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YeeD is an essential partner for YeeE-mediated thiosulfate uptake in bacteria and regulates thiosulfate ion decomposition [1]

['Mai Ikei', 'Division Of Biological Science', 'Graduate School Of Science', 'Technology', 'Nara Institute Of Science', 'Ikoma', 'Nara', 'Ryoji Miyazaki', 'Keigo Monden', 'Yusuke Naito']

Date: 2024-05

Abstract Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving a membrane protein YeeE (TsuA) in Escherichia coli was characterized. YeeE-like proteins are conserved in some bacteria, archaea, and eukaryotes. However, the precise function of YeeE, along with its potential partner protein in the thiosulfate ion uptake pathway, remained unclear. Here, we assessed selective thiosulfate transport via Spirochaeta thermophila YeeE in vitro and characterized E. coli YeeD (TsuB) as an adjacent and essential protein for YeeE-mediated thiosulfate uptake in vivo. We further showed that S. thermophila YeeD possesses thiosulfate decomposition activity and that a conserved cysteine in YeeD was modified to several forms in the presence of thiosulfate. Finally, the crystal structures of S. thermophila YeeE-YeeD fusion proteins at 3.34-Å and 2.60-Å resolutions revealed their interactions. The association was evaluated by a binding assay using purified S. thermophila YeeE and YeeD. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.

Citation: Ikei M, Miyazaki R, Monden K, Naito Y, Takeuchi A, Takahashi YS, et al. (2024) YeeD is an essential partner for YeeE-mediated thiosulfate uptake in bacteria and regulates thiosulfate ion decomposition. PLoS Biol 22(4): e3002601. https://doi.org/10.1371/journal.pbio.3002601 Academic Editor: Lotte Søgaard-Andersen, Max Planck Institute for Terrestrial Microbiology: Max-Planck-Institut fur terrestrische Mikrobiologie, GERMANY Received: January 25, 2024; Accepted: March 26, 2024; Published: April 24, 2024 Copyright: © 2024 Ikei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 8J4C and 8K1R. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Funding: This work was supported by JSPS/MEXT KAKENHI (Grant No. JP21H05157 to T.M., Grant Nos. JP22K15075, JP20K15733 to Mu.I, Grant No. JP22K15061, JP22H05567 to R.M., and Grant Nos. JP22H02567, JP22H02586, JP21H05155, JP21H05153, JP21K19226, JP21KK0125 to T.T.), MEXT as “Program for Promoting Researches on the Supercomputer Fugaku” (Development and application of large-scale simulation-based inferences for biomolecules JPMXP1020230119) and HPCI project (hp200064 and hp230209) to T.M., PRESTO (JPMJPR20E1 to Mu.I) from the Japan Science and Technology Agency (JST), and private research foundations (the Chemo-Sero-Therapeutic Research Institute, Naito Foundation, Takeda Science Foundation, G-7 Scholarship Foundation, the Sumitomo Foundation, the Institute for Fermentation (Osaka), Yamada Science Foundation, KOSÉ Cosmetology Research Foundation, and Japan Foundation for Applied Enzymology) to T.T. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: BLI, biolayer interferometry; DDM, dodecyl β-maltoside; DTT, dithiothreitol; IAA, iodoacetamide; IPTG, isopropyl β-D-thiogalactopyranoside; LCP, lipidic cubic phase; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MD, molecular dynamics; MS, mass spectrometry; NEM, N-Ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; SA, sinapinic acid; SSM, solid-supported membrane

Introduction From bacteria to eukaryotes, sulfur is a vital element for cellular activities. For example, sulfur-containing biomolecules, such as L-cysteine, L-methionine, thiamine, glutathione, and biotin, play a variety of essential roles in cells [1]. Bacteria and plants can utilize L-cysteine as a source of sulfate, but they also have sulfur assimilation pathways to synthesize L-cysteine from inorganic sulfur compounds. In bacteria, L-cysteine is important not only as a component for protein synthesis but also as a reducing agent against oxidative stress [2]. There are 2 pathways for bacterial L-cysteine synthesis, which use O-acetylserine as a precursor, called the sulfate and thiosulfate pathways [3]. In the sulfate pathway, sulfate ion is first decomposed into sulfide ion through phosphorylation by 2 molecules of ATP and reduction by 4 molecules of NADPH. Subsequently, L-cysteine is synthesized