

Altered protein S-glutathionylation depicts redox imbalance triggered by transition metal oxide nanoparticles in a breastfeeding system

Nanosafety has become a public concern following nanotechnology development. By now, attention has seldom been paid to breastfeeding system, which is constructed by mammary physiological structure and derived substances (endogenous or exogenous), cells, tissues, organs, and individuals (mother and child), connecting environment and organism, and spans across mother-child dyad. Thus, breastfeeding system is a center of nutrients transport and a unique window of toxic susceptibility in the mother-child dyad. We applied metabolomics combined with redox proteomics to depict how nanoparticles cause metabolic burden via their spontaneous redox cycling in lactating mammary glands. Two widely used nanoparticles [titanium dioxide (nTiO2) and zinc oxide (nZnO)] were exposed to lactating mice via intranasal administration. Biodistribution and biopersistence of nTiO2 and nZnO in mammary glands destroyed its structure, reflective of significantly reduced claudin-3 protein level by 32.1% (P < 0.01) and 47.8% (P < 0.01), and significantly increased apoptosis index by 85.7 (P < 0.01) and 100.3 (P < 0.01) fold change, respectively. Airway exposure of nTiO2 trended to reduced milk production by 22.7% (P = 0.06), while nZnO significantly reduced milk production by 33.0% (P < 0.01). Metabolomics analysis revealed a metabolic shift by nTiO2 or nZnO, such as increased glycolysis (nTiO2: fold enrichment = 3.31, P < 0.05; nZnO: fold enrichment = 3.68, P < 0.05), glutathione metabolism (nTiO2: fold enrichment = 5.57, P < 0.01; nZnO: fold enrichment = 4.43, P < 0.05), and fatty acid biosynthesis (nTiO2: fold enrichment = 3.52, P < 0.05; nZnO: fold enrichment = 3.51, P < 0.05) for tissue repair at expense of lower milk fat synthesis (35.7% reduction by nTiO2; 51.8% reduction by nZnO), and finally led to oxidative stress of mammary glands. The increased GSSG/GSH ratio (57.5% increase by nTiO2; 105% increase by nZnO) with nanoparticle exposure confirmed an alteration in the redox state and a metabolic shift in mammary glands. Redox proteomics showed that nanoparticles induced S-glutathionylation (SSG) modification at Cys sites of proteins in a nanoparticle type-dependent manner. The nTiO2 induced more protein SSG modification sites (nTiO2: 21; nZnO:16), whereas nZnO induced fewer protein SSG modification sites but at deeper SSG levels (26.6% higher in average of nZnO than that of nTiO2). In detail, SSG modification by nTiO2 was characterized by Ltf at Cys423 (25.3% increase), and Trf at Cys386;395;583 (42.3%, 42.3%, 22.8% increase) compared with control group. While, SSG modification by nZnO was characterized by Trfc at Cys365 (71.3% increase) and Fasn at Cys1010 (41.0% increase). The discovery of SSG-modified proteins under airway nanoparticle exposure further supplemented the oxidative stress index and mammary injury index, and deciphered precise mechanisms of nanotoxicity into a molecular level. The unique quantitative site-specific redox proteomics and metabolomics can serve as a new technique to identify nanotoxicity and provide deep insights into nanoparticle-triggered oxidative stress, contributing to a healthy breastfeeding environment.

Formation of Co(II), Ni(II), Zn(II) complexes of alternative metal binding heptapeptides and nitrilotriacetic acid: Discovering new potential affinity tags

Using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) a new series of alternative metal binding-5 (amb5) heptapeptides that provide various combinations of His, Cys and Asp as potential metal binding sites are compared for their relative formation of complexes with the metal ions Co(II), Ni(II) and Zn(II). These amb5 peptides have the general primary structure acetyl-Aa1-Aa2-Gly3-Pro4-Tyr5-Aa6-Aa7, where Aa1,2,6,7 are the four amino acids that include the His, Cys, and/or Asp potential metal binding sites. The study includes two polyhistidine heptapeptides; the His-tag and His-tag with Pro4, because they represent the widely applied His affinity tag used in expression cloning and purification of recombinant proteins with immobilized metal affinity chromatography (IMAC). We compare these amb5 heptapeptides for their formation of the amb5+M(II) and amb5+M(II)+NTA complexes, where M = Co, Ni, or Zn, and NTA is nitrilotriacetic acid as a model for the binding of amb5 in a IMAC column. Overall, the results show that the inclusion of Asp at positions 1, 2, and 7; i.e., ac-Asp1-Asp2-Gly3-Pro4-Tyr5-Aa6-Asp7, generally favors the formation of amb5+Ni(II) and amb5+Ni(II)+NTA complexes, while the inclusion of Cys at positions 2 and 6; i.e., ac-Aa1-Cys2-Gly3-Pro4-Tyr5-Aa6-Cys7, with Aa1 = His, Cys or Asp, favors the formation of amb5+Zn(II) complexes. The comparison with the His-tag and competition between Ni(II) and Zn(II) for forming the amb5+M(II)+NTA complexes, showed the structure ac-Asp1-Asp2-Gly3-Pro4-Tyr5-His6-Aa7, with Aa7 = Cys or Asp, were the most selective for Ni(II) and forming the amb5+Ni(II)+NTA complex. Collision-induced dissociation of the single negatively-charged [amb5+Ni(II)+NTA]− shows it competitively dissociates into [amb5−3H+Ni(II)]− or [NTA−3H+Ni(II)]− while the doubly-charged [amb5+Ni(II)+NTA]2− complex dissociates into [NTA−3H+Ni(II)]− and [amb5J−H]−, indicating these complexes are realistic mimics for the complex inside an IMAC column.

Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H2O2. This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete.

Interrogation of 3D-swapped structure and functional attributes of quintessential Sortase A from Streptococcus pneumoniae.

The anchoring of the surface proteins to the cell wall in gram-positive bacteria involves a peptide ligation reaction catalyzed by transpeptidase sortase. Most bacterial genomes encode multiple sortases with dedicated functions. Streptococcus pneumoniae (Sp) carries four sortases; a housekeeping sortase (SrtA), and three pilin specific sortases (SrtC1, C2, C3) dedicated to the biosynthesis of covalent pilus. Interestingly, SrtA, meant for performing housekeeping roles, is also implicated in pilus assembly of Sp. The allegiance of SpSrtA to the pathogenic pilus assembly makes it an ideal target for clinical inhibitor development. In this paper, we describe biochemical characterization, crystal structure and peptide substrate preference of SpSrtA. Transpeptidation reaction with a variety of substrates revealed that the enzyme preferred elongated LPXTG sequences and transferred them equally well to both Ala- and Gly-terminated peptides. Curiously, crystal structure of both wild type and an active site (Cys to Ala) mutant of SpSrtA displayed inter-twined 3D-swapped dimers in which each protomer generated a classic eight stranded beta-barrel "sortase fold". Size-exclusion chromatography and sedimentation equilibrium measurements revealed predominant presence of a dimer in equilibrium with its monomer. The crystal structure-based Cys-Cys distance mapping with defined chemical cross-linkers established the existence of 3D-swapped structure in solution. The swapping in SpSrtA, unprecedented for sortase family, may be physiologically relevant and meant to perform regulatory functions.

Isothiocyanate can be used as a highly specific recognition site for fluorescent cysteine probes

Cysteine (Cys) is an essential biothiol and amino acid molecule, playing important roles in many physiological and pathological events. As a biomarker of many diseases, Cys has important clinical significance. Accordingly, it is of great significance to develop an efficient and specific detecting method for Cys in life systems. We report herein a new approach that isothiocyanate (ITC) can be used as an effective new specific recognition site of Cys to develop effective probes for the detection of Cys in life systems. To demonstrate the effectiveness of this approach, ITC was attached to an easy-to-prepare coumarin fluorophore and a novel molecular probe (CM-NCS) was synthesized. As expected, the probe could respond rapidly, selectively, and sensitively to Cys and could be well used for the detection of Cys in live cells and zebrafish. All the results demonstrated that the new recognition site (ITC) would provide a good new choice to develop highly Cys-selective probes.

In silico allicin induced S-thioallylation of SARS-CoV-2 main protease

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused due to new coronavirus infection with 3716075 deaths across the world as reported by the World Health Organization (WHO). SARS-CoV-2 main protease (Mpro) plays a vital role in the replication of coronavirus and thus an attractive target for the screening of inhibitors for the therapy of COVID-19. The preclinical drugs ebselen and PX-12 are potent inhibitors of SARS-CoV-2 Mpro and covalently modifies the active site Cys-145 residue of Mpro through selenosulfide/disulfide. In the current report, using virtual screening methods, reactive sulfur species allicin is subjecting for covalent docking at the active site of SARS-CoV-2 Mpro using PX-12 as a benchmark reference compound. The results indicate that allicin induces dual S-thioallylation of Cys-145 and Cys-85/ Cys-156 residues of SARS-CoV-2 Mpro. Using density functional theory (DFT), Gibbs free energy change (DG) is calculated for the putative reactions between N-acetylcysteine amide thiol and allicin/allyl sulfenic acid. The overall reaction is exergonic and allyl disulfide of Cys-145 residue of Mpro is involved in a sulfur mediated hydrogen bond. The results indicate that allicin causes dual S-thioallylation of SARS-CoV-2 Mpro which may be of interest for treatment and attenuation of ongoing coronavirus infection.

