©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DsbA-DsbB Interaction through Their Active Site Cysteines
EVIDENCE FROM AN ODD CYSTEINE MUTANT OF DsbA (*)

(Received for publication, April 17, 1995; and in revised form, May 31, 1995)

Satoshi Kishigami (1), Eiko Kanaya (2)(§), Masakazu Kikuchi (2)(§), Koreaki Ito (1)(¶)

From the  (1)Department of Cell Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01 and the (2)Protein Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Formation of disulfide bonds in Escherichia coli envelope proteins is facilitated by the Dsb system, which is thought to consist of at least two components, a periplasmic soluble enzyme (DsbA) and a membrane-bound factor (DsbB). Although it is believed that DsbA directly oxidizes substrate cysteines and DsbB reoxidizes DsbA to allow multiple rounds of reactions, direct evidence for the DsbA-DsbB interaction has been lacking. We examined intracellular activities of mutant forms of DsbA, DsbA30S and DsbA33S, in which one of its active site cysteines (Cys or Cys, respectively) has been replaced by serine. The DsbA33S protein was found to dominantly interfere with the disulfide bond formation and to form intermolecular disulfide bonds with numerous other proteins when cells were grown in media containing low molecular weight disulfides such as GSSG. In the absence of added GSSG, DsbA33S protein remained specifically disulfide-bonded with DsbB. These in vivo results not only confirm the previous findings that Cys of DsbA is hyper-reactive in vitro but provide evidence that DsbA indeed interacts selectively with DsbB. We propose that the Cys-mediated DsbA-DsbB complex represents an intermediate state of DsbA-DsbB recycling reaction that has been fixed because of the absence of Cys on DsbA.


INTRODUCTION

After translocation across the cytoplasmic membrane of prokaryotic cells or the endoplasmic reticulum membrane of eukaryotic cells, secretory proteins are folded into functional conformations. One characteristic feature of the folding of secretory proteins is that many of them must acquire correctly positioned disulfide bonds(1) . Remarkable progress has been achieved recently in our understanding of how protein disulfides are formed in the periplasmic space of living prokaryotic cells. In Escherichia coli, mutational inactivation of either dsbA(2, 3) , dsbB(4, 5) , or dsbC(6) gene pleiotropically impairs disulfide bond formation of envelope proteins. DsbA and DsbC are soluble proteins of the periplasm while DsbB is a cytoplasmic membrane protein with four transmembrane segments(7) . Among these proteins, the structure (8) and biochemical reactivities(9, 10, 11, 12, 13, 14, 15) of the DsbA protein have been characterized in considerable details. It directly oxidizes secretory proteins. The role of DsbB has been proposed to reoxidize the reduced form of DsbA to enable it to work again(4, 16) . Assuming the DsbA-DsbB circuit, it is still unknown what are the acceptors of electrons that are obligatorily liberated upon oxidation of the thiols. Little is known either about DsbC, which, according to a proposal(16) , might be specialized in isomerization of disulfide bonds.

The Dsb proteins all contain a Cys-X-X-Cys motif seen characteristically for the redox active sites in the thioredoxin-like oxidoreductases(17) . The one in DsbA is Cys-Pro-His-Cys. DsbB contains a set of thioredoxin-like motifs as well as additional cysteine residues, among which 4 essential Cys residues facing the periplasm have been identified(7) .

It was shown that mutational alterations of either Cys or Cys inactivate the enzymatic activity of DsbA in vivo(18) or in vitro(10, 13) . Cys is exposed to the solvent, is very reactive, and has a low pK value, whereas Cys is shielded from the solvent(9, 10, 11, 12) .

In this work, we examined in vivo behaviors of the active site mutants of DsbA that we constructed previously(13) . The results indicate that the mutant form of DsbA (DsbA33S) in which Cys was replaced by serine exhibited three unusual phenotypes. First, it dominantly interferes with the functioning of the DsbA-DsbB system. Second, it is disulfide-bonded with a number of cellular proteins when cells are grown in the presence of small molecule disulfides such as GSSG. Finally, it forms an intermolecular disulfide bond with DsbB irrespective of the presence or absence of GSSG in the medium. We suggest that DsbA preferentially interacts with DsbB, and the lack of Cys makes the interaction abortive with respect to the catalytic turnover normally required for the functioning of the system.