from sulfide ion and O-acetylserine by O-acetylserine sulfhydrylase-A (CysK). In the thiosulfate pathway, S-sulfocysteine is synthesized from thiosulfate ion and O-acetylserine by O-acetylserine sulfhydrylase-B (CysM); S-sulfocysteine is then reduced by 1 NADPH molecule, and L-cysteine is synthesized. The sulfate and thiosulfate ions used in these pathways are taken up from the environment by transporters on the cytoplasmic membrane. Sulfate and thiosulfate ions are trapped by periplasmic proteins Sbp [4] and CysP [5], respectively. Both sulfate and thiosulfate ions are then passed on to a complex formed by the inner membrane proteins CysU and CysW and the cytosolic protein CysA (CysUWA) (also called CysTWA) [3] and transported into the cytoplasm by the CysUWA complex using the energy from ATP hydrolysis. A bacterial membrane protein, YeeE, has been identified as mediating thiosulfate uptake based on growth complementation assay of Escherichia coli cells, and the structure of Spirochaeta thermophila YeeE (StYeeE) has been determined by X-ray crystallography [6]. YeeE was proposed to be almost entirely buried in the membrane and shows a unique hourglass-like structure. An electron density near a conserved cysteine residue on the outside surface was assigned to a thiosulfate ion, which seemed to be connected to the sulfur atom of the cysteine via a S─H─S hydrogen bond [6]. In addition, the binding of thiosulfate ions to purified YeeE protein was shown by isothermal titration calorimetry experiments. In the center of YeeE, 3 conserved, functionally important cysteines, including the thiosulfate-interacting one, are arranged side by side and perpendicular to the membrane. Based on these structural features and in vivo functional analysis using a series of mutants, thiosulfate ion was proposed to be transported from the environment to the cytoplasm while transiently interacting with the 3 conserved cysteine residues by relays mediated via hydrogen bonds [6], independently of the thiosulfate pathway described above. However, it has not been elucidated whether YeeE alone transports thiosulfate ions. Moreover, no YeeE-associated protein has been clearly defined, although 1 candidate gene is yeeD, which resides within the same operon that includes yeeE [7], and the regulatory mechanism of the alternative, sophisticated thiosulfate uptake by YeeE remains unclear. Recently, in addition to the E. coli yeeE gene, yeeD was shown to be involved in thiosulfate uptake and the two were named tsuA and tsuB, respectively [8]. YeeD is a cytoplasmic protein that belongs to the TusA (tRNA 2-thiouridine synthesizing protein A) protein family [9]. TusA plays various roles in sulfate transfer activities in cells, such as thiomodification of tRNA [10], molybdenum cofactor biosynthesis [11], and dissimilatory sulfur and tetrathionate oxidation [12]. The cysteine residue in a Cys-Pro-X-Pro (CPxP) motif of TusA receives activated sulfur from the L-cysteine desulfurase IscS [13]. Although YeeD possesses the CPxP motif, it cannot complement TusA function [11]. Therefore, YeeD is thought to have a sulfate-related yet distinct function from TusA in bacteria. In some bacteria, such as gram-positive Corynebacterium species YeeE and YeeD are encoded as 1 polypeptide, implying that they function together in the thiosulfate uptake pathway. However, the enzymatic activity of YeeD and the functional cooperativity of YeeE and YeeD have not been well characterized. In this study, first, we measured thiosulfate uptake activity using StYeeE-reconstituted liposomes to clarify YeeE function. Second, according to an E. coli growth complementation assay, we found that E. coli YeeD (EcYeeD) plays an essential role in YeeE function in vivo. Third, we demonstrated a thiosulfate decomposition activity in purified S. thermophila YeeD (StYeeD). Fourth, direct interaction between StYeeE and StYeeD proteins was detected, and substrate thiosulfate ions weakened the binding of these proteins. Fifth, the crystal structures of StYeeE–YeeD complex were determined at 3.34 Å and 2.60 Å resolutions. Critical residues of YeeD for both its activity and interaction with YeeE were defined. Based on these results, detailed mechanisms for the functional cooperation between YeeE and YeeD in thiosulfate uptake are discussed.

