Conserved Role of the Linker α-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB

Abstract

In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the α-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying α-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved. In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the α-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying α-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved. Disulfide bonds formation is a critical step in protein folding. By covalently cross-linking amino acids far apart in the protein primary structure, the formation of native disulfides is associated with the increased stability and structural complexity typical of many secreted proteins. In bacterial cells disulfide bond formation occurs in the periplasmic space. The soluble enzyme DsbA 3The abbreviations used are: Dsb, disulfide bond protein; tPA, tissue plasminogen activator. is maintained in an oxidized state by the membrane protein DsbB, and catalyzes the rapid, but rather indiscriminate oxidation of cysteines in substrate proteins. DsbB recycles DsbA by transferring electrons to molecular oxygen or other electron acceptors through quinones and the membrane enzymes of the respiratory chain. Non-correctly paired cysteines are rearranged by the soluble enzyme DsbC, which is maintained in a reduced state by the membrane protein DsbD, which transfers electrons from thioredoxin in the cytoplasm to DsbC in the periplasm (1Nakamoto H. Bardwell J.C. Biochim. Biophys. Acta. 2004; 1694: 111-119Crossref PubMed Scopus (151) Google Scholar, 2Kadokura H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (447) Google Scholar, 3Collet J.F. Bardwell J.C. Mol. Microbiol. 2002; 44: 1-8Crossref PubMed Scopus (178) Google Scholar). The DsbA-DsbB and DsbC-DsbD systems transfer electrons in opposite directions, i.e. from and into the periplasm, respectively. The presence of cross-talk between the oxidation and isomerization pathways would be expected to result in a wasteful, futile cycle that would be detrimental for the cell. For this reason, large kinetic barriers must have evolved to prevent non-physiological interactions between the Dsb enzymes; therefore, allowing the co-existence of the two pathways in the same cellular environment (4Rozhkova A. Stirnimann C.U. Frei P. Grauschopf U. Brunisholz R. Grutter M.G. Capitani G. Glockshuber R. EMBO J. 2004; 23: 1709-1719Crossref PubMed Scopus (109) Google Scholar). The structural features of DsbC that prevent its misoxidation by DsbB are not well understood. The catalytic domains of DsbC and DsbA are structurally similar, and they are both thioredoxin motif proteins (1Nakamoto H. Bardwell J.C. Biochim. Biophys. Acta. 2004; 1694: 111-119Crossref PubMed Scopus (151) Google Scholar). However, whereas DsbA is a monomer, DsbC is a homodimer with each subunit comprising the catalytic thioredoxin domain connected via a long α-helical linker to a domain responsible for dimerization and substrate binding (5McCarthy A.A. Haebel P.W. Torronen A. Rybin V. Baker E.N. Metcalf P. Nat. Struct. Biol. 2000; 7: 196-199Crossref PubMed Scopus (218) Google Scholar). Earlier genetic and biochemical studies by Bardwell and coworkers (6Bader M.W. Hiniker A. Regeimbal J. Goldstone D. Haebel P.W. Riemer J. Metcalf P. Bardwell J.C. EMBO J. 2001; 20: 1555-1562Crossref PubMed Scopus (92) Google Scholar) suggested that the dimerization of the thioredoxin domains is responsible for preventing oxidation by DsbB. Recently we constructed a series of chimeric proteins comprising the DsbC dimerization domain linked to either TrxA or to DsbA. Several of these chimeras catalyzed disulfide isomerization and oxidation in vivo, suggesting that dimerization of thioredoxin domains is not sufficient to confer protection from the action of DsbB (7Segatori L. Paukstelis P.J. Gilbert H.F. Georgiou G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10018-10023Crossref PubMed Scopus (41) Google Scholar). Comparison of the structure of the Escherichia coli DsbC with that of its homologue DsbG suggests that the α-linker that connects the dimerization and the catalytic domains may play a role in determining the specificity of the enzyme for substrate proteins (8Heras B. Edeling M.A. Schirra H.J. Raina S. Martin J.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8876-8881Crossref PubMed Scopus (87) Google Scholar). Interestingly, in DsbG as well as in the DsbCs from both E. coli and Haemophilus influenza (9Zhang M. Monzingo A.F. Segatori L. Georgiou G. Robertus J.D. