The Oxidase DsbA Folds a Protein with a Nonconsecutive Disulfide

One of the last unsolved problems of molecular biology is how the sequential amino acid information leads to a functional protein. Correct disulfide formation within a protein is hereby essential. We present periplasmic ribonuclease I (RNase I) from Escherichia coli as a new endogenous substrate for the study of oxidative protein folding. One of its four disulfides is between nonconsecutive cysteines. In general view, the folding of proteins with nonconsecutive disulfides requires the protein disulfide isomerase DsbC. In contrast, our study with RNase I shows that DsbA is a sufficient catalyst for correct disulfide formation in vivo and in vitro. DsbA is therefore more specific than generally assumed. Further, we show that the redox potential of the periplasm depends on the presence of glutathione and the Dsb proteins to maintain it at–165 mV. We determined the influence of this redox potential on the folding of RNase I. Under the more oxidizing conditions of dsb– strains, DsbC becomes necessary to correct non-native disulfides, but it cannot substitute for DsbA. Altogether, DsbA folds a protein with a nonconsecutive disulfide as long as no incorrect disulfides are formed. One of the last unsolved problems of molecular biology is how the sequential amino acid information leads to a functional protein. Correct disulfide formation within a protein is hereby essential. We present periplasmic ribonuclease I (RNase I) from Escherichia coli as a new endogenous substrate for the study of oxidative protein folding. One of its four disulfides is between nonconsecutive cysteines. In general view, the folding of proteins with nonconsecutive disulfides requires the protein disulfide isomerase DsbC. In contrast, our study with RNase I shows that DsbA is a sufficient catalyst for correct disulfide formation in vivo and in vitro. DsbA is therefore more specific than generally assumed. Further, we show that the redox potential of the periplasm depends on the presence of glutathione and the Dsb proteins to maintain it at–165 mV. We determined the influence of this redox potential on the folding of RNase I. Under the more oxidizing conditions of dsb– strains, DsbC becomes necessary to correct non-native disulfides, but it cannot substitute for DsbA. Altogether, DsbA folds a protein with a nonconsecutive disulfide as long as no incorrect disulfides are formed. Disulfide bond formation is an essential step in the folding of many proteins. Anfinsen et al. (1Anfinsen C.B. Kim E. Sela M. White Jr., F.H. Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1309-1314Crossref PubMed Scopus (897) Google Scholar) have stated that “From chemical and physical studies of reformed enzyme, it may be concluded that the information for correct pairing of half-cystine residues in disulfide linkage, and for the assumption of the native secondary and tertiary structures, is contained in the amino acid sequence itself”. The reformed enzyme is bovine pancreatic ribonuclease A, a protein with four disulfides (three between nonconsecutive cysteines and one between consecutive). However, the rate of in vitro disulfide bond formation was much slower than the rates observed in living cells, and the yield of active enzyme was low (much of the protein containing the incorrect disulfide bonds). The explanation for the fast in vivo rates is resolved with the discovery that an enzyme is required to catalyze efficient disulfide bond formation (2Peek J.A. Kim R.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6210-6214Crossref PubMed Scopus (184) Google Scholar, 3Kamitani S. Kim Y. Ito K. EMBO J. 1992; 11: 57-62Crossref PubMed Scopus (225) Google Scholar, 4Bardwell J.C. Kim K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (830) Google Scholar, 5De Lorenzo F. Kim R.F. Steers E. Jr. Givol D. Anfinsen B. J. Biol. Chem. 1966; 241: 1562-1567Abstract Full Text PDF PubMed Google Scholar), protein-disulfide isomerase (PDI). PDI is present in the lumen of the endoplasmic reticulum and is composed of four thioredoxin-like domains. This essential enzyme has the ability to catalyze both the oxidation of new disulfides and the isomerization of existing disulfides (6Freedman R.B. Kim P. Ruddock L.W. EMBO Rep. 2002; 3: 136-140Crossref PubMed Scopus (169) Google Scholar). In Escherichia coli, the periplasmic protein DsbA is the catalyst of disulfide formation (4Bardwell J.C. Kim K. Beckwith J. Cell. 1991; 67: 581-589Abstract Full Text PDF PubMed Scopus (830) Google Scholar, 7Messens J. Kim J.F. Int. J. Biochem. Cell Biol. 2006; 38: 1050-1062Crossref PubMed Scopus (132) Google Scholar). DsbA, however, is not a very specific oxidant and can introduce non-native disulfide bonds, which need to be corrected. To correct these non-native disulfides, the cell uses two protein disulfide isomerases, DsbC (8Missiakas D. Kim C. Raina S. EMBO J. 1994; 13: 2013-2020Crossref PubMed Scopus (193) Google Scholar, 9Shevchik V.E. Kim G. Robert-Baudouy J. EMBO J. 1994; 13: 2007-2012Crossref PubMed Scopus (132) Google Scholar) and DsbG (10Bessette P.H. Kim J.J. Gilbert H.F. Georgiou G. J. Biol. Chem. 1999; 274: 7784-7792Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 11Andersen C.L. Kim A. Missiakas D. Raina S. Mol. Microbiol. 