Structural and functional characterization of the oxidoreductase a-DsbA1 from wolbachia pipientis

The &alpha;-proteobacterium Wolbachia pipientis is a highly successful intracellular endosymbiont of invertebrates that manipulates its host\u27s reproductive biology to facilitate its own maternal transmission. The fastidious nature of Wolbachia and the lack of genetic transformation have hampered analysis of the molecular basis of these manipulations. Structure determination of key Wolbachia proteins will enable the development of inhibitors for chemical genetics studies. Wolbachia encodes a homologue (&alpha;-DsbA1) of the Escherichia coli dithiol oxidase enzyme EcDsbA, essential for the oxidative folding of many exported proteins. We found that the active-site cysteine pair of Wolbachia &alpha;-DsbA1 has the most reducing redox potential of any characterized DsbA. In addition, Wolbachia &alpha;-DsbA1 possesses a second disulfide that is highly conserved in &alpha;-proteobacterial DsbAs but not in other DsbAs. The &alpha;-DsbA1 structure lacks the characteristic hydrophobic features of EcDsbA, and the protein neither complements EcDsbA deletion mutants in E. coli nor interacts with EcDsbB, the redox partner of EcDsbA. The surface characteristics and redox profile of &alpha;-DsbA1 indicate that it probably plays a specialized oxidative folding role with a narrow substrate specificity. This first report of a Wolbachia protein structure provides the basis for future chemical genetics studies.<br /

Structural and biochemical characterization of Chlamydia trachomatis DsbA reveals a cysteine-rich and weakly oxidising oxidoreductase

Copyright © 2016 Christensen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character. In this study we show that the truncated soluble form of the predicted membrane anchored protein C. trachomatis DsbA (CtDsbA) has oxidase activity and redox properties broadly similar to other characterized DsbA proteins. However CtDsbA is distinguished from other DsbAs by having six cysteines, including a second disulfide bond, and an unusual dipeptide sequence in its catalytic motif (Cys-Ser-Ala-Cys). We report the 2.7 Å crystal structure of CtDsbA revealing a typical DsbA fold, which is most similar to that of DsbA-II type proteins. Consistent with this, the catalytic surface of CtDsbA is negatively charged and lacks the hydrophobic groove found in EcDsbA and DsbAs from other enterobacteriaceae. Biochemical characterization of CtDsbA reveals it to be weakly oxidizing compared to other DsbAs and with only a mildly destabilizing active site disulfide bond. Analysis of the crystal structure suggests that this redox character is consistent with a lack of contributing factors to stabilize the active site nucleophilic thiolate relative to more oxidizing DsbA proteins

The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis

open20openRosado, Leonardo Astolfi; Wahni, Khadija; Degiacomi, Giulia; Pedre, Brandã¡n; Young, David; De la Rubia, Alfonso G.; Boldrin, Francesca; Martens, Edo; Marcos-Pascual, Laura; Sancho-Vaello, Enea; Albesa-Jové, David; Provvedi, Roberta; Martin, Charlotte; Makarov, Vadim; Versã©es, Wim; Verniest, Guido; Guerin, Marcelo; Mateos, Luis M.; Manganelli, Riccardo; Messens, JorisRosado, Leonardo Astolfi; Wahni, Khadija; Degiacomi, Giulia; Pedre, Brandã¡n; Young, David; De la Rubia, Alfonso G.; Boldrin, Francesca; Martens, Edo; Marcos-Pascual, Laura; Sancho-Vaello, Enea; Albesa-Jové, David; Provvedi, Roberta; Martin, Charlotte; Makarov, Vadim; Versã©es, Wim; Verniest, Guido; Guerin, Marcelo; Mateos, Luis M.; Manganelli, Riccardo; Messens, Jori