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 1512-1518Crossref PubMed Scopus (14) Google Scholar), the α-helical linker serves to place the CXXC active sites within the two thioredoxin domains directly facing each other, indicating that this is a conserved feature of bacterial disulfide isomerase enzymes. These observations, together with our data using DsbC chimeras (7Segatori L. Paukstelis P.J. Gilbert H.F. Georgiou G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10018-10023Crossref PubMed Scopus (41) Google Scholar), suggested that the α-helical linker may play a pivotal role in the function of DsbC enzymes. Here we show that deletions in the α-helical linker abolish the ability of DsbC to avoid oxidation by DsbB and that this phenomenon is conserved among members of this enzyme family. However, the DsbC mutant enzymes retained the ability to catalyze the rearrangement of disulfide bonds in substrate proteins expressed in the E. coli periplasm, indicating that the reduction of the enzyme by DsbD is not compromised by the deletions. Strains and Plasmids—The bacterial strains and plasmids used in this study are listed in Table 1. The putative from H. influenzae, Pseudomonas aeruginosa, Vibrio Erwinia chrysanthemi, and Yersinia from the of bacterial cells and into D. Beckwith J. J. PubMed Scopus Google Scholar). The α-helix of DsbC from E. H. influenzae, and by and into the a C-terminal For protein the putative and DsbC with and into and into E. coli and and L. H.F. J. PubMed Google dsbA L. J.L. S. Georgiou G. Bardwell J.C. J.F. 2004; PubMed Scopus Google L. J.L. S. Georgiou G. Bardwell J.C. J.F. 2004; PubMed Scopus Google J. Bardwell J.C. Georgiou G. J. 2001; PubMed Scopus Google C-terminal from E. coli in with in J.C. PubMed Scopus Google from H. in from in from P. in from V. cholerae in from E. in from E. coli in from E. coli in from E. coli in from E. coli in from E. coli in from H. in from P. in from V. cholerae in from E. in from in from E. coli in from E. coli in from E. coli in from E. coli in from in from in from in from in from H. in from H. in from H. in from H. in in a In the of the DsbC and the on the folding of a of the tissue plasminogen containing disulfide bonds comprising the catalytic and domains of the E. coli and E. coli with the and with at in of with and from to of and to a of vtPA with for by and at The in as to the of cells and with a the of by at the soluble protein by the protein by the in with to a protein of of the to of the same containing of plasminogen and at and the in in E. coli M. D. Beckwith J. J. Biol. PubMed Scopus Google Scholar) with the DsbC and a the M. D. Beckwith J. J. Biol. PubMed Scopus Google Scholar). in and as M. D. Beckwith J. J. Biol. PubMed Scopus Google Scholar). study the in of the E. coli and with the in with and and in containing amino and with and the to a of cells and with and in a The of as E. Beckwith J. J. Mol. Biol. PubMed Scopus Google Scholar). For of E. coli with plasmids in and in amino with and as of to the same on the of containing the same with and and at for and the of the E. coli and or the α-helix the the in and the plasmids into E. coli and as (7Segatori L. Paukstelis P.J. Gilbert H.F. Georgiou G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10018-10023Crossref PubMed Scopus (41) Google Scholar). proteins used in this study as by DsbB as M. Bardwell J.C. PubMed Scopus Google Scholar, M. Bardwell J. J. Biol. PubMed Scopus Google Scholar), with the that a with used in place of a The of the DsbB For the of E. coli H. H. Bardwell J.C. Beckwith J. 2004; PubMed Scopus Google Scholar) carrying a dsbA H. H. Bardwell J.C. Beckwith J. 2004; PubMed Scopus Google Scholar), in with with the and the cells at DsbA from the periplasmic by a by on a as B. Glockshuber R. J. Biol. 2002; PubMed Scopus Google Scholar). or α-helix by in for at by on with the oxidation of DsbC by DsbB in reduced DsbC or DsbC in at with in The by the addition of DsbB to a of between DsbA and DsbB using reduced DsbA and DsbB. and the by with to a The proteins by and with in reduced DsbC with in as H. M. H. Bardwell J.C. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). and oxidized of the proteins by and with The of reduction in the presence of as A. J. Biol. PubMed Google Scholar). The of in the as a of the of reduced and the is expressed as the of the of the to the E. A. Biochem. Biochem. Mol. Biol. PubMed Scopus Google Scholar). The of reduced, as by and Gilbert Gilbert H.F. PubMed Scopus Google Scholar). The protection of from to a study J. H. U. PubMed Scopus Google Scholar). The of with or of each of the DsbC of DsbC long α-helix linker acids the dimerization domain to the catalytic domain in the E. coli DsbC, the two protein domains structurally and (5McCarthy A.A. Haebel P.W. Torronen A. Rybin V. Baker E.N. Metcalf P. Nat. Struct. Biol. 2000; 7: 196-199Crossref PubMed Scopus (218) Google Scholar). We constructed a series of mutant DsbC of or amino acids within the α-helical linker and to the proteins and respectively. residues are in a α-helical the of each amino in the DsbC linker would be expected to the catalytic domain to be by with to the dimerization domain. As a result, the active which in DsbC are facing each other, in the would be expected to be by an is by the of amino acids The the deletions in the D. Beckwith J. J. PubMed Scopus Google Scholar), the of the the used in these the E. coli DsbC and the α-helix to as by with a for the C-terminal Disulfide in oxidation of protein cysteines by DsbA is and may the formation of disulfides between cysteines in the these residues may not be connected in the of the i.e. the native state M. D. Beckwith J. J. Biol. PubMed Scopus Google Scholar, A. Bardwell J.C. PubMed Scopus Google Scholar). For this the folding of proteins is by the isomerization of bonds. The folding of native the periplasmic or the and have to on the action of DsbC. In proteins are to isomerization to active M. D. Beckwith J. J. Biol. PubMed Scopus Google Scholar). we that of DsbC from in a in the of active a of tissue plasminogen containing to cells DsbC from the J. Georgiou G. Microbiol. PubMed Google Scholar) We that of the DsbC containing deletions in the α-helix linker the folding of However, and result in of the active vtPA to the DsbC. the other of or amino acids in active vtPA and to with DsbC and with the native substrate i.e. and a of to and of the role of DsbA as the primary of many proteins to in dsbA As a result, dsbA phenotypes as or of H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (447) Google Scholar). The kinetic between the and as we DsbC to as an of the proteins. the of the α-helical linker of DsbC in the of this the DsbC expressed in a and ability to the associated with of disulfide bond formation In E. coli in the than that of its of DsbC from a did not result in a in with the of the enzyme is maintained in a reduced by DsbD, and as a protein J.C. PubMed Scopus Google Scholar). However, of one or more amino acids within the α-helical linker of DsbC to enzymes that could the formation of active We that the ability of the to the of with the disulfide isomerase as in the vtPA and In other and the mutant proteins that a of active vtPA and could in to of the from the expressed whereas and the opposite and The ability of the and to folding in the of DsbA suggests that these DsbC are of at of the function of DsbA. with not in a mutant not revealing that the of the DsbC is on by DsbB. The of the by cells on depends on the in the cells and of DsbC from did not However, with the the DsbC α-helix with with or with and the in data that the of DsbC is on the presence of DsbB, we the ability of and to be oxidized by DsbB in proteins C-terminal and could be to by protein that the DsbC are that the structure of the mutant proteins not by the deletions within the α-helix not in the respiratory as a of electrons from DsbB. of the reduced of each DsbC by DsbB in the presence of as the and by the addition of DsbB H. M. H. Bardwell J.C. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). of with the oxidized and reduced proteins by In this DsbA oxidized of with DsbB. In DsbC oxidation by DsbB of the protein found in the reduced state of with DsbB. of one or more amino acids in the α-helix increased its to oxidation by DsbB. with the in in deletions of two or amino acids in greater to and of and found in the oxidized state with and of and respectively. The disulfide isomerase of the DsbC α-helix in the of reduced by the in by active as a function of In this an that to the oxidation of is by an in the of of as more native enzyme Gilbert H.F. PubMed Scopus Google Scholar). In this DsbC isomerase to the enzyme The of the enzymes by reduction in the presence of which in the of and a in A. J. Biol. PubMed Google Scholar). The than that of the enzyme In to the data in deletions of or amino acids for the did not result in disulfide isomerase and to the and that the and isomerase in not the catalytic of the enzymes but rather the of in interactions with DsbB, and The of the chimeras on the protection of from The DsbC the of to an to the DsbC suggesting that in the α-helical linker not the of are expressed as from a of isomerization enzyme are expressed