1997; 26: 121-132Crossref PubMed Scopus (91) Google Scholar). The structural and enzymatic properties of both enzymes and their recycling by the inner membrane protein DsbD are reported in a recent review (7Messens J. Kim J.F. Int. J. Biochem. Cell Biol. 2006; 38: 1050-1062Crossref PubMed Scopus (132) Google Scholar). Today, the native substrates of DsbG are not known. Most of the substrates in the study of oxidative protein folding in E. coli are eukaryotic in origin (12Rietsch A. Kim P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (194) Google Scholar, 13Sun X.X. Kim C.C. J. Biol. Chem. 2000; 275: 22743-22749Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 14Zapun A. Kim D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (220) Google Scholar). In the periplasm of E. coli, there are more than 300 proteins with at least two cysteines, suggesting that they are potential physiological substrates for DsbA (15Hiniker A. Kim J.C. Trends Biochem. Sci. 2004; 29: 516-519Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). DsbA can properly fold the vast majority of these proteins. However, some of these proteins, like the peptidoglycan amidase MepA and the phytase AppA contain multiple disulfides, some of which are formed between cysteines in nonconsecutive positions (16Lim D. Kim S. Forsberg C.W. Jia Z. Nat. Struct. Biol. 2000; 7: 108-113Crossref PubMed Scopus (149) Google Scholar, 17Marcyjaniak M. Kim S.G. Sabala I. Bochtler M. J. Biol. Chem. 2004; 279: 43982-43989Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Work on AppA and MepA suggests that the formation of these nonconsecutive disulfides is dependent on DsbC (18Hiniker A. Kim J.C. J. Biol. Chem. 2004; 279: 12967-12973Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 19Berkmen M. Kim D. Beckwith J. J. Biol. Chem. 2005; 280: 11387-11394Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We have determined the structure of another DsbC substrate (18Hiniker A. Kim J.C. J. Biol. Chem. 2004; 279: 12967-12973Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), the periplasmic ribonuclease (RNase I) (20Padmanabhan S. Kim K. Chu C.Y. Lim R.W. Lim L.W. Arch. Biochem. Biophys. 2001; 390: 42-50Crossref PubMed Scopus (14) Google Scholar). We show that RNase I contains four disulfides, three consecutive and one non-consecutive and is therefore a physiologically relevant model protein to study oxidative protein folding in E. coli. Through studies of wild type and disulfide mutants of RNase I as substrates for the Dsb oxidoreductases, we show that a protein with a nonconsecutive disulfide may in vivo as well as in vitro fold to its proper structure with DsbA alone and that the dependence on DsbC is linked to the redox state of the environment. Mutagenesis, Expression, and Purification—RNase I disulfide mutants are constructed. Wild type DsbA, DsbC, DsbG, RNase I 2The abbreviations used are: RNase I, ribonuclease I; ruRNase I, reduced and unfolded RNase I; scRNase I, scrambled RNase I; GdnHCl, guanidine hydrochloride; Mes, 4-morpholineethanesulfonic acid., and the RNase I mutants are expressed and purified. The details can be found in the supplemental “Experimental Procedures” data. Reduced and Unfolded RNase I (ruRNase I)—RNase I (∼10 mg/ml) was reduced and unfolded in a buffer solution containing 50 mm Tris/HCl, pH 8.7, 50 mm NaCl, 6 m GdnHCl, and 100 mm dithiothreitol. The mixture was incubated for 2 h at room temperature. To remove the excess of GdnHCl and dithiothreitol, the mixture was dialyzed against 50 mm Tris/HCl, pH 8.7, 0.6 m GdnHCl, 50 mm NaCl, and 1 mm dithiothreitol at 15 °C. The dialysis buffer was argon-flushed for 30 min. All 8 cysteines were found to react with Ellman reagent indicating that all were reduced (21Wright S.K. Kim R.E. Anal. Biochem. 