Andrographolide and its fluorescent derivative inhibit the main proteases of 2019-nCoV and SARS-CoV through covalent linkage

The coronavirus disease 2019 (COVID-19) pandemic caused by 2019 novel coronavirus (2019-nCoV) has been a crisis of global health, whereas the effective vaccines against 2019-nCoV are still under development. Alternatively, utilization of old drugs or available medicine that can suppress the viral activity or replication may provide an urgent solution to suppress the rapid spread of 2019-nCoV. Andrographolide is a highly abundant natural product of the medicinal plant, Andrographis paniculata, which has been clinically used for inflammatory diseases and anti-viral therapy. We herein demonstrate that both andrographolide and its fluorescent derivative, the nitrobenzoxadiazole-conjugated andrographolide (Andro- NBD), suppressed the main protease (Mpro) activities of 2019-nCoV and severe acute respiratory syndrome coronavirus (SARS-CoV). Moreover, Andro-NBD was shown to covalently link its fluorescence to these proteases. Further mass spectrometry (MS) analysis suggests that andrographolide formed a covalent bond with the active site Cys145 of either 2019-nCoV Mpro or SARS-CoV Mpro. Consistently, molecular modeling analysis supported the docking of andrographolide within the catalytic pockets of both viral Mpros. Considering that andrographolide is used in clinical practice with acceptable safety and its diverse pharmacological activities that could be beneficial for attenuating COVID-19 symptoms, extensive investigation of andrographolide on the suppression of 2019-nCoV as well as its application in COVID-19 therapy is suggested.

S-glutathionylation proteome profiling reveals a crucial role of a thioredoxin-like protein in interspecies competition and cariogenecity of Streptococcus mutans.

S-glutathionylation is an important post-translational modification (PTM) process that targets protein cysteine thiols by the addition of glutathione (GSH). This modification can prevent proteolysis caused by the excessive oxidation of protein cysteine residues under oxidative or nitrosative stress conditions. Recent studies have suggested that protein S-glutathionylation plays an essential role in the control of cell-signaling pathways by affecting the protein function in bacteria and even humans. In this study, we investigated the effects of S-glutathionylation on physiological regulation within Streptococcus mutans, the primary etiological agent of human dental caries. To determine the S-glutathionylated proteins in bacteria, the Cys reactive isobaric reagent iodoacetyl Tandem Mass Tag (iodoTMT) was used to label the S-glutathionylated Cys site, and an anti-TMT antibody-conjugated resin was used to enrich the modified peptides. Proteome profiling identified a total of 357 glutathionylated cysteine residues on 239 proteins. Functional enrichment analysis indicated that these S-glutathionylated proteins were involved in diverse important biological processes, such as pyruvate metabolism and glycolysis. Furthermore, we studied a thioredoxin-like protein (Tlp) to explore the effect of S-glutathionylation on interspecies competition between oral streptococcal biofilms. Through site mutagenesis, it was proved that glutathionylation on Cys41 residue of Tlp is crucial to protect S. mutans from oxidative stress and compete with S. sanguinis and S. gordonii. An addition rat caries model showed that the loss of S-glutathionylation attenuated the cariogenicity of S. mutans. Taken together, our study provides an insight into the S-glutathionylation of bacterial proteins and the regulation of oxidative stress resistance and interspecies competition.

PH dependent chelation study of Zn(II) and Ni(II) by a series of hexapeptides using electrospray ionization – Ion mobility – Mass spectrometry

Presented here are the results of a study characterizing the selective metal chelating performance of the alternative metal binding (amb) peptide: acetyl-His1-Cys2-Gly3-Pro4-His5-Cys6-OH, and eight ambs with systematic modifications to His, Cys, and carboxyl C-terminus metal chelating sites. The results show that from the divalent metal ions of zinc, nickel, cobalt, magnesium and calcium, the ambs most extensively formed complexes with zinc and nickel. The ambs, which retained both Cys2 and Cys6 in their primary structure, exhibited the greatest formation of zinc complexes. The replacement of His1 and His5 residues with two additional Cys with the amidation of the C-terminus also increased the zinc chelation at pH 7.0. Density functional theory indicated that these modifications might be disrupting the hydrogen bonding between the His-Cys and carboxylate terminus making the 4Cys more available for chelation. Nickel chelation was generally lower than zinc because of competition from the Cys to form disulfide bonds in the presence of nickel. The two ambs that formed the highest number of nickel complexes both included the amidated C-termini and either 4Cys or 2Cys-2His. Comparison with recent published results of six heptapeptide ambs, which have the same primary structures, but with the inclusion of Tyr5 before the final two residues, indicates the inclusion of the Tyr5 residue increases the zinc chelation at pH 7.0 from 25%, of total observed species, to 70%. These types of studies may pave the way to discoveries of new therapeutics suitable as enzyme inhibitors or chelators for diseases associated with metal homeostasis misbalances, or as new peptide tags for recombinant protein purification.

