Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme inNeisseria meningitidis (original) (raw)
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Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis
Journal of Molecular Biology, 2009
Neisseria meningitidis encodes three DsbA oxidoreductases (NmDsbA1–NmDsbA3) that are vital for the oxidative folding of many membrane and secreted proteins, and these three enzymes are considered to exhibit different substrate specificities. This has led to the suggestion that each N. meningitidis DsbA (NmDsbA) may play a specialized role in different stages of pathogenesis; however, the molecular and structural bases of the different roles of NmDsbAs are unclear. With the aim of determining the molecular basis for substrate specificity and how this correlates to pathogenesis, we undertook a biochemical and structural characterization of the three NmDsbAs. We report the 2.0-Å-resolution crystal structure of the oxidized form of NmDsbA1, which adopted a canonical DsbA fold similar to that observed in the structures of NmDsbA3 and Escherichia coli DsbA (EcDsbA). Structural comparisons revealed variations around the active site and candidate peptide-binding region. Additionally, we demonstrate that all three NmDsbAs are strong oxidases with similar redox potentials; however, they differ from EcDsbA in their ability to be reoxidized by E. coli DsbB. Collectively, our studies suggest that the small structural differences between the NmDsbA enzymes and EcDsbA are functionally significant and are the likely determinants of substrate specificity.
Crystal Structure of Neisseria meningitidis DsbD c-terminal domain in the reduced form
2018
The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S5, Tables S1 and S2, and Movie S1. The atomic coordinates and structure factors (codes 6DPS, 6DNV, 6DNU, and 6DNL) have been deposited in the Protein Data Bank (http://wwpdb.org/). The NMR assignments have been deposited in the BRMB under accession numbers 27012 (His-n-DsbD Ox), 27013 (His-n-DsbD Red), 27014 (His-c-DsbD Ox), and 27015 (His-c-DsbD Red).
Characterization of DsbD in Neisseria meningitidis
Molecular Microbiology, 2011
Proper periplasmic disulfide bond formation is important for folding and stability of many secreted and membrane proteins, and is catalysed by three DsbA oxidoreductases in Neisseria meningitidis. DsbD provides reducing power to DsbC that shuffles incorrect disulfide bond in misfolded proteins as well as to the periplasmic enzymes that reduce apo-cytochrome c (CcsX) or repair oxidative protein damages (MrsAB). The expression of dsbD, but not other dsb genes, is positively regulated by the MisR/S two-component system. Quantitative real-time PCR analyses showed significantly reduced dsbD expression in all misR/S mutants, which was rescued by genetic complementation. The direct and specific interaction of MisR with the upstream region of the dsbD promoter was demonstrated by electrophoretic mobility shift assay, and the MisR binding sequences were mapped. Further, the expression of dsbD was found to be induced by dithiothrietol (DTT), through the MisR/S regulatory system. Surprisingly, we revealed that inactivation of dsbD can only be achieved in a strain carrying an ectopically located dsbD, in the dsbA1A2 double mutant or in the dsbA1A2A3 triple mutant, thus DsbD is indispensable for DsbAcatalysed oxidative protein folding in N. meningitidis. The defects of the meningococcal dsbA1A2 mutant in transformation and resistance to oxidative stress were more severe in the absence of dsbD.
Identification of a cation transport pathway inNeisseria meningitidisPorB
Proteins: Structure, Function, and Bioinformatics, 2013
Neisseria meningitidis is the main causative agent of bacterial meningitis. In its outer membrane, the trimeric Neisserial porin PorB is responsible for the diffusive transport of essential hydrophilic solutes across the bilayer. Previous molecular dynamics simulations based on the recent crystal structure of PorB have suggested the presence of distinct solute translocation pathways through this channel. Although PorB has been electrophysiologically characterized as anion-selective, cation translocation through nucleotide-bound PorB during pathogenesis is thought to be instrumental for host cell death. As a result, we were particularly interested in further characterizing cation transport through the pore. We combined a structural approach with additional computational analysis. Here, we present two crystal structures of PorB at 2.1 and 2.65 Å resolution. The new structures display additional electron densities around the protruding loop 3 (L3) inside the pore. We show that these electron densities can be identified as monovalent cations, in our case Cs 1 , which are tightly bound to the inner channel. Molecular dynamics simulations reveal further ion interactions and the free energy landscape for ions inside PorB. Our results suggest that the crystallographically identified locations of Cs 1 form a cation transport pathway inside the pore. This finding suggests how positively charged ions are translocated through PorB when the channel is inserted into mitochondrial membranes during Neisserial infection, a process which is considered to dissipate the mitochondrial transmembrane potential gradient and thereby induce apoptosis.
Journal of Biological Chemistry, 2018
The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S5, Tables S1 and S2, and Movie S1. The atomic coordinates and structure factors (codes 6DPS, 6DNV, 6DNU, and 6DNL) have been deposited in the Protein Data Bank (http://wwpdb.org/). The NMR assignments have been deposited in the BRMB under accession numbers 27012 (His-n-DsbD Ox), 27013 (His-n-DsbD Red), 27014 (His-c-DsbD Ox), and 27015 (His-c-DsbD Red).
Journal of Molecular Biology, 2008
The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a sulfenic acid intermediate on the catalytic Cys with the concomitant release of the product-methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the sulfenic acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the sulfenic acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed. Abbreviations used: Ac-Met-S-SO-NHMe, L-methionine S-sulfoxide with its amino and carboxylic ends linked by amide bonds to an acetyl and a methyl amine group, respectively; Ac-Met-R,S-SO-NHMe, mixture of Ac-Met-S-SO-NHMe and its diastereomer Ac-Met-R-SO-NHMe; MetSO, L-methionine sulfoxide; Met-S-SO, L-methionine-S-sulfoxide; Met-R-SO, L-methionine-R-sulfoxide; Msr, methionine sulfoxide reductase; nmMsrA, the MsrA domain from Neisseria meningitidis PilB used in this study; nmMsrA