MATERIALS AND METHODS

Biological Materials

E. coli K12 strain CU141 (19) was used as a dsb strain. Strains SS140 and SS141 were CU141 derivatives into which the dsbA33::Tn5 mutation (2) or the dsbB::kan5 (4) mutation, respectively, had been introduced by P1 transduction. Plasmid pSS18 carried dsbA and was constructed by cloning the 1.0-kilobase EcoRI-HindIII fragment of the pUC19-derived dsbA plasmid (13) into pMW119, a pSC101-based lac promoter vector (Nippon Gene). Plasmids pSS19 and pSS20 were similar to pSS18, except for the presence of the dsbA30S or the dsbA33S mutation (13) that caused a Cys to Ser or a Cys to Ser substitution in DsbA, respectively. pSS39 carried dsbB; the dsbB region of the chromosome was amplified by polymerase chain reaction using primers 5`AAACTGCGCACTCTATGC and 5`CGGGGATCCTTAGCGACCGAACAGATC, digested with NsiI and BamHI, and cloned into PstI-BamHI cleaved pSTV29 (a pACYC184-derived lac promoter vector from Takara Shuzo). pSS42 encoded a variant of DsbB in which its carboxyl-terminal 10 amino acid residues had been replaced by a sequence of 20 amino acids, IKLIDEEQKLISEEDLLRKR, including a Myc epitope sequence; a 68-base pair EcoRV-KpnI fragment from a vector (pTYE006) carrying a Myc-encoding sequence()was inserted into pSS39 that had been digested with DraI and KpnI. The DsbB`-Myc protein retained the activity to complement the dsbB::kan5 mutation.

Analysis of the Formation of Intramolecular Disulfide Bond in -Lactamase and Intermolecular Mixed Disulfides Involving DsbA

Cells were grown at 37 °C either in peptone broth (20) or M9/glucose/amino acid medium (2) with or without GSSG at a specified concentration. To overproduce products of genes placed under the lac promoter control, isopropyl--D-thiogalactopyranoside was added to 2 mM. Bla()was constitutively synthesized from the plasmids. A portion of exponentially growing cultures was mixed with an equal volume of 10% trichloroacetic acid to denature and precipitate whole cell proteins, which were collected by centrifugation (microcentrifuge for 2 min), washed with acetone, and dissolved in 1% SDS, 1 mM EDTA, 50 mM Tris-HCl (pH 8.1) that contained 5 mM iodoacetamide. After separation by SDS-PAGE in the absence of any reducing reagent(2) , polypeptides reactive with antiserum against Bla (5 Prime 3 Prime, Inc.), antiserum against DsbA(14) , or monoclonal antibody against c-Myc (Ab-1, Oncogene Science, Inc.) were visualized by immunoblotting (14) . In the case of the Myc antibody, the second antibodies used were against mouse IgG.


RESULTS

DsbA33S Mutant Protein Exhibits Dominant Negative Phenotype

Plasmids overexpressing either wild-type DsbA protein, the DsbA30S mutant protein, or the DsbA33S mutant protein were introduced into the dsbA-disrupted cells. Disulfide bond formation was assessed by examining electrophoretic mobility of Bla, which normally contains one intramolecular disulfide bond in SDS-PAGE under non-reducing conditions. Consistent with the previous biochemical results(10, 13) , both DsbA30S and DsbA33S were inactive in supporting Bla oxidation in vivo (Fig. 1, lanes2 and 3). The same plasmids were then introduced into wild-type cells. It was found that cells overproducing DsbA33S were defective in disulfide bond formation of Bla (Fig. 1, lane6), indicating that DsbA with a single cysteine at residue 30 is not only inactive but also interfering with the normal functioning of the Dsb system.