Discussion By the growth complementation assay, we have demonstrated that YeeD is a required enzyme for growth when thiosulfate is solely imported via YeeE. YeeD’s substrate was identified as the thiosulfate ion, and the uptake activity for thiosulfate ions was measured using YeeE-reconstituted liposomes. In addition, the interaction between YeeE and YeeD was revealed by both binding assay and X-ray crystallography. By mutational analysis of YeeD, a conserved cysteine residue was found to be critical for YeeE’s activity in vivo as well as thiosulfate ion decomposition activity. Compared with other TusA family proteins, YeeD shows some similarities and differences. Although conserved cysteine residues in other TusA family proteins, corresponding to C17 in StYeeD, are important for their own activities [10,20], TusA could not decompose thiosulfate ions [24]. Metallosphaera cuprina TusA was reported to exist as both monomer and dimer forms [12]. Similarly, StYeeD appeared to be monomers and dimers in gel filtration (S2C Fig). StYeeD may exploit this feature to transiently form a dimer in the thiosulfate decomposition reaction (discussed below). The conserved cysteine residues in YeeD and TusA are important for binding to their respective partners (Fig 4) [12,18], which seems to be a common feature in TusA family proteins. Meanwhile, YeeD is unique in that its interaction partner, YeeE, is a membrane protein, unlike other TusA family proteins. Previously, the conserved cysteine residue of PA1006 was reported to be persulfide-modified [20]. Furthermore, StYeeD has several different sulfur-related modification statuses, including persulfide modification, perthiosulfonic acid, and thiosulfonate (Fig 2D and 2F–2H). In the case of DsrE3 and TusA, the conserved cysteines can possess thiosulfonate, but the modifications were detected only with their substrate tetrathionate [12]. Along with the fact that YeeE only allows thiosulfate to pass through, YeeD also seems to be specialized for thiosulfate reactivity. Both can contribute to the efficient uptake of thiosulfate. Since YeeE alone can transport thiosulfate ions but was unable to rescue the growth deficiency in vivo, cooperation between YeeE and YeeD is crucial. In this study, we have unveiled a sophisticated pathway for thiosulfate uptake in the bacterial membrane. An open question is whether there are other proteins that directly receive sulfur compounds from YeeD in sulfur assimilation pathways. Based on our findings, we propose detailed mechanisms for thiosulfate ion uptake by YeeE and YeeD (S6A Fig). From the previous crystal structure of StYeeE, the transportation of thiosulfate ions across the membrane was proposed to be relayed by the first-to-third cysteine residues [6]. Our crystal structure of the StYeeE and StYeeD complex now further shows that YeeD is positioned to interact with the YeeE cytoplasmic side, which we propose to be the exit of the thiosulfate pathway on YeeE; the conserved cysteine residue of YeeD can function as a “fourth cysteine.” Therefore, the interactions between the two can facilitate the delivery of a thiosulfate ion from YeeE to YeeD: The conserved cysteine residue of YeeD can directly capture a thiosulfate ion passed through YeeE. This transport scheme of thiosulfate ions by YeeE and YeeD recalls a group-translocation originally proposed by Mitchell and Moyle [25]. From our X-ray crystal structure, YeeE and YeeD appear to work as a 1:1 complex. The fact that YeeE and YeeD are encoded as a single polypeptide in some species supports this idea. After receiving the thiosulfate ion, YeeD’s conserved cysteine would be thiosulfonated. Our BLI method analysis in the presence of thiosulfate showed that the interaction between StYeeD and StYeeE was weakened, possibly due to thiosulfonation on YeeD. The thiosulfonated YeeD may therefore dissociate from YeeE and be released into the cytoplasm. This dissociation model agrees well with our MD simulation results. Since YeeD can decompose thiosulfate by itself, it would do so after dissociating from YeeE. Of additional note, since the existence of StYeeD dimers in solution was suggested by gel filtration chromatography, StYeeD may form a transient dimer after being released from StYeeE. We suggest a plausible mechanism for decomposition of thiosulfate ion by YeeD (S6B Fig). While the reducing power for this series of reactions is thought to be supplied from the cytoplasm, a reducing environment, the electron route