as are expressed as are expressed as The from a of isomerization enzyme in a In and in of DsbC proteins are among are more than DsbC homologues in the data which between and identity with the E. coli We the ability of DsbCs to be reduced by DsbD, avoid oxidation by DsbB, is conserved among members of the enzyme family. from the H. influenzae, P. aeruginosa, E. chrysanthemi, pseudotuberculosis, and V. each with E. coli DsbC an amino sequence identity from to by of The the and to a C-terminal and expressed from the of the V. cholerae the other DsbCs in the periplasmic at a to the E. coli enzyme not As in the DsbC homologues as as the E. coli enzyme in the folding of vtPA into its active The V. cholerae DsbC a of active vtPA but this is to the of that enzyme in the E. coli of the DsbCs could the of or in not also suggesting that the ability to avoid oxidation by DsbB must be a conserved property among DsbC of H. and DsbC of active with and the H. and DsbC proteins in and vtPA as in The with and with of dsbA cells with the and in and as in The H. influenzae, P. aeruginosa, E. chrysanthemi, pseudotuberculosis, and V. enzymes to as by by and from the E. coli The and for the of from the V. cholerae which did not with substrate the enzymes and in the of As a result, the enzymes isomerase with as substrate with the E. enzyme a to the E. coli DsbC. the protein disulfide isomerase is at a isomerase with the bacterial to a as in Table The V. cholerae DsbC to be an with as the whereas the other enzymes in that and The of the DsbCs on the protection of from and and The is an of the of the enzymes. Interestingly, the Vibrio as well as the Pseudomonas DsbCs of DsbCs from bacterial are expressed as from a of isomerization enzyme is the of the of as a function of are expressed as are expressed as not using to not are expressed as The from a of isomerization enzyme is the of the of as a function of not using to not in a the bacterial in this DsbCs from H. and to the of in the α-helical linker on the of the In to the E. coli DsbC, we constructed a series of carrying deletions of amino acids and of the H. and proteins. The the deletions in the The ability of the H. and DsbC mutant enzymes to assist the folding of vtPA in the periplasm of E. coli of the DsbC mutant enzymes the folding of the of one or amino acids in the α-helical linker of either disulfide isomerase in the formation of DsbC with isomerase to the whereas deletions of two or amino acids in the same region of the a of in the of active vtPA We also the ability of H. and DsbC to the of a dsbA As in of the DsbC in a dsbA therefore, as of oxidation in the periplasm of E. For both Haemophilus and Yersinia the of two and amino acids to than the of one or amino the in the E. coli DsbC isomerase and is also in the DsbCs In this study we the of deletions in the α-helical linker of the DsbC on its ability to oxidation by DsbB and on the of disulfide bond isomerization in and an in In to the a amino in the α-helical linker DsbC to the of disulfide bond formation in dsbA cells. The of and on the α-helical linker and on by DsbB in Examination of the of oxidation of DsbCs by DsbB in the presence of a electron in revealed that in the linker DsbC more to oxidation of did not abolish the disulfide isomerase of the mutant DsbC enzymes. the mutant enzymes catalyzed disulfide isomerization of both vtPA and the native E. coli protein in a DsbD-dependent manner, with reduced and As of disulfide bond rearrangement by the mutant DsbCs on reduction by The enzymes that a ability to assist the folding of proteins with multiple disulfides in a that a in that a of the enzyme is oxidized by DsbB at the of reduced DsbC that is to function as an the α-helical linker of as the catalyst for both protein oxidation in a dsbA and for disulfide bond For this to enzyme that oxidized by DsbB must at state with enzyme that reduced by In this a of oxidized DsbC would be expected to be directly reduced by DsbD they transfer disulfides into periplasmic protein of the reduced may oxidation by DsbB in disulfide result in an electron from which is used for the reduction of DsbD by to the mutant DsbC, to DsbB, and to the quinones and molecular would be expected to in an that could the However, of the mutant DsbCs in a dsbA did not with the not the electron in the cycle is therefore, not a on the at the interactions of DsbC family enzymes with DsbD and DsbB we the in and in function of homologues from H. influenzae, P. aeruginosa, V. E. chrysanthemi, and the of amino among these enzymes could be by E. coli DsbD and the folding of vtPA to from with the