Figure 1: Complementing and interfering activities of active site mutants of DsbA. Strain SS140 (dsbA::Tn5, lanes1-3) and CU141 (dsbA, lanes 4-6) were transformed with a plasmid carrying bla (for Bla) as well as wild-type or mutant forms of dsbA as indicated below. Plasmids used were: lanes1 and 4, pSS18 (dsbA); lanes2 and 5, pSS19 (dsbA30S); lanes3 and 6, pSS20 (dsbA33S). Cells were grown in M9 medium. After separation of whole cell proteins by SDS-PAGE under non-reducing conditions, Bla was visualized by immunoblotting. red and ox indicate reduced and oxidized forms of Bla, respectively. A faintband that overlaps the lower edge of the oxidized Bla is a nonspecific background.



DsbA33S Mutant Protein Is Disulfide-bonded with Other Proteins in the Presence of Exogenously Added Small Molecule Disulfides

Fig. 2shows similar electropherograms as shown above, but probed with anti-DsbA antibodies. In cells (with intact dsbA on the chromosome) overproducing DsbA33S, a number of higher molecular mass bands were found to react with anti-DsbA (Fig. 2A, lane4). Most of these higher molecular mass bands disappeared under reducing conditions (Fig. 2A, lane6), suggesting that they represented mixed disulfides between DsbA and other proteins. Results of non-reducing/reducing two-dimensional SDS-PAGE (data not shown) suggested that the band of about 41 kDa (arrow in Fig. 2) represented a dimeric form of DsbA.()Cells overproducing DsbA or DsbA30S contained only a few cross-reacting proteins (Fig. 2A, lanes2 and 3), some of which (e.g. a band of about 33 kDa) were unrelated to the disulfide cross-linking we were addressing since they persisted under the reducing conditions (Fig. 2A, lanes5 and 6) and they were observed even in the dsbA-disrupted cell (data not shown).


Figure 2: DsbA33S is disulfide-bonded with other proteins in the presence of small molecule disulfides. A, cells of CU141 (dsbA) that carried pMW119 (vector; lanes1 and 5), pSS18 (dsbA; lane2), pSS19 (dsbA30S; lane3) or pSS20 (dsbA33S; lanes4 and 6) were grown in peptone medium. B, cells of CU141/pSS20 (lanes1 and 3) and SS140 (dsbA::Tn5)/pSS20 (lanes2 and 4) were grown in M9 medium supplemented with (lanes3 and 4) or without (lanes1 and 2) 10 mM GSSG. In all cases, except for lanes5 and 6 of A, whole cell proteins were separated by non-reducing SDS-PAGE, and proteins reactive with anti-DsbA were visualized. Samples for lanes5 and 6 were separated after reduction with 5% -mercaptoethanol. The arrow indicates a putative dimer of DsbA, and the arrowhead indicates the DsbA33S-DsbB complex.



When cells were grown on minimal salt medium rather than on broth, most of the higher molecular mass cross-reacting polypeptides were not produced (Fig. 2B, lane1), with an exception of a protein of about 36 kDa (arrowhead in Fig. 2). Evidence presented in the following section shows that the 36-kDa protein represents a DsbA33S-DsbB complex. The difference between the broth and the salt media examined with respect to the production of the cross-linked DsbA complexes appeared to be due to the presence of small molecule disulfides such as cystine and GSSG in the former. This was clearly demonstrated by the results presented in Fig. 2B, in which effects of GSSG addition to the M9 medium were examined. Thus, even in M9 medium, inclusion of GSSG enhanced the formation of the intermolecular disulfide bonds involving DsbA33S (Fig. 2B, compare lanes1 and 3). GSSG was effective at concentration of 1 mM (data not shown) or 10 mM (Fig. 2B). It was found additionally that the disulfide-mediated intermolecular cross-linking was more extensive in the absence of chromosomal dsbA function than in its presence (Fig. 2B, compare lanes3 and 4).