remains unknown and needs to be elucidated in a future study. In the presence of thiosulfate ion, StYeeD incorporated thiosulfate, but StYeeD(C17A) did not (Fig 2E and 2G), suggesting that the thiosulfate ion directly binds to the cysteine residue of YeeD. Our MS results also suggest that there are StYeeD molecules with perthiosulfonic acid (-S-SO 3 -) and persulfide modifications (-S-S-). Taking this together with the detection of H 2 S in the presence of StYeeD and thiosulfate ion (Fig 2H), it would be reasonable for sulfide ion (S2-) to be released from a persulfide-modified version of StYeeD (-S-S-) (S6B Fig). Although sulfonic acid (-SO 3 -) formation by the oxidization of a cysteine residue is an irreversible process, perthiosulfonic oxidization of a cysteine residue (-S-SO 3 -) is reversible as shown by Doka and colleagues [19]. Therefore, even if -S-SO 3 - is formed, sulfite ion (SO 3 2-) will be released. In our experimental condition for the thiosulfate decomposition assay, reductant β-ME is present to facilitate these reactions. In the cell, there could be other acceptor proteins that take S or SO 3 atoms from YeeD protein. Such proteins should be identified in a future study. While the significance of the StYeeD dimer in this study has not been demonstrated, if it were involved in this series of reactions, the scheme depicted in the S6B Fig box would likely occur. Because StYeeD does not form disulfide bonds in its dimer (S2C and S2D Fig), 1 StYeeD molecule in a dimer can be thiosulfonated and the other can have an active -SH group, which can take the SO 3 from thiosulfonated YeeD (-S-S 2 O 3 -). Through this process, a persulfide-modified form (-S-S-) can be generated. In some bacteria, including E. coli, YeeD has 2 cysteine residues. In species having YeeD with 2 cysteine residues, the abovementioned process could be carried out by 1 YeeD molecule using these 2 cysteine residues. Another possibility is that the nonconserved cysteine (C39) of EcYeeD may not serve as the decomposition active center but may interact with YeeE and/or enhance its activity. In agreement with these ideas, our growth complementation assay showed that both cysteines are important for the growth of E. coli cells. In the 2.60-Å structure of StYeeE-StYeeD, the electron density is faint on the N-terminal side of the CPxP motif, raising the possibility that some electrons may be attracted to the CPxP motif (Fig 3D). If that happens, the CPxP motif could make the region electron-rich and exhibit high reducing power. Previously, the CPxP architecture was proposed to stabilize an α-helix-1 by capping it [17]. In the TusA family proteins, the formation of CPxP may also provide high reducing power, conferring on them the unique property of selective decomposition of their substrates. In conclusion, we revealed that YeeE and YeeD cooperatively contribute to the thiosulfate uptake pathway and possess unprecedented regulation mechanisms. Our findings also deepen the understanding of the functions of TusA family proteins.

Materials and methods Strains and plasmids For the growth complementation assay, we used plasmids derived from pAZ061, which is based on pET-16b-TEV [26] and possesses E. coli yeeE and yeeD genes between BamHI and XhoI sites amplified from E. coli genomic DNA (JCM 20135, RIKEN BRC) using primer set 5′-AAATTTATATTTTCAAGGATCCCATATGTTTTCAATGATATTAAG-3′ and 5′-GGCTTTGTTAGCAGCCCTCGAGTCAGGCTTTTTGAACGG-3′ (S2A Fig), and the E. coli MG1655 ΔcysPUWA ΔyeeE::kan (DE3) strain, which cannot grow on a minimum medium with thiosulfate ion as the single sulfur source [6]. pAZ061 derivatives having mutations in the yeeD region were prepared by site-directed mutagenesis. The DNA sequence encoding EcYeeD was deleted from pAZ061 using primers 5′-GTTTGGGTTAGGCATCGCTTCCCCAACGGCC-3′ and 5′-GGCCGTTGGGGAAGCGATGCCTAACCCAAAC-3′. The plasmid used to express StYeeE (1-328aa, UniProt ID: G0GAP6) with C-terminal GSSGENLYFQGEDVE-His 8 sequence (pKK550) was prepared as in [6]. For the expression of the StYeeE-StYeeD fusion protein, we modified pKK550; the resulting plasmid, pAZ150, encodes StYeeE (1–330 aa)-AATPTPVAEAAPSSAEDRVLPFQVATGAVALQTAPRVKKA-StYeeD (1–80 aa, UniProt ID: G0GAP7)-GSSGENLYFQGEDVE-His 6 . The mutations in the StYeeE and StYeeD regions were introduced by site-directed mutagenesis. For the expression of StYeeD, the DNA sequence encoding StYeeD (1–80 aa) was inserted into a modified pCGFP-BC plasmid. The resulting plasmid, pNT015, expresses StYeeD (1–80 aa)-GSSGENLYFQGEDVE-His 6. The pNT015 derivatives were prepared by Gene Synthesis and Mutagenesis (SC1441, GenScript) or site-directed mutagenesis. Growth complementation tests of E. coli cells E. coli MG1655 ΔcysPUWA ΔyeeE::kan (DE3) cells harboring pAZ061 or its derivatives were cultured in LB medium containing 50 μg/ml ampicillin and 25 μg/ml kanamycin for 16 h at 37°C. The culture was diluted 100-fold and cultured for another 8 h at 37°C, after which the cells were precipitated by centrifugation and washed twice with S-free medium (42 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 8.6 mM NaCl, 19 mM NH 4 Cl, 1 mM MgCl 2 , 0.2% (w/v) glucose, 0.01% (w/v) thiamine hydrochloride, and 0.1 mM CaCl 2 ). Subsequent passage cultures in S-free medium containing 500 μM Na 2 S 2 O 3 were started at OD 600 = 0.2. The ΔOD 600 of the culture was measured every 30 min with OD-Monitor C&T (TAITEC) for 24 h. Measurement was performed 3 times for each transformant. Protein expression and purification StYeeE and StYeeE(C22A)-StYeeD proteins were purified as follows. E. coli C41(DE3) cells transformed with plasmids expressing these proteins were cultured in 2.5 l of LB medium containing 50 μg/ml ampicillin until the OD 600 reached 0.4. Isopropyl β-D-thiogalactopyranoside (IPTG) was then added to 1 mM, and the cells were cultured at 30°C for 17 h. Subsequent procedures were performed at 4°C or on ice. The cells were collected by centrifugation as pellets, suspended in a buffer (10 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM EDTA-Na (pH 8.0), 2 mM Na 2 S 2 O 3 , 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM β–mercaptoethanol (β-ME)), and disrupted with a Microfluidizer Processor M-110EH (Microfluidics International). The suspension was centrifuged (10,000 rpm for 20 min, himac R13A rotor), and the collected supernatant was further ultracentrifuged (40,000 rpm for 60 min, Beckman 45Ti rotor) to obtain the membrane fraction, which was flash-frozen in liquid nitrogen and stored at −80°C until purification. The membrane fraction was resuspended in solubilization buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM Na 2 S 2 O 3 , 10 mM imidazole-HCl (pH 8.0), 5% (v/v) glycerol, 1 mM β-ME, 0.1 mM PMSF) containing 1% (w/v) n-dodecyl β-maltoside (DDM) and stirred at 4°C for 60 min. The insoluble fraction was removed by ultracentrifugation (45,000 rpm for 30 min, Beckman 70Ti rotor), and the supernatant was mixed with 5 ml of Ni-NTA agarose (QIAGEN) pre-equilibrated with solubilization buffer for 60 min. Then, 100 ml of wash buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM Na 2 S 2 O 3 , 50 mM imidazole-HCl (pH 8.0), 5% (v/v) glycerol, 1 mM β-ME, 0.1 mM PMSF, 0.1% (w/v) DDM) was added to the column. Next, 5 ml of elution buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM Na 2 S 2 O 3 , 200 mM imidazole-HCl (pH 8.0), 5% (v/v) glycerol, 1 mM β-ME, 0.1 mM PMSF, and 0.1% (w/v) DDM) was added to the column 6 times, and the fractions with target proteins were pooled. To remove the His-tag, TEV(S219V) protease [27] was added with a protein weight ratio of 10 (proteins):1 (TEV) and the protein solution was dialyzed overnight against dialysis buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM Na 2 S 2 O 3 , 0.1% (w/v) DDM, 1 mM β-ME). Ni-NTA agarose (2.5 ml) pre-equilibrated with dialysis buffer was added to the sample and stirred for 60 min to remove TEV proteases. The flow-through fraction was collected, concentrated with an Amicon Ultra 50K NMWL (Merck Millipore), and ultracentrifuged (45,000 rpm for 30 min, himac S55A2 rotor). The sample was then applied to a Superdex 200 increase 10/300 GL column (Cytiva) equilibrated with a buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM Na 2 S 2 O 3 , 0.1% (w/v) DDM, 1 mM β-ME). The fractions with target proteins were collected and pooled. StYeeE(C22A)-StYeeD(L45A) protein was purified as for StYeeE and StYeeE(C22A)-YeeD except that the buffer lacked Na 2 S 2 O 3 . StYeeD purification was performed as follows. E. coli C41(DE3) cells containing StYeeD-encoding plasmids were inoculated into 25 ml of LB medium containing 50 μg/ml ampicillin and cultured overnight at 37°C. The culture was then added to 0.5 l of LB medium containing 50 μg/ml ampicillin and allowed to grow until OD 600 = 0.4. After the addition of IPTG to 1 mM, the cells were cultured at 30°C for 17 h. The cells were collected by centrifugation (4,500 rpm for 10 min, himac R9A2), flash-frozen in