The uncharged surface features surrounding the active site of<i>Escherichia coli</i>DsbA are conserved and are implicated in peptide binding

Guddat, L.W.; Bardwell, James C.A.; Zander, Thomas; Martin, Jennifer L.

doi:10.1002/pro.5560060603

Cited by 88 publications

(99 citation statements)

References 43 publications

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“…3A). This groove is used by EcDsbA to interact with its partner protein DsbB (49) and has been proposed to be important for substrate binding interactions (54). The altered groove and the lack of a hydrophobic patch in SaDsbA suggest a differing substrate specificity to EcDsbA and indicates that SaDsbA may be unable to interact with DsbB-like quinone oxidoreductases.…”

Section: Resultsmentioning

confidence: 99%

Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

Heras

Kurz

Jarrott

et al. 2008

Journal of Biological Chemistry

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In Gram-negative bacteria, the introduction of disulfide bonds into folding proteins occurs in the periplasm and is catalyzed by donation of an energetically unstable disulfide from DsbA, which is subsequently re-oxidized through interaction with DsbB. Gram-positive bacteria lack a classic periplasm but nonetheless encode Dsb-like proteins. Staphylococcus aureus encodes just one Dsb protein, a DsbA, and no DsbB. Here we report the crystal structure of S. aureus DsbA (SaDsbA), which incorporates a thioredoxin fold with an inserted helical domain, like its Escherichia coli counterpart EcDsbA, but it lacks the characteristic hydrophobic patch and has a truncated binding groove near the active site. These findings suggest that SaDsbA has a different substrate specificity than EcDsbA. Thermodynamic studies indicate that the oxidized and reduced forms of SaDsbA are energetically equivalent, in contrast to the energetically unstable disulfide form of EcDsbA. Further, the partial complementation of EcDsbA by SaDsbA is independent of EcDsbB and biochemical assays show that SaDsbA does not interact with EcDsbB. The identical stabilities of oxidized and reduced SaDsbA may facilitate direct re-oxidation of the protein by extracellular oxidants, without the need for DsbB.The formation of native disulfide bonds through air oxidation of cysteine pairs is a slow reaction and organisms ranging from bacteria to humans encode enzymatic systems to catalyze the process. In eukaryotes, oxidative folding in the endoplasmic reticulum is primarily catalyzed by protein-disulfide isomerases that are reoxidized by Ero1p/Erv2p proteins (reviewed in Ref. 1). In Escherichia coli, dithiol oxidation takes place in the periplasm through the action of the Dsb 3 (Disulfide bond) family of proteins. Dsb proteins form two distinct pathways, the DsbA-DsbB (oxidative) pathway that introduces disulfides indiscriminately, and the DsbC/DsbG-DsbD (isomerization) pathway that shuffles incorrect disulfides (2, 3).Probably the best-studied Dsb protein is EcDsbA, the primary disulfide catalyst in E. coli (reviewed in Refs. 2, 4). This promiscuously oxidizing protein is a 21-kDa monomer containing a CPHC active site in a thioredoxin (TRX) fold with an inserted helical domain of ϳ80 residues. Upon catalyzing disulfide bond formation in substrate proteins, reduced EcDsbA relies on EcDsbB, a quinone reductase, to recover its catalytically active, and higher energy, oxidized form (5

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Section: Resultsmentioning

confidence: 99%

Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

Heras

Kurz

Jarrott

et al. 2008

Journal of Biological Chemistry

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“…This large family accommodates thioredoxin-like, glutaredoxin-like and PDI-like proteins, as well as members of the bacterial Dsb family [14,26,42,43], all of which contain one or more copies of the highly conserved thioredoxin fold, i.e. a β-α-β-α-β-α-β-β-α structure (as shown in Figures 1 and 2) encompassing a reactive -Cys-Xaa-Xaa-Cys-tetrapeptide [9,34,[44][45][46].…”

Section: Thioredoxin and The Thioredoxin Foldmentioning

confidence: 99%

The protein disulphide-isomerase family: unravelling a string of folds

Ferrari

Söling

1999

Biochemical Journal

403

359

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The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum. These proteins are characterized by the presence of one or more domains of roughly 95-110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys-tetrapeptide. In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins,

