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• W2056365671 abstract "ScoI and TlpA bind by cross-linking study (View Interaction: 1, 2) The eukaryotic, mitochondrial Sco1 protein and its prokaryotic homologs (ScoI, ScoC, SenC, PrrC, BsSco) share a similar domain structure. They consist of an N-terminal membrane anchor, which directs the protein to the inner mitochondrial membrane or the bacterial cytoplasmic membrane, and a hydrophilic, globular, thioredoxin-like domain that protrudes into the mitochondrial intermembrane space or the periplasm of Gram-negative bacteria. Two cysteines in a CXXXC motif are functionally important. Originally thought to play a unified role as copper chaperone in the synthesis of the binuclear Cu–Cu cofactor (CuA center) on subunit II (CoxII) of heme-copper cytochrome oxidases, several Sco1 family members – though still in minority – execute diverse functions, depending on the organism investigated [1, 2]. For example, humans have two variants of which Sco1 binds Cu+, employing the two cysteine thiols and a histidine imidazole as the ligands, whereas Sco2 is a protein dithiol:disulfide oxidoreductase, using the CXXXC motif for a thioredoxin-like activity. Yet, both functions are needed for assembly of the aa 3-type cytochrome oxidase in human mitochondria [3]. Regarding prokaryotic Sco1-like proteins, a certain non-exclusiveness in CuA synthesis became evident from the fact that 12% of the analyzed genomes carry genes for CoxII but not for Sco1-like proteins, and 6% have genes for Sco but not for CoxII [2]. On the one hand, this implies a Sco-independent biogenesis of the CuA center in some bacteria, and on the other hand, Sco seems to exert different functions in other bacteria. A new Cu+ chaperone, PCuAC, was shown to metallate in vitro the CuA site on subunit II of the ba 3-type oxygen reductase of Thermus thermophilus [4]. In these experiments, the Sco1-like protein served as a reductant for two conserved cysteines in CoxII that take part in CuA binding [5]. Furthermore, both Sco1- and PCuAC-like proteins were found in some bacteria to co-participate in forming active cbb 3-type oxidase which carries only a high-spin heme-CuB center on subunit I but lacks CuA [6-9]. Finally, protein thiol:disulfide oxidoreductase activity has been demonstrated for Sco1-like proteins from Rhodobacter sphaeroides and Pseudomonas putida [10, 11]. One of the substrates was Cu2+, which became reduced to Cu+, an activity that had been reported also for the BsSco protein from Bacillus subtilis [12]. Regardless of whether Sco1-like proteins act as copper chaperones or disulfide reductases, their active-site cysteines must be in the reduced, dithiol form as a prerequisite for liganding copper or reducing target proteins. This requires another, hitherto unknown reductase that converts the oxidized, disulfide form of Sco back into the reduced, dithiol form. Particularly in bacteria, targeting of Sco to the outer side of the cytoplasmic membrane exposes it to an oxidizing environment. By analogy with periplasmic systems such as the dithiol:disulfide exchange relay (Dsb [13]) or the cytochrome c biogenesis pathway (Ccm [14]), Sco-like proteins may become oxidized after translocation and folding and then re-reduced before copper can bind. Likewise, if Sco is a disulfide reductase, it must be re-reduced before starting a new round of reaction. A candidate that might act as a reductase for Sco is the thioredoxin-like protein TlpA which possesses a CXXC motif in the active site. TlpA was originally discovered in the Gram-negative α-proteobacterium Bradyrhizobium japonicum [15]. B. japonicum knock-out mutants of the tlpA gene (bll1380) and of the scoI gene (blr1131), which encodes a Sco1 homolog, share similar phenotypes, including defects in the aa 3-type cytochrome oxidase, symbiotic nitrogen fixation, and nitrate respiration [15, 16]. Furthermore, the TlpA and ScoI topologies in the membrane look alike which may facilitate an interaction between their periplasmic thioredoxin domains. While TlpA clearly acts as a protein-disulfide reductase [17], the genuine target protein has remained elusive. ScoI was shown to bind Cu2+, provided that the CXXXC motif was in the reduced form, but it had no disulfide reduction activity on non-physiological model substrates [16]. Prompted by these properties, we wanted to determine the redox potential of ScoI and then – if it were less negative than that of TlpA – to test whether TlpA is capable of reducing oxidized ScoI. The results presented here support an electron