1998; 265: 8-14Crossref PubMed Scopus (102) Google Scholar). Scrambled RNase I (scRNase I)—To induce the formation of incorrect disulfides in ruRNase I (scrambled RNase I), the sample of ruRNase I was incubated with 50 μm CuCl2 and 2 mm H2O2 for 30 min at 25 °C. Excess of CuCl2 and H2O2 were removed by a final dialysis against 50 mm Tris, pH 8.7, 0.6 m GdnHCl, and 50 mm NaCl. In Vitro Folding—RNase I was refolded by mixing ruRNase I or scRNase I with Dsb protein (DsbA and/or DsbC and/or DsbG) in folding buffer solution to obtain a final concentration of 5 μm RNase I and 20 μm Dsb protein. The folding mixture was incubated for 3 min at 15 °C. All buffer solutions were argon-flushed for 15 min. Details about the folding buffer solution preparation are in the supplemental data. RNase I Activity Assay—The RNA hydrolysis activity was measured in a methylene blue RNase I assay (22Greiner-Stoeffele T. Kim M. Hahn U. Anal. Biochem. 1996; 240: 24-28Crossref PubMed Scopus (44) Google Scholar). The absorbance change was followed in function of time at 659 nm. The measured RNase activities are the initial velocities determined on the first 10% of the progress curves. A linear response was observed for RNase I concentrations between 9 and 36 nm. More details can be found in the supplemental “Experimental Procedures” data. Dsb-refolded RNase I for Circular Dichroism (CD) Spectroscopy and Crystallization—After 3-min refolding at 15 °C, the reaction was stopped by injecting the sample on a Ni2+-Sepharose column equilibrated in 50 mm Hepes/NaOH, pH 8.0. As a control for spontaneous folding in redox buffer solution, reduced and unfolded RNase I in the absence of Dsb proteins was injected. RNase I eluted with 1 m NaCl in the same buffer solution. His-tagged Dsb proteins were eluted with 1 m imidazole in the same buffer solution. The RNase activity of the flow-through, the 1 m NaCl elution fraction, and the imidazole fraction were measured. No RNase activity was detected in the flow-through of the column. Spontaneous folding of ruRNase yielded 10% of wild type activity, and DsbA-catalyzed folding yielded 83% of wild type activity. RNase I wild type, ruRNase I, scRNase I, DsbA-refolded ruR-Nase I, DsbA/C-refolded scRNase I were dialyzed against 50 mm boric acid, pH 8.0, 100 mm KF buffer solution for CD spectroscopy or against 20 mm Tris, pH 7.5, and 20 mm NaCl for crystallization. Samples for crystallization were concentrated to 8 and 9.5 mg/ml. Samples for CD were diluted to 0.2 mg/ml, and for each sample 10 scans were recorded between 200 and 250 nm in a J-715 spectropolarimeter (Jasco). Crystal Structure Determination—Crystals of DsbA-refolded RNase I were grown in hanging drop vapor diffusion method under the conditions as described (20Padmanabhan S. Kim K. Chu C.Y. Lim R.W. Lim L.W. Arch. Biochem. Biophys. 2001; 390: 42-50Crossref PubMed Scopus (14) Google Scholar). X-ray data were collected at European Molecular Biology Laboratory (EMBL) beamline X12 at the DESY synchrotron (Hamburg, Germany). The crystals were first soaked for 1 min in a cryoprotectant solution containing 0.1 m Mes, pH 6.1, 0.1 m ammonium acetate, 10% glycerol, 1 mm MgCl2 and 28% PEG3350 and subsequently flash-frozen in the cryostream. Data were integrated and scaled the of Z. Kim 1997; PubMed Scopus Google Scholar) The structure was and Kim P. R.W. J. M. T. Biol. 1998; PubMed Scopus Google Scholar) from the of the in vivo protein Data and data. In the initial of a was with the of the structure was the was in of are in of DsbA-refolded RNase cell a of measured of bond of Data in a new containing the mutants and the were by wild type E. coli. were grown at in with their specific This was diluted 100 in a chemical J. in Molecular Scholar), that the contained and The was grown at it an of the of the and cell were by in 15 at °C. The of the from a with an nm of 1 was in buffer solution containing 10 mm Tris, pH 7.5, and mm The sample was for 30 min on and subsequently diluted with containing 1 mm to Kim Microbiol. 2005; PubMed Scopus Google Scholar). another 15 min on the of a was and argon-flushed for 15 min. of the redox potential in the periplasm of wild type E. coli and in the dsb– was measured at a sample of with a The was with redox solution and for to The chemical redox potential at 1 m was to a redox potential at pH the were to pH to obtain from a of 50 mm and 50 mm in a buffer solution of 50 mm pH mm and 1 mm and on the with a glutathione redox was with a redox potential to mV. the redox potential of the glutathione was measured. The linear between and was used to obtain the glutathione redox of the periplasmic All of RNase I for RNA the structure of E. coli RNase I at Data This structure shows an RNase three consecutive and one nonconsecutive disulfides The the nonconsecutive and the disulfides of RNase I are All four disulfides be formed for RNase I to function We the disulfide one by one by the cysteines by and measured RNA hydrolysis with methylene blue RNA as a substrate (22Greiner-Stoeffele T. Kim M. Hahn U. Anal. Biochem. 