DsbA33S Protein Preferentially Forms Mixed Disulfides with DsbB

The apparent molecular weight of the major DsbA33S-cross-linked polypeptide formed in minimal medium (without GSSG) was similar to the sum of those of DsbA and DsbB. It indeed represented a complex between DsbA33S and DsbB as the following lines of evidence indicated. First, this particular product fractionated with membranes (data not shown). Second, it was undetectable when DsbA33S was overproduced in the dsbB::kan5 cells (Fig. 3A, compare lanes1 and 2). Third, when both DsbA33S and DsbB were overproduced, the intensity of this product increased (Fig. 3A, compare lanes5 and 6). Finally, when a DsbB`-Myc fusion protein that was 10 amino acids longer than the normal DsbB was expressed from a plasmid, a new band of higher apparent molecular mass was created (Fig. 3A, lane4). A band that precisely coincided with this new band was stained by antibody against the Myc epitope but only when DsbA33S was synthesized simultaneously (Fig. 3B, lane2). Two-dimensional SDS-PAGE (not shown) indicated that, upon reduction, mobility of the Myc-cross-reacting material was shifted to that of DsbB`-Myc (see Fig. 3B, lane1).()These results, taken together, indicate that the DsbA33S protein specifically interacts with DsbB to form disulfide-bonded heterodimers.


Figure 3: Demonstration of a DsbA33S-DsbB complex. A, cells of CU141 (for all lanes except lane2) or SS141 (dsbB::kan5; lane2) that carried plasmids as indicated below were grown in M9/glycerol medium and analyzed by non-reducing SDS-PAGE and immunoblotting with anti-DsbA serum, as described in the legend to Fig. 2. Plasmids carried were: lanes1 and 2, pSS20 (dsbA33S); lane3, pSS18 (dsbA) and pSS42 (dsbB`-myc); lane4, pSS20 and pSS42; lane5, pSS20 and pSTV29 (vector); lane6, pSS20 and pSS39 (dsbB). The upper and lowerarrowheads indicate the DsbA33S-DsbB`-Myc complex and DsbA33S-DsbB complex, respectively. B, the same samples as used in lanes 3-6 of A were electrophoresed in lanes1-4, respectively, and stained with anti-Myc antibody. The arrowhead indicates the DsbA33S-DsB`-Myc complex, and the asterisk indicates the DsB`-Myc protein.



Effects of GSSG on the Activities of DsbA33S Mutant Protein

Previous studies showed that purified DsbA33S mutant protein retains activities to bind glutathione as well as to reduce oxidized insulin(10) . We also noted that the mutant form of DsbA can stimulate oxidative folding of a mutant human lysozyme in the presence of GSSG(13) . On the other hand, Bardwell et al. (4) showed that GSSG phenotypically suppresses the dsbB disruption, probably by reoxidizing the reduced form of DsbA. We examined effects of GSSG on the in vivo complementation and interference activities of DsbA33S. Inclusion of 1 mM GSSG in the medium partially restored the Bla-oxidizing ability of DsbA33S (Fig. 4, compare lanes2 and 6). GSSG at 1 mM almost completely abolished the dominant negative effect of DsbA33S (Fig. 4, lane7). This concentration of GSSG only slightly enhanced disulfide bond formation in the dsbB::kan5 strain (Fig. 4, lane8), while 10 mM GSSG effectively suppressed the dsbB mutation (Fig. 4, lane12). GSSG up to 10 mM did not restore the defective disulfide bond formation in the dsbA-disrupted strain ((4) ),()indicating that GSSG can help the functioning of DsbA in the absence of DsbB but cannot substitute for the DsbA function itself(4) . The effective suppression of the dominant interference by 1 mM GSSG might be due to activation of the DsbA33S form of DsbA. Such an unusual mode of action of DsbA should be independent of the DsbB function.