Structure and mechanisms of the DsbB–DsbA disulfide bond generation machine

AbstractAll organisms possess specific cellular machinery that introduces disulfide bonds into proteins newly synthesized and transported out of the cytosol. In E. coli, the membrane-integrated DsbB protein cooperates with ubiquinone to generate a disulfide bond, which is transferred to DsbA, a periplasmic dithiol oxido-reductase that serves as the direct disulfide bond donor to proteins folding oxidatively in this compartment. Despite the extensive accumulation of knowledge on this oxidation system, molecular details of the DsbB reaction mechanisms had been controversial due partly to the lack of structural information until our recent determination of the crystal structure of a DsbA–DsbB–ubiquinone complex. In this review we discuss the structural and chemical nature of reaction intermediates in the DsbB catalysis and the illuminated molecular mechanisms that account for the de novo formation of a disulfide bond and its donation to DsbA. It is suggested that DsbB gains the ability to oxidize its specific substrate, DsbA, having very high redox potential, by undergoing a DsbA-induced rearrangement of cysteine residues. One of the DsbB cysteines that are now reduced then interacts with ubiquinone to form a charge transfer complex, leading to the regeneration of a disulfide at the DsbB active site, and the cycle can begin anew

Properties of the thioredoxin fold superfamily are modulated by a single amino acid residue

The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins

The 1.2 A resolution crystal structure of TcpG, the Vibrio cholerae DsbA disulfide-forming protein required for pilus and cholera-toxin production

The enzyme TcpG is a periplasmic protein produced by the Gram-negative pathogen Vibrio cholerae. TcpG is essential for the production of ToxR-regulated proteins, including virulence-factor pilus proteins and cholera toxin, and is therefore a target for the development of a new class of anti-virulence drugs. Here, the 1.2 Å resolution crystal structure of TcpG is reported using a cryocooled crystal. This structure is compared with a previous crystal structure determined at 2.1 Å resolution from data measured at room temperature. The new crystal structure is the first DsbA crystal structure to be solved at a sufficiently high resolution to allow the inclusion of refined H atoms in the model. The redox properties of TcpG are also reported, allowing comparison of its oxidoreductase activity with those of other DSB proteins. One of the defining features of the Escherichia coli DsbA enzyme is its destabilizing disulfide, and this is also present in TcpG. The data presented here provide new insights into the structure and redox properties of this enzyme, showing that the binding mode identified between E. coli DsbB and DsbA is likely to be conserved in TcpG and that the [beta]5-[alpha]7 loop near the proposed DsbB binding site is flexible, and suggesting that the tense oxidized conformation of TcpG may be the consequence of a short contact at the active site that is induced by disulfide formation and is relieved by reduction

Protein disulphide isomerase-assisted functionalization of proteinaceous substrates

Protein disulphide isomerase (PDI) is an enzyme that catalyzes thiol-disulphide exchange reactions among a broad spectrum of substrates, including proteins and low-molecular thiols and disulphides. As the first protein-folding catalyst reported, the study of PDI has mainly involved the correct folding of several cysteine-containing proteins. Its application on the functionalization of protein-based materials has not been extensively reported. Herein, we review the applications of PDI on the modification of proteinaceous substrates and discuss its future potential. The mechanism involved in PDI functionalization of fibrous protein substrates is discussed in detail. These approaches allow innovative applications in textile dyeing and finishing, medical textiles, controlled drug delivery systems and hair or skin care products.We thank to FCT 'Fundacao para a Ciencia e Tecnologia' (scholarship SFRH/BD/38363/2007) for providing Margarida Fernandes the grant for PhD studies

The name's bond.......disulfide bond

A repeating theme in the structural biology of disulfide oxidants and isomerases is the extraordinary architectural similarity between functionally related proteins from prokaryotes and eukaryotes. The recently determined structure of full-length yeast protein disulfide isomerase (PDI) reveals a U-shaped molecule with two redox-active sites. It bears a remarkable resemblance to the V-shaped, but dimeric, bacterial disulfide isomerases DsbC and DsbG. Similarly, the much-anticipated structure of the bacterial membrane protein DsbB, the redox partner of DsbA, comprises a flexible redox loop embedded in an antiparallel four-helix bundle. This architecture is similar to that of soluble eukaryotic Ero1p and Erv2p proteins, the redox partners of PDI. Importantly, the DsbB crystal structure is a complex with DsbA, providing our first view of the molecular interactions between these two proteins