E. coli DsbC expressed The structure of the E. coli revealed that the between the two residues from the thioredoxin domain of DsbC and to have for disulfide P.W. Goldstone D. Katzen F. Beckwith J. Metcalf P. EMBO J. 2002; PubMed Scopus Google Scholar). and are the residues responsible for the binding between E. coli DsbC and P.W. Goldstone D. Katzen F. Beckwith J. Metcalf P. EMBO J. 2002; PubMed Scopus Google Scholar). revealed that these residues are conserved among the putative DsbCs in the data residues in and data not In the bacterial disulfide to have a conserved mechanism for interactions with DsbB. DsbB to with thioredoxin family proteins (6Bader M.W. Hiniker A. Regeimbal J. Goldstone D. Haebel P.W. Riemer J. Metcalf P. Bardwell J.C. EMBO J. 2001; 20: 1555-1562Crossref PubMed Scopus (92) Google Scholar, S. M. Glockshuber R. E. EMBO J. PubMed Scopus Google Scholar, L. Beckwith J. J. 2000; PubMed Scopus Google Scholar), and as oxidize the active of DsbC in the state (6Bader M.W. Hiniker A. Regeimbal J. Goldstone D. Haebel P.W. Riemer J. Metcalf P. Bardwell J.C. EMBO J. 2001; 20: 1555-1562Crossref PubMed Scopus (92) Google Scholar). However, the of the enzymes from used in this study to oxidation by the E. coli DsbB and and data not In with our the E. coli DsbC, deletions in the α-helical linker of the H. influenza and DsbCs the mutant enzymes to the phenotypes by dsbA cells These that the linker region and the of the active sites in DsbC enzymes evolved to oxidation in the periplasm of bacterial cells. Interestingly, the of deletions in the linker of the E. H. and the same of in oxidation and isomerization of either or amino acids in in disulfide isomerase and In of or amino acids of dsbA phenotypes but or vtPA which folding depends on disulfide isomerization of in isomerization and oxidation on the linker not from the catalytic of the enzymes of the E. coli enzymes revealed in isomerization as in the well the DsbC in the than the E. coli enzyme and and did not with the ability of the enzymes to disulfide bond isomerization in For and the in a of vtPA and in from the of reduction of could not the in that the dimerization region and the thioredoxin catalytic domain of DsbC are to and are on J. Biol. 2000; PubMed Scopus Google Scholar), which is also by our of DsbC is that a in the linker in in either of these of an amino in the linker would be expected to the α-helix and a of the of the and the catalytic the active by amino to the of of the The reduction in the of the α-helix amino is and would result in an of the between the binding the by the dimerization (5McCarthy A.A. Haebel P.W. Torronen A. Rybin V. Baker E.N. Metcalf P. Nat. Struct. Biol. 2000; 7: 196-199Crossref PubMed Scopus (218) Google Scholar) and the catalytic is that the of the linker by may be the for the in the in and in of the be that the two DsbC family enzymes are coli DsbC and have linker However, in enzymes the catalytic sites are to each other and at with to the long of we that the of linker deletions is more to from the of the two active sites with to each a of the active sites from the in DsbC a of the motif by would be expected to in the to the enzyme to the of the proteins with DsbB and is not a of the active sites by in would to a protein that is more reduced by DsbD and more to oxidation by DsbB with the mutant the active sites would be expected to be by would be to the two active sites in the both with as is the with the DsbC The of the DsbC in However, we a in addition to the on the of the catalytic the the α-helix to be more or the ability of the protein to in the presence of its the of the catalytic in with these the DsbC to with both DsbD and DsbB. of these have to the of and which is in In large kinetic barriers prevent the of electrons between enzymes of the Dsb interactions between and are in to of than interactions (4Rozhkova A. Stirnimann C.U. Frei P. Grauschopf U. Brunisholz R. Grutter M.G. Capitani G. Glockshuber R. EMBO J. 2004; 23: 1709-1719Crossref PubMed Scopus (109) Google Scholar). (6Bader M.W. Hiniker A. Regeimbal J. Goldstone D. Haebel P.W. Riemer J. Metcalf P. Bardwell J.C. EMBO J. 2001; 20: 1555-1562Crossref PubMed Scopus (92) Google Scholar) that that abolish the dimerization of DsbC the protein to as a in a manner, to the that the structure of the enzyme is responsible for its resistance to oxidation by DsbB. However, that is not the of the enzyme that the of the DsbC active with the catalytic of DsbB more features that to the and of the active sites in the structure of the We and for with the of the DsbC for with the phytase and for the with