transfer from TlpA to ScoI. Soluble, N-terminally Strep-tagged derivatives of wild-type and mutant ScoIs were expressed and purified as described [16] except for an additional, last purification step by cation exchange on a resource S column (GE Healthcare, Glattbrugg, Switzerland) using buffer S (50 mM succinic acid -NaOH, 0.2 mM ethylenediaminetetraacetate (EDTA), pH 5.3). A linear salt gradient was developed with buffer S containing 1 M NaCl. For disulfide-trapping, wild-type and mutant ScoIs were purified in the presence of 1 mM dithiothreitol (DTT). ScoI-C74S and -C78S were expressed from plasmids pRJ8336 and pRJ8335, respectively, which had been constructed similarly to the previously described plasmid pRJ8331 that encodes wild-type ScoI [16]. The C74 and C78 codons were replaced by serine codons (TCG) via QuikChange mutagenesis (Stratagene, La Jolla, USA). The soluble form of TlpA-C110S (residues 38–221) lacking its N-terminal membrane anchor was purified as MalE fusion protein [18], using expression plasmid pMalpTlpA-C110S (our laboratory collection). The periplasmic extract was prepared as described [17]. The soluble form of wild-type TlpA (TlpAsol, residues 36–221) fused to the Escherichia coli OmpA signal sequence was expressed and purified as reported previously [17]. Protein concentrations were determined via the molar extinction coefficients at 280 nm (ScoI: 16,060 M−1 cm−1, TlpA: 17,270 M−1 cm−1, DsbA: 21,740 M−1 cm−1; TrxDsbA: 14,500 M−1 cm−1). For production of ScoIox, ScoI was incubated with diamide (10 mM final concentration) after the first purification step (Strep-Tactin, IBA, Göttingen, Germany) and incubated overnight in 100 mM Tris–HCl buffer at pH 8. Diamide was removed by cation exchange chromatography during the second purification step (Resource S column; GE Healthcare). ScoIox (2.6 μM) was incubated overnight at 25 °C with a 3-fold excess (7.8 μM) of TlpAred in buffer A to reach equilibrium. The reaction was acid-quenched (final concentration 10% formic acid, v/v), the reaction products were separated via RP-HPLC at 60 °C (see above) with a linear acetonitrile gradient from 41-to-45%, and the peak areas were quantified with PeakFit V.4.12 (SeaSolve). All experiments were performed in buffer B (50 mM sodium phosphate, pH 7.0, 1 mM EDTA) at room temperature. Preparations of ScoIred and TlpAred had been obtained after incubation in 10 mM DTT in buffer A for >1 h. DTT was removed using a HiTrap desalting column (GE Healthcare) equilibrated with buffer B. Disulfide exchange between ScoIox and TlpAred was analyzed in an SX18.MV stopped-flow instrument (Applied Photophysics, Leatherhead, UK) and initiated by 1:1 mixing (initial protein concentration 2 μM each; final concentrations 1 μM). The reaction was followed via fluorescence decrease above 320 nm. Disulfide exchange between ScoIred and DsbAox was initiated by manual mixing (final concentration 2 μM each) and followed by fluorescence increase at 327 nm, due to generation of DsbAred, in a QM-7/2003 spectrofluorimeter (PTI, Seefeld, Germany). Rate constants for the forward and reverse reactions (k 2 and k −2, respectively) were fitted with Berkeley Madonna™ version 8.3, and the k 2/k −2 ratios were fixed based on the calculated equilibrium constants (K eq = k 2/k −2). K eq values used were 17.7 for the oxidation of ScoI by DsbA, and 1760 for the reduction ScoI by TlpA. To calculate K eq according to Eq. (3), we applied a value of −256 mV for the redox potential of TlpA, which is the mean of two published redox potentials [17, 22]. Proteins were reduced with 1 mM DTT, and 0.5-ml solution containing between 0.3 and 1 mg/ml protein was desalted against 10 mM Tris–HCl, pH 8, using a Nap-5 column (GE Healthcare). The eluates were concentrated with an Amicon Ultra 4 spin filter (Millipore, Zug, Switzerland) with a 10-kDa molecular mass cut-off. TlpA-C110S was activated with 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) by addition of 750 μl of 0.3 mM DTNB, 10 mM Tris–HCl, pH 8, to 750 μl of reduced, desalted TlpA-C110S and incubation at room temperature for 15 min. The solution was concentrated with an Amicon Ultra 4 spin filter and desalted with a Nap-5 column as above. Reactions were performed in 10 mM Tris–HCl, pH 8 containing 2.5 μM each of activated TlpA-C110S and one of the ScoI variants, and stopped by adding 0.4 mM iodoacetamide (final concentration). 