Topological analysis of SARS CoV-2 main protease.

There is an urgent necessity of effective medication against severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), which is producing the COVID-19 pandemic across the world. Its main protease (M) represents an attractive pharmacological target due to its involvement in essential viral functions. The crystal structure of free M shows a large structural resemblance with the main protease of SARS CoV (nowadays known as SARS CoV-1). Here, we report that average SARS CoV-2 M is 1900% more sensitive than SARS CoV-1 M in transmitting tiny structural changes across the whole protein through long-range interactions. The largest sensitivity of M to structural perturbations is located exactly around the catalytic site Cys-145 and coincides with the binding site of strong inhibitors. These findings, based on a simplified representation of the protein as a residue network, may help in designing potent inhibitors of SARS CoV-2 M.

Modulation of the Metal(loid) Specificity of Whole-Cell Bioreporters by Genetic Engineering of ZntR Metal-Binding Loops.

Bacterial cell-based biosensors, or whole-cell bioreporters (WCBs), are an alternative tool for the quantification of hazardous materials. Most WCBs share similar working mechanisms. In brief, the recognition of a target by sensing domains induces a biological event, such as changes in protein conformation or gene expression, providing a basis for quantification. WCBs targeting heavy metal(loid)s employ metalloregulators as sensing domains and control the expression of genes in the presence of target metal(loid) ions, but the diversity of targets, specificity, and sensitivity of these WCBs are limited. In this study, we genetically engineered the metal-binding loop (MBL) of ZntR, which controls the znt-operon in Escherichia coli. In the MBL of ZntR, three Cys sites interact with metal ions. Based on the crystal structure of ZntR, MBL sequences were modified by sitedirected mutagenesis. As a result, the metal-sensing properties of WCBs differed depending on amino acid sequences and the new selectivity to Cr or Pb was observed. Although there is room for improvement, our results support the use of currently available WCBs as a platform to generate new WCBs to target other environmental pollutants including metal(loid)s.

How the Local Environment of Functional Sites Regulates Protein Function.

Proteins form complex biological machineries whose functions in the cell are highly regulated at both the cellular and molecular levels. Cellular regulation of protein functions involves differential gene expressions, post-translation modifications, and signaling cascades. Molecular regulation, on the other hand, involves tuning an optimal local protein environment for the functional site. Precisely how a protein achieves such an optimal environment around a given functional site is not well understood. Herein, by surveying the literature, we first summarize the various reported strategies used by certain proteins to ensure their correct functioning. We then formulate three key physicochemical factors for regulating a protein's functional site, namely, (i) its immediate interactions, (ii) its solvent accessibility, and (iii) its conformational flexibility. We illustrate how these factors are applied to regulate the functions of free/metal-bound Cys and Zn sites in proteins.

Hydrogen Sulfide Positively Regulates Abscisic Acid Signaling through Persulfidation of SnRK2.6 in Guard Cells

The phytohormone abscisic acid (ABA) plays pivotal roles in triggering stomatal closure and facilitating adaptation of plants to drought stress. Hydrogen sulfide (H2S), a small signaling gas molecule, is involved in ABA-dependent stomatal closure. However, how H2S regulates ABA signaling remains largely unclear. Here, we show that ABA induces the production of H2S catalyzed by L-CYSTEINE DESULFHYDRASE1 (DES1) in guard cells, and H2S in turn positively regulates ABA signaling through persulfidation of Open Stomata 1 (OST1)/SNF1-RELATED PROTEIN KINASE2.6 (SnRK2.6). Two cysteine (Cys) sites, Cys131 and Cys137, which are exposed on the surface of SnRK2.6 and close to the activation loop, were identified to be persulfidated, which promotes the activity of SnRK2.6 and its interaction with ABA response element-binding factor 2 (ABF2), a transcription factor acting downstream of ABA signaling. When Cys131, Cys137, or both residues in SnRK2.6 were substituted with serine (S), H2S-induced SnRK2.6 activity and SnRK2.6–ABF2 interaction were partially (SnRK2.6C131S and SnRK2.6C137S) or completely (SnRK2.6C131SC137S) compromised. Introduction of SnRK2.6C131S, SnRK2.6C137S, or SnRK2.6C131SC137S into the ost1-3 mutant could not rescue the mutant phenotype: less sensitivity to ABA- and H2S-induced stomatal closure and Ca2+ influx as well as increased water loss and decreased drought tolerance. Taken together, our study reveals a novel post-translational regulatory mechanism of ABA signaling whereby H2S persulfidates SnRK2.6 to promote ABA signaling and ABA-induced stomatal closure.