liquid nitrogen, and stored at −80°C until use. The next procedures were performed at 4°C or on ice. The frozen pellets were suspended in 25 ml of sonication buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole-HCl (pH 8.0), 1 mM β-ME, 0.1 mM PMSF), sonicated for 10 min on ice with a Q500 sonicator (QSONICA), and centrifuged (15,000 rpm for 30 min, himac R13A). Ni-NTA agarose (2.5 ml) pre-equilibrated with the sonication buffer was added to the supernatant and rotated for 1 h. After the flow-through fraction was removed, the resin was washed with 125 ml of a buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole-HCl (pH 8.0), 1 mM β-ME, 0.1 mM PMSF). Next, 5 ml of a buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 200 mM imidazole-HCl (pH 8.0), 1 mM β-ME, 0.1 mM PMSF) was added, and the elution fraction was collected. The elution step was repeated 6 times. The fractions with target proteins were pooled and concentrated using an Amicon Ultra 3K NMWL (Merck Millipore). The sample was ultracentrifuged (45,000 rpm × 30 min, himac S55A2 rotor) and loaded onto a Superdex 200 increase 10/300 GL column pre-equilibrated with YeeD gel filtration buffer (10 mM Tris-HCl (pH 8.0) and 300 mM NaCl). The fractions with StYeeD proteins were concentrated using an Amicon Ultra 3K NMWL. MALDI-TOF MS For StYeeD(WT) sample preparation for MS (Fig 2D), StYeeD(WT) protein was mixed with gel filtration buffer, so that the final concentration of StYeeD(WT) was 48.2 μM and the total volume was 200 μl, and incubated for 2.5 h at 37°C. The sample was then ultracentrifuged, and the buffer was exchanged with MS buffer (10 mM Tris-HCl (pH 8.0)) using a NAP-5 Column (Cytiva). The final concentration of StYeeD(WT) for MS analysis was 17.5 μM. For StYeeD(C17A) sample preparation for MS (Fig 2E), 48.2 μM StYeeD(C17A) in gel filtration buffer was incubated with or without 500 μM thiosulfate for 2.5 h at 37°C in a total volume of 200 μl. After the incubation, the sample was ultracentrifuged, and the buffer was exchanged with MS buffer using a NAP-5 Column. The final concentrations of StYeeD(C17A) were 12.3 μM for without thiosulfate and 16.6 μM for with thiosulfate. For the DTT condition of StYeeD(WT) (Fig 2F), DTT was added to StYeeD(WT) to give final concentrations of 401 μM and 16.7 mM, respectively, and a total volume of 288 μl. The sample was then incubated for 10 min at 37°C and ultracentrifuged. A 48-μl aliquot was taken and the buffer was exchanged with MS buffer using a NAP-5 Column. The concentration of resultant DTT-treated StYeeD(WT) was 10.96 μM. For the thiosulfate or hydrogen peroxide condition of StYeeD(WT), the DTT-treated StYeeD(WT) sample was processed as follows. First, to remove DTT, the sample was applied to a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated with gel filtration buffer. Next, the sample after gel filtration was concentrated using Amicon ultra 3K NMWL. For the thiosulfate condition (Fig 2G), the sample was then mixed with thiosulfate so that the final concentrations of StYeeD(WT) and thiosulfate were 48.2 μM and 500 μM, respectively, and the total volume was 200 μl. After incubating for 2.5 h at 37°C, the sample was ultracentrifuged, and the buffer was exchanged with MS buffer using a NAP-5 Column. The final concentration of StYeeD(WT) for MS analysis was 14.0 μM. For the hydrogen peroxide condition (Fig 2H), the sample after gel filtration and concentration was mixed with hydrogen peroxide so that the final concentrations of StYeeD(WT) and hydrogen peroxide were 48.2 μM and 0.1% (w/v), respectively, and the total volume was 200 μl. After incubating for 10 min at room temperature, the sample was ultracentrifuged, and the buffer was exchanged with MS buffer using a NAP-5 Column. The final concentration of StYeeD(WT) for MS analysis was 23.8 μM. Samples for MALDI-TOF analysis were prepared by the sinapinic acid (SA) double layer method [28]. In brief, 1 μl matrix solution A (saturated solution of SA in ethanol) was deposited onto the MALDI target and allowed to dry. Matrix solution B (saturated solution of SA in TA30 solvent (1:2 (v/v) acetonitrile:0.1% (v/v) trifluoroacetic acid in water)) and protein solution were mixed at a ratio of 1:24 (v/v). Matrix solution B/protein mixture was then deposited onto the matrix spot and allowed to dry. The dried samples were analyzed by Autoflex-II (Bruker Daltonics) with a linear positive mode and acquisition mass range of 3,000 to 20,000 Da. Theoretical m/z values were