7.5 μl of each reaction was analyzed by reducing and non-reducing (i.e., without DTT/β-mercaptoethanol) 15% Tris-tricine sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Coomassie staining. As controls, individual proteins were incubated in 10 mM Tris–HCl, pH 8. Fig. 1 a demonstrates that both redox forms of ScoI could be separated on a C18 RP-HPLC column. We first performed a redox equilibrium titration of ScoIred with different amounts of DsbAox. Fig. 1b shows a graph in which the proportion of ScoIox, determined via the peak area of ScoIox relative to the sum of the peak areas of ScoIox plus ScoIred, was plotted against the total DsbAox concentration (DsbAox and DsbAred could not be separated under the applied conditions). A fit according to Eq. (4), with correction for a small (17%) fraction of air-oxidized ScoIred in the absence of DsbAox, yielded an equilibrium constant of 17.7, which translates into a redox potential of −159 mV for ScoI (Eq. (3)). This indicated that ScoI from B. japonicum was more oxidizing than any other member of the ScoI protein family characterized so far [1]. We next investigated the thiol-disulfide exchange reaction between ScoI and its presumed redox partner TlpA. The redox potentials of ScoI (−160 mV; from above) and TlpA (−256 mV) [17, 22] argued for TlpAred being a strong reductant of ScoIox (predicted equilibrium constant K eq = 1760). Fig. 2 a shows that this assumption held true. When ScoIox was incubated with a 3-fold molar excess of TlpAred, quantitative reduction of ScoIox was observed. Specifically, the chromatogram showed only a single peak for ScoIred and an expected 2:1 ratio between the TlpAred and TlpAox peaks at the end of the reaction. Previous studies on the E. coli periplasm with respect to mechanisms underlying the coexistence of the oxidizing DsbA/DsbB and reducing DsbC/DsbD pathways of disulfide bond formation and isomerization, respectively, had shown that disulfide exchange reactions between proteins belonging to different pathways (e.g. the oxidation of DsbC by DsbA), are kinetically restricted, with disulfide exchange rate constants below 103 M−1 s−1, whereas physiological disulfide exchange reactions (e.g. the oxidation of DsbA by DsbB) proceed fast, with rate constants in the range of 104 to 106 M−1 s−1 [24, 25]. To test if ScoI is a physiological substrate of TlpA, we investigated the reduction kinetics of ScoIox by TlpAred at pH 7.0 and 25 °C with stopped-flow fluorescence: As demonstrated previously, the intrinsic tryptophan fluorescence of TlpA decreased upon formation of its active-site disulfide bond [17]. Since the soluble, periplasmic domain of ScoI lacks tryptophan, we could follow ScoIox reduction by TlpAred via the decrease of TlpA's tryptophan fluorescence: Fig. 2b shows the fluorescence trace of a reaction initiated by mixing equimolar amounts of ScoIox and TlpAred (final initial concentrations 2 μM). Using the known ratio between the forward (k 2) and reverse (k −2) reaction (K eq = 1760 = k 2/k −2), we obtained rate constants of 9.4 × 104 M−1 s−1 and 53 M−1 s−1 for k 2 and k −2, respectively. Hence, the rate of ScoIox reduction by TlpAred was in a range typically observed for physiologically relevant disulfide exchange reactions, which provided a strong hint that TlpA is a reductant of ScoI in vivo. Finally, as a negative control, we also measured the rate constant of oxidation of B. japonicum ScoIred with the non-physiological oxidant DsbAox from E. coli. Fig. 2c shows that this reaction proceeded almost three orders of magnitude slower (k 2 = 200 M−1 s−1) than the reduction of ScoIox by TlpAred. The crystal structure of the soluble domain of B. japonicum TlpA [26] and an inspection of its active site (C107XXC110; numbering refers to native TlpA with its membrane anchor) had revealed that C107 is exposed to the protein surface and, therefore, is the likely nucleophile for reduction of target proteins such as ScoI. No structure is available of B. japonicum ScoI. In the structure of the ScoI-homologous B. subtilis BsSco protein, however, the more N-terminally located active-site cysteine C45 is less surface-exposed than C49 [27]. By analogy, assuming structural conservation, we consider the more C-terminally located C78 in the C74XXXC78 motif of B. japonicum ScoI as the likely target for nucleophilic attack by C107 of TlpA. Of concern was the fast disulfide exchange between TlpAred and ScoIox (see above), which might preclude the trapping of a mixed disulfide intermediate. Therefore, we used TlpA-C110S and either wild-type ScoI, or ScoI-C74S, or ScoI-C78S as the interacting partners. Soluble TlpA-C110S was activated with DTNB, incubated with soluble, reduced ScoI, and iodoacetamide was finally added to quench