1996; 240: 24-28Crossref PubMed Scopus (44) Google Scholar). The disulfide is within 1 between the and we this the RNA hydrolysis activity to wild between and be for the correct of the RNA substrate by A is which is found on this in the structure of K. Kim S. M. T. E. E. F. M. J. Mol. Biol. 2001; PubMed Scopus Google Scholar). The disulfide is between the nonconsecutive cysteines and at the of the which the residues and this disulfide the of the the of residues on the of and on 6 in the formation of or the protein be the consecutive disulfide on the of 6 reduced the RNA hydrolysis activity to 10% of wild As the consecutive and the nonconsecutive disulfides, on both of the 6 are essential for the of the residues of the active and of the for RNA hydrolysis is the this disulfide reduced the activity to of wild The that RNase I is an endogenous enzyme that depends on the formation of a nonconsecutive disulfide for correct folding it a model to the ability of Dsb proteins to catalyze the formation of a nonconsecutive disulfide in vitro and in In DsbA a with a the presence of a nonconsecutive DsbA on its unfolded RNase I. We have the specific of DsbA, DsbC, and DsbG in the folding of unfolded RNase I (ruRNase I) and activity and structure A and The of and Anfinsen E. Kim C.B. J. Biol. Chem. 1961; Full Text PDF PubMed Google Scholar) on bovine pancreatic RNase A shows spontaneous folding and disulfide formation by molecular to between spontaneous folding and folding of RNase I, we within 3 min at 15 °C, pH Under these in glutathione redox buffer solution, the yield of refolded RNase I was than 10% in all the RNase I was refolded by DsbA, as by and CD spectroscopy A and In the structure of DsbA-refolded RNase I to be to the in vivo protein of for that DsbA can a protein with a nonconsecutive the refolding by DsbC in about the same of functional RNase I as refolding by DsbA. DsbC can function as an in this with the of et al. A. Kim D. Raina S. Creighton T.E. Biochemistry. 1995; 34: 5075-5089Crossref PubMed Scopus (220) Google Scholar). We scRNase I in the presence of CuCl2 and RNase I with incorrect disulfides A. Kim J.F. J.C. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). No cysteines were detected with Ellman reagent (21Wright S.K. Kim R.E. Anal. Biochem. 1998; 265: 8-14Crossref PubMed Scopus (102) Google Scholar), and scRNase I its activity. DsbA was to scRNase I, and with the of the isomerase activity of DsbC RNase I its activity and structure A and DsbC to an essential in the of incorrect disulfides, which may in a more oxidizing environment. the DsbG, which has to have isomerase activity (10Bessette P.H. Kim J.J. Gilbert H.F. Georgiou G. J. Biol. Chem. 1999; 274: 7784-7792Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), not catalyze the refolding of scrambled or RNase I. DsbA and DsbC Work in the influence of the redox potential on the refolding of RNase I. The redox potential the in vitro refolding of ruRNase I very and the of refolding of ruRNase I by DsbA and DsbC alone was found within the same The yield of refolded ruRNase I was under the DsbA was with DsbC and this the redox potential As DsbA and DsbC to in to the of the refolding of ruRNase I. We a with DsbC on scRNase I not the refolding by DsbC was found to be redox In DsbC on scRNase I and on ruRNase I a redox potential but the refolding is DsbC and DsbA are In DsbA to RNase I the for the folding of RNase I in and of the were constructed. To for structural of RNase I, RNA hydrolysis activity in periplasmic was used were with in the presence of 1 mm to Kim Microbiol. 2005; PubMed Scopus Google Scholar), and the concentration was to 2 as the RNase from E. coli is RNase I Kim E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). DsbG has no on the RNase I activity, suggesting that this is not in the folding of RNase I. The in vivo of DsbG in E. coli is (7Messens J. Kim J.F. Int. J. Biochem. Cell Biol. 2006; 38: 1050-1062Crossref PubMed Scopus (132) Google Scholar). DsbD shows of the RNase I activity. In the absence of DsbC is to function as an isomerase DsbC is not indicating that DsbA and/or DsbC are the folding DsbC can be as a folding catalyst a no activity. This with DsbA as the folding In a no RNase activity was the dependence of this protein on DsbA and suggesting that DsbC cannot substitute for DsbA. In a the activity to wild type, indicating that DsbA can well catalyze the formation