calculated with the equation of m/z = (M + n) / n, where M is molecular mass and n is the charge (n = 1 was adopted). Measurement of enzyme activity of StYeeD The release of H 2 S due to chemical decomposition of thiosulfate ions by StYeeD was monitored by HSip-1 (Dojindo, SB21-10). The reaction solution contained 19.6 μM HSip-1, 24.1 μM YeeD, and 70 μM β-ME in the YeeD gel filtration buffer. As negative controls, 24.1 μM BSA was used instead of YeeD, or no protein was added to the solution. The reaction was started by adding 5 μl of 100 mM Na 2 S 2 O 3 at a 1:200 [v/v] ratio (final, 500 μM Na 2 S 2 O 3 ), and the solution was incubated at 37°C. The fluorescence of HSip-1 was measured using a fluorescence spectrophotometer (F-7000, Hitachi), at an excitation wavelength of 491 nm and an emission wavelength of 521 nm, every 30 min until 150 min. Crystallization Purified StYeeE(C22A)-StYeeD fusion protein was concentrated to 21.5 mg/ml by Amicon Ultra 50K NMWL and crystallized using the lipidic cubic phase (LCP) method, as previously performed in the case of purified StYeeE [6]. Around 15 μl of 21.5 mg/ml StYeeE(C22A)-StYeeD was mixed with 4.3 μl of a buffer (20 mM Tris-HCl (pH 8.0), 300 mM Na 2 S 2 O 3 , 0.1% (w/v) DDM, 1 mM β-ME) and incubated on ice for 20 min. Then, 16 μl of the sample was mixed with 24 μl of monoolein (M-239, Funakoshi) in an LCP syringe (Art Robbins Instruments) for 10 min. After 30 min of incubation at 20°C, 30 nl of the mixed samples were spotted onto MRC under oil crystallization plates (Hampton Research) using the Crystal Gryphon protein crystallization aliquot system (Art Robbins Instruments) with 3 μl of buffers (18 to 24% (v/v) pentaerythritol-propoxylate (5/4 PO/OH), 100 mM 2-morpholinoethanesulfonic acid (MES)-NaOH (pH 7.0), and 300 mM NaCl) covering them. The plate was incubated at 20°C for 7 d. For StYeeE(C22A)-StYeeD(L45A) fusion protein, the purified protein was concentrated to 16.5 mg/ml and crystallized using the LCP method. The crystals appeared using 0.35 M ammonium formate, 0.1 M Tris-HCl (pH 8.0 to 9.0), and 22 to 42% (v/v) 1,4-Butanediol as covering solutions. The plate was incubated at 20°C for 14 d. The crystals that appeared were harvested by Crystal Mounts and Loops (MiTegen) without using cryoprotectant, directly flash-frozen in liquid nitrogen, and stored in liquid nitrogen until X-ray diffraction experiments. Data collection and structural determination X-ray diffraction experiments of StYeeE(C22A)-StYeeD and StYeeE(C22A)-StYeeD(L45A) were performed at beamline BL32XU of SPring-8. Data were collected with the automated data collection system ZOO [29]. The complete data sets were obtained by combining multiple small wedge datasets from hundreds of tiny crystals with sizes of approximately 20 μm. The principle of this method has been described in detail [30]. Data processing was performed with KAMO [30] using XDS [31] programs. For StYeeE(C22A)-StYeeD dataset, the initial phase was determined by molecular replacement using the StYeeE crystal structure (PDB ID: 6LEO) as a template by PHASER [32], and a StYeeD structure predicted by AlphaFold2 [15,16] was manually fitted to the density map using COOT [33]. Structure refinement was performed using COOT [33] and PHENIX [34] iteratively until R work /R free reached 0.265/0.310 at 3.34 Å resolution. For StYeeE(C22A)-StYeeD(L45A) dataset, the initial phase was determined by molecular replacement using the StYeeE(C22A)-StYeeD structure as a template by MOLREP [35]. Refinement of the structure was performed using COOT [33] and PHENIX [34] in a iterative way until R work /R free reached 0.206/0.255 at 2.60 Å resolution. Figures of the structures were prepared using PyMOL (https://pymol.org/2/) and Chimera [36]. Interaction analysis between StYeeE and StYeeD by the BLI method BLI method [23] was performed to analyze the interaction between StYeeE and StYeeD-His 6 proteins using the Octet N1 System (Sartorius) at room temperature. A Ni-NTA biosensor was hydrated with Octet buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 0.1% (w/v) DDM, 1 mM β-ME) for 10 min and mounted in the Octet N1 System. The biosensor was first dipped in Octet buffer, and the initial baseline was measured for 30 s. Next, the biosensor was dipped in 4.82 μM StYeeD-His 6 protein solution in Octet buffer for 120 s for loading. The solution was then changed to Octet buffer for 30 s to measure the baseline, after which, the biosensor was submerged in 11.05 μM StYeeE