Figure 4: Suppression of the DsbA33S phenotypes by GSSG. Cells as indicated below were grown in M9 medium supplemented with GSSG at the final concentration of 0 (lanes 1-4), 1 (lanes 5-8), or 10 (lanes 9-12) mM. Disulfide bond formation of Bla was examined by non-reducing SDS-PAGE followed by immunoblotting. Strains used were: lanes 1, 5, and 9, CU141/pMW119 (vector); lanes 2, 6, and 10, SS140 (dsbA::Tn5)/pSS20 (dsbA33S); lanes 3, 7, and 11, CU141/pSS20; lanes 4, 8, and 12, SS141 (dsbB::kan5)/pMW119. It should be noted that the lower bands in lanes 2-4 largely represent the background protein of unknown identity (see also Fig. 1).




DISCUSSION

Like in any other thioredoxin-like oxidoreductases, the two cysteine residues of DsbA are of vital importance for the physiological functions of this enzyme. Detailed studies established that the two cysteines have different reactivities. The amino-terminally located Cys has unusually low pK(12) and is very reactive with alkylating reagents as well as with small molecule sulfhydryls(9, 10) . The mixed disulfide between GSH and Cys of DsbA is very unstable(10) . Although similar instability has been noted for the oxidized form of wild-type DsbA (9, 10, 11) , DsbA in the periplasmic space of living cells is mostly in the oxidized state, provided that DsbB is functional(5, 21) . In contrast to Cys, Cys residue is buried inside the protein and inaccessible from the solvent(9, 10, 12) . Apparently, the asymmetric nature of the two cysteines contributes to the catalytic reactivity of this unusual enzyme(16) .

It is shown here that the DsbA33S mutant protein, in which only the reactive Cys residue remains, is disulfide-bonded with a number of cellular proteins in the presence of low molecular weight disulfides. It might be possible that the reactive Cys residue first forms mixed disulfides with cystine or glutathione added exogenously. This binding may activate the protein. Due possibly to the polypeptide-binding nature of DsbA(8) , the disulfide bond may be then transferred to a free cysteine on the substrate protein. Repeating this cycle might lead to the formation of protein disulfides in the presence of GSSG(13) .

Although the formation of disulfide-bonded complexes between DsbA33S and general protein substrates requires the presence of GSSG or other small molecule disulfides, DsbA33S is preferentially disulfide-bonded with DsbB in the absence of GSSG. It should be noted that our method of detection, in which cellular proteins were first denatured by trichloroacetic acid and then dissolved in SDS in the presence of iodoacetamide, precludes the possibility of artificial formation of the disulfide-bonded complexes after cell disruption(21) . It seems likely that DsbA has specific affinity to DsbB and that Cys of DsbA can react with one of the four essential cysteines of DsbB without mediation of GSSG.

The high reactivity of Cys of DsbA with a cysteine residue of DsbB will contribute to the dominant interference by the DsbA33S mutant protein, which should sequester the DsbB protein in the heterodimeric complex. More importantly, the high reactivity of the cysteine residues on the DsbA and the DsbB protein will contribute to the proposed (4, 16) reoxidation of the reduced form of DsbA by DsbB. We suppose it likely that the DsbA33S-DsbB complex we observed here represents an intermediate state of DsbA-DsbB recycling reaction that has been fixed because of the absence of the partner cysteine, Cys, on DsbA.


FOOTNOTES

*
This work was supported by grants from the ministry of Education, Science and Culture, Japan, from Yamada Science Foundation, and from Mitsubishi Kasei Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, 1916 Noji-cho, Kusatsu, Shiga 525, Japan.

To whom correspondence should be addressed. Tel.: 81-75-751-4015; Fax: 81-75-771-5699 or 81-75-761-5626; kito{at}virus.kyoto-u.ac.jp

T. Yoshihisa, personal communication.

The abbreviations used are: Bla, -lactamase; PAGE, polyacrylamide gel electrophoresis.

Note that the 41-kDa band in lane4 of Fig. 2 actually contained two components, the DsbA dimer and an unrelated background protein that was observed in broth-grown cells (lanes 1, 2, 3, 5, and 6).

In the experiments reported in Fig. 3B, whole cell proteins were first precipitated by trichloroacetic acid. Although this treatment was necessary to reduce the background stainings as well as to avoid a possible artifact during the sample manipulations (21), it proved to reduce the recovery of the free form of DsbB`-Myc protein (asterisk in lane1 of Fig. 3B). The acid treatment did not appear to lower the recovery of DsbA33S-DsB`-Myc complex.