of the required nonconsecutive in a more oxidizing periplasmic environment. dsb– a More redox potential of the periplasm is not known. to our this is the first time that the redox potential of the periplasm of E. coli is The redox potential is as as are In the wild type dsb– strains, the pH was found to be which is the pH for RNase I The measured redox potential was to a glutathione redox potential at pH “Experimental each at least 10 were measured. The redox potential of the periplasm of E. coli was found to be 10 DsbC the redox to a more oxidizing DsbA the redox potential of the periplasm to a more oxidizing to 30 is about the in redox potential observed between a and an cell Kim Biol. 2001; PubMed Scopus Google Scholar). As in the and strains, the more Dsb proteins are the more oxidizing the periplasm Altogether, our data that Dsb proteins are therefore of the redox of the the activity of RNase I in a is of the absence of the isomerase activity of DsbC. A explanation is that a redox potential in a to the formation of non-native non-native disulfides cannot be by DsbA, which a activity A and This new well with the that no activity is detected in a the redox potential is and is in with our in vitro and the Dsb the of the determined glutathione the of a redox buffer in the as it in the one of the required for the of glutathione or the glutathione Kim R.K. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar) the redox potential of the periplasm to mV. that glutathione the of a redox in the However, it has no on RNase I activity As the formation of correct disulfides in RNase I is by DsbA and DsbC and not by its redox environment. The cell to have to a by the presence of and the Dsb proteins. one of the redox has on the redox of the folding is that there a between the disulfide and the that contain nonconsecutive disulfides on DsbC for proper proteins with consecutive disulfides show no dependence on DsbC (12Rietsch A. Kim P. Georgiou G. Beckwith J. J. Bacteriol. 1997; 179: 6602-6608Crossref PubMed Scopus (194) Google Scholar, A. Kim J.C. J. Biol. Chem. 2004; 279: 12967-12973Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 19Berkmen M. Kim D. Beckwith J. J. Biol. Chem. 2005; 280: 11387-11394Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, J.C. Kim Biochemistry. 1997; PubMed Scopus Google Scholar). on RNase I are and in with this and (18Hiniker A. Kim J.C. J. Biol. Chem. 2004; 279: 12967-12973Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) reported that show no RNase I activity and as concluded of RNase I. However, in to their we RNase I activity in a that DsbA is in folding a protein with a nonconsecutive disulfide in a more oxidizing environment. The is to from a used to RNase activity in et al. M. Kim D. Beckwith J. J. Biol. Chem. 2005; 280: 11387-11394Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) the same as for phytase a nonconsecutive disulfide containing enzyme of E. coli. AppA in a and shows activity in a However, the in vitro of DsbA to AppA was not and the redox potential the folding was not that the nonconsecutive disulfide bond of AppA it dependent on DsbC. data show that there is another to these The observed of AppA activity in a be of the of the redox potential the formation of incorrect RNase I, the of the folding DsbA with DsbC as well in vivo as in vitro in the folding in a more oxidizing environment. the DsbC on its not catalyze the folding of RNase I in in vitro it was found to be as as DsbA. This the in chemical between the of the periplasm and a buffer solution with an in vivo periplasm with the correct redox buffer solution at in the presence of its Dsb proteins to be of the folding are not at but are at a In a cell at is a the rates of the between DsbA and and between DsbC and and with all be more relevant for proper folding than the in the oxidative folding of the in vivo is the that has the of redox in dsb– In a the redox potential and in this more oxidizing incorrect disulfides be under this the protein from the presence of DsbC to correct non-native As for of in dsb– strains, the redox potential change of the periplasm be dsb– from E. coli in with RNase I we in vitro and in vivo that the presence of a nonconsecutive RNase I folds to its native structure in the presence of DsbA DsbA more specific than assumed. in a more oxidizing incorrect disulfide are the of DsbC as an isomerase is essential. As the oxidative folding that proteins with non-consecutive disulfides DsbC has to be in a more or We and for their We the of synchrotron time at the European Molecular Biology Laboratory (EMBL) at the (Hamburg, Germany). with