free thiols. Reaction products were analyzed with non-reducing and reducing SDS–PAGE. The occurrence of a band at 38 kDa, reflecting the sum of the molecular masses of TlpA and ScoI, indicated that a mixed disulfide had formed (Fig. 3 ). This band was not seen in reducing SDS–PAGE. We observed disparate reactivities with the mutant ScoI variants. Little heterodisulfide formed at a slow rate between TlpA-C110S and ScoI-C78S. By contrast, the TlpA-C110SScoI–C74S heterodisulfide formed rapidly and stayed remarkably stable (Fig. 3), suggesting that C78 is indeed the preferred target, as was rationalized before. The disulfide between TlpA-C110S and wild-type ScoI was apparently resolved over time by the still intact C74. Homodimers of the individual proteins did not form under these conditions. The 38-kDa heterodisulfide from the reaction between TlpA-C110S and ScoI-C74S was subjected to tryptic digestion followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) (data not shown). A large tryptic peptide with the mass of 5146.8 Da was detected which can only be explained as being the disulfide-bridged product of tryptic peptides from the active sites (underscored) of TlpA-C110S (97TLLVNLWATWCVPSRK112; 1888.3 Da) and ScoI-C74S (62GKPTLIFFGYTHSPDVCPTSLFEISEVLR90; 3255.8 Da). Almost 20 years after the discovery of TlpA in B. japonicum [15], we can now present a substrate: ScoI. Since the active site of TlpA protrudes into the periplasm, it was clear that any given substrate must be periplasmic or at least facing the periplasm, as in the case of ScoI. Three lines of evidence support the reducing role of TlpA on ScoI. (i) TlpA has a substantially more negative redox potential than ScoI. In fact, the E o′ of ScoI (−160 mV) falls into a range that is characteristic for oxidizing proteins such as DsbA (−122 mV) [19]. Therefore, the purported role of Sco1-like proteins as disulfide reductases in some organisms [1, 2] most likely does not apply to B. japonicum ScoI. (ii) Oxidized ScoI was efficiently and rapidly reduced by the dithiol form of TlpA. The thermodynamics and kinetics of the interaction between TlpA and ScoI provided strong evidence that the physiologically active state of ScoI is its reduced form, and that TlpA reduces ScoI if the latter became oxidized in the oxidizing environment of the periplasm. The data are fully consistent with a function of ScoI as Cu+- or Cu2+-binding protein or copper chaperone [16], because only the dithiol form of its active site is expected to interact with copper ions. (iii) We demonstrated the physical interaction between TlpA and ScoI by trapping the transiently formed heterodisulfide. Furthermore, the preferentially reacting cysteines on TlpA (C107) and ScoI (C78) were identified. Their reactivity is largely determined by the surface topology and accessibility which we knew already for C107 of TlpA from the crystal structure [26] and may now infer also for C78 of ScoI. To the best of our knowledge, TlpA is the first identified reductant for any member of the widespread family of prokaryotic Sco1-like proteins. Only the mitochondrial Cox17 protein had been shown to transfer two electrons concomitantly with Cu+ transfer to human Sco1 but not to Sco2 [28]. Being a disulfide reductase destined to enable copper binding to ScoI, B. japonicum TlpA assumes a position that is reminiscent of the bacterial CcmG protein which is committed to keep apocytochrome c in the reduced form prior to the covalent heme ligation [14]. However, studies with Neisseria gonorrhoeae [29] and E. coli [30] have shown an expanded scope of TlpA-like proteins in the tolerance to reactive oxygen species. It cannot be excluded, therefore, that TlpA also reduces B. japonicum substrates other than ScoI. Targets of this kind ought to be accessible from the periplasm, and they might even have a CXXXC motif like that of ScoI. Incidentally, the periplasmic CuA binding site on CoxII of the aa 3-type cytochrome oxidase fulfills these requirements [16]. This work was supported by the for Scientific Research, the NCCR Structural Biology Program, and the ETH Zurich. Help and advice from Serge Chesnov of the Functional Genomics Center Zurich and from Chasper Puorger are gratefully acknowledged. We thank Sarah Landolt for constructing ScoI expression plasmids." @default.
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• W2056365671 title "Thioredoxin-like protein TlpA from <i>Bradyrhizobium japonicum</i> is a reductant for the copper metallochaperone ScoI" @default.
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