S. Kishigami, E. Kanaya, M. Kikuchi, and K. Ito, unpublished results.


ACKNOWLEDGEMENTS

We thank Jim Bardwell and Jon Beckwith for providing the dsbB::kan5 mutant, Tohru Yoshihisa for the plasmid carrying the Myc sequence and helpful comments and discussion, Yoshinori Akiyama for helpful discussion throughout the work, and Kiyoko Mochizuki and Kuniko Ueda for technical support.


REFERENCES
  1. Bardwell, J. C. A., and Beckwith, J.(1993)Cell 74, 769-771 [Medline] [Order article via Infotrieve]
  2. Kamitani, S., Akiyama, Y., and Ito, K.(1992)EMBO J. 11, 57-62 [Abstract]
  3. Bardwell, J. C., McGovern, K., and Beckwith, J.(1991)Cell 67, 581-589 [Medline] [Order article via Infotrieve]
  4. Bardwell, J. C., Lee, J. O., Jander, G., Martin, N., Belin, D., and Beckwith, J.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1038-1042 [Abstract]
  5. Missiakas, D., Georgopoulos, C., and Raina, S.(1993)Proc. Natl. Acad. Sci. U. S. A. 90, 7084-7088 [Abstract]
  6. Missiakas, D., Georgopoulos, C., and Raina, S.(1994)EMBO J. 13, 2013-2020 [Abstract]
  7. Jander, G., Martin, N. L., and Beckwith, J.(1994)EMBO J. 13, 5121-5127 [Abstract]
  8. Martin, J. C. L., Bardwell, J. C. A., and Kuriyan, J.(1993)Nature 365, 464-468 [CrossRef][Medline] [Order article via Infotrieve]
  9. Zapun, A., Bardwell, J. C. A., and Creighton, T. E.(1993)Biochemistry 32, 5083-5092 [Medline] [Order article via Infotrieve]
  10. Zapun, A., Cooper, L., and Creighton, T. E.(1994)Biochemistry 33, 1907-1914 [Medline] [Order article via Infotrieve]
  11. Wunderlich, M., Jaenicke, R., and Glockshuber, R.(1993)J. Mol. Biol. 233, 559-566 [CrossRef][Medline] [Order article via Infotrieve]
  12. Nelson, J. W., and Creighton, T. E.(1994)Biochemistry 33, 5974-5983 [Medline] [Order article via Infotrieve]
  13. Kanaya, E., Anaguchi, H., and Kikuchi, M.(1994)J. Biol. Chem. 269, 4273-4278 [Abstract/Free Full Text]
  14. Akiyama, Y., Kamitani, S., Kusukawa, N., and Ito, K.(1992)J. Biol. Chem. 267, 22440-22445 [Abstract/Free Full Text]
  15. Akiyama, Y., and Ito, K. (1993)J. Biol. Chem. 268, 8146-8150 [Abstract/Free Full Text]
  16. Bardwell, J. C. A. (1994)Mol. Microbiol. 14, 199-205 [Medline] [Order article via Infotrieve]
  17. Holmgren, A. (1989)J. Biol. Chem. 264, 13963-13966 [Free Full Text]
  18. Yu, J., McLaughlin, S., Freedman, R. B., and Hirst, T. R.(1993)J. Biol. Chem. 268, 4326-4330 [Abstract/Free Full Text]
  19. Akiyama, Y., Ogura, T., and Ito, K.(1994)J. Biol. Chem. 269, 5218-5224 [Abstract/Free Full Text]
  20. Ito, K., Wittekind, M., Nomura, M., Shiba, K., Yura, T., Miura, A., and Nashimoto, H. (1983)Cell 32, 789-797 [Medline] [Order article via Infotrieve]
  21. Kishigami, S., Akiyama, Y., and Ito, K.(1995)FEBS Lett. 364, 55-58 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.