solution in Octet buffer for 120 s to measure the association of StYeeE. Finally, the buffer was exchanged to Octet buffer, and the dissociation of StYeeE from the biosensor was measured for 120 s. Biosensors without StYeeD-His 6 proteins were measured with the same procedure and used as references. After the reference data were subtracted from StYeeD-His 6 protein data, the association rate constant (k a ), dissociation rate constant (k d ), and affinity constant (K D ) were calculated by a local fitting method. Measurements were performed 3 times for each protein. Reconstitution of proteoliposomes A mixture of 0.8 mg/ml StYeeE and 4 mg/ml E. coli total lipid extract (Avanti Polar Lipids) in a buffer (20 mM HEPES-HCl (pH 7.0), 300 mM NaCl, 5% (v/v) glycerol, and 0.1% (w/v) DDM) was rotated at 4°C for 1 h. Next, the detergent was removed using SM2-beads (Bio-Rad). The resulting solution was ultracentrifuged, and the precipitates were suspended in a buffer (25 mM HEPES-NaOH (pH 7.5) and 100 mM NaCl). The reconstituted proteoliposome samples were flash-frozen in liquid nitrogen and stored at −80°C until measurements. Measurement of ion transport activity The SSM method was used to detect thiosulfate uptake activity of the StYeeE-reconstituted proteoliposomes. Frozen proteoliposomes were thawed on ice and sonicated using a bath sonicator (VELVO-CLEAR, VS-50R) for 10 s 3 times before use. Measurement was performed using SURFE2R N1 (Nanion Technologies) as described [14]. First, 50 μl of 0.5 mM 1-octadecanethiol (dissolved in isopropanol) was applied to N1 Single Sensors (Nanion Technologies, Nr. 2-03-35002-000) and incubated for 30 min at room temperature. Next, the sensors were washed twice with isopropanol, washed twice with distilled water, and dried. Then, 1.5 μl of 7.5 μg/μl 1,2-diphytanoyl-sn-glycero-3-phosphocholin (dissolved in n-decane) was applied to the sensor, followed by 50 μl of nonactivating buffer (B buffer: 140 mM NaCl, 4 mM MgCl 2 , 25 mM HEPES, 25 mM MES, KOH (pH 6.7), with or without Na 2 SO 4 ). Proteoliposomes were pipetted toward the sensor beneath the B buffer surface, and the sensor was centrifuged (2,000g for 30 min, 25°C) to adsorb the liposomes onto the sensor surface. The resultant sensors were mounted in SURFE2R N1, and the sensors were rinsed with buffer B before each measurement. The current change on the sensor was monitored while B buffer and A buffer (140 mM NaCl, 4 mM MgCl 2 , 25 mM HEPES, 25 mM MES, KOH (pH 6.7), with Na 2 S 2 O 3 ) were exchanged. For each condition, measurement was performed four times, and the 3 results with least noise were analyzed. MD simulations of StYeeE-StYeeD complex All-atom MD simulations of the StYeeE-StYeeD complex were performed in POPC lipid bilayers and 150 mM NaCl solution, where 2 intermediate states, StYeeE-StYeeD-SH and StYeeE-StYeeD-S-S 2 O 3 –, were examined. The initial structure of the protein-membrane complex was constructed using the CHARMM-GUI membrane builder [37]. The system size is 100 Å × 100 Å × 120 Å, and the total number of atoms is approximately 111,600. The CHARMM C36m force field parameters were used for proteins and lipids [38], and the topology and parameters for S-S 2 O 3 –-modified Cys were derived from the CGenFF parameters [39]. The system was equilibrated using 2,000-step energy minimization, followed by equilibration in the NVT and NPT ensembles for 3.5 ns using the positional restraints on the proteins and lipids. The production run was then carried out for 1 μs in the NPT ensemble at T = 310 K and P = 1 atm with the time step of 3.5 fs using the RESPA integrator with hydrogen-mass repartitioning and bond-length constraining [40] and the Bussi thermostat and barostat for temperature and pressure control [41]. The particle-mesh Ewald method was used for the calculation of long-range electrostatic interaction [42]. All simulations were performed using GENESIS [43,44].

Acknowledgments We thank Kayo Abe for secretarial assistance, Akira Sasaki for liposome preparation, Rie Kurata for performing mass spectrometry, Naoki Sakai for helping with the data analysis, the beamline scientists at BL32XU of SPring-8 (Hyogo, Japan) for helping with data collection, and Nanion Technologies for assistance with initial data collection. The synchrotron radiation experiments were performed at BL32XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal Nos. 2020A2564, 2021A2745, 2022A2738, 2023A2727).

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