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“…To date, all domains that have a thioredoxin-like fold and interact with proteins and whose structure has been resolved include a cis-proline at the beginning of strand ␤ 4 . This cis-proline has been implicated in substrate binding for several thioredoxin superfamily members, including DsbA, thioredoxin, glutaredoxin, and glutathione Stransferase (27)(28)(29)(30)43). A proline can be found in a similar position in the model structure for the bЈ domain of PDI (16) and is conserved in the a, bЈ, and aЈ domains of all of the catalytically active human PDI family members with the exception of the bЈ domain of ERp72 (see Refs.…”

Section: The I272w Mutation In the Bј Domain Of Human Pdi Completely mentioning

confidence: 99%

Three Binding Sites in Protein-disulfide Isomerase Cooperate in Collagen Prolyl 4-Hydroxylase Tetramer Assembly

Koivunen

Salo

Myllyharju

et al. 2005

Journal of Biological Chemistry

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Protein-disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b , and a . It is a ubiquitous protein folding catalyst that in addition functions as the ␤-subunit in vertebrate collagen prolyl 4-hydroxylase (C-P4H) ␣ 2 ␤ 2 tetramers. We report here that point mutations in the primary peptide substrate binding site in the b domain of PDI did not inhibit C-P4H assembly. Based on sequence conservation, additional putative binding sites were identified in the a and a domains. Mutations in these sites significantly reduced C-P4H tetramer assembly, with the a domain mutations generally having the greater effect. When the a or a domain mutations were combined with the b domain mutation I272W tetramer assembly was further reduced, and more than 95% of the assembly was abolished when mutations in the three domains were combined. The data indicate that binding sites in three PDI domains, a, b , and a , contribute to efficient C-P4H tetramer assembly. The relative contributions of these sites were found to differ between Caenorhabditis elegans C-P4H ␣␤ dimer and human ␣ 2 ␤ 2 tetramer formation.Protein-disulfide isomerase (PDI 1 ; EC 5.3.4.1) is a ubiquitous catalyst of disulfide bond formation and rearrangement during protein folding in the endoplasmic reticulum (ER). In addition, it functions as a subunit in two enzyme complexes, the collagen prolyl 4-hydroxylases (C-P4Hs; EC 1.14.11.2) (1, 2) and microsomal triglyceride transfer protein (3). The C-P4Hs, which are crucial for the synthesis of extracellular collagen macromolecules (4), are tetrameric enzymes consisting of two catalytic ␣ subunits and two ␤ subunits identical to PDI (5). The role of PDI in this tetramer is to keep the highly insoluble ␣ subunits in solution and in a catalytically active, nonaggregated conformation (6, 7). In addition, PDI retains the tetramer within the ER, since the ␣ subunits lack the ER retrieval signal. The human C-P4Hs have at least three isoenzymes, ␣(I) 2 ␤ 2 , ␣(II) 2 ␤ 2 , and ␣(III) 2 ␤ 2 , in which the ␣ subunits differ but the ␤ subunit is always PDI (8 -11).The PDI polypeptide comprises four domains, a, b, bЈ, and aЈ, of which the first and last contain the -CGHC-catalytic site required for thiol-disulfide exchange activity (12) (see Fig. 1). The a and b domains have a thioredoxin fold (13,14), and the bЈ and aЈ domains are also thought on the basis of homology to have the same fold. In addition, there is a short linker region x between the bЈ and aЈ domains (15), and the C terminus of the polypeptide contains a highly acidic 29-amino acid extension c, which is not critical for any of the major functions of the protein except ER retrieval (16). The principle substrate binding site of PDI is located in the bЈ domain (17), which by itself is capable of binding short peptide substrates. We have recently identified a putative hydrophobic pocket in the bЈ domain by structural homology modeling and sequence alignments and shown by site-directed mutagenesis that this pocket contains several residues that...

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The uncharged surface features surrounding the active site ofEscherichia coliDsbA are conserved and are implicated in peptide binding

Cited by 88 publications

References 43 publications

Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide

The protein disulphide-isomerase family: unravelling a string of folds

Three Binding Sites in Protein-disulfide Isomerase Cooperate in Collagen Prolyl 4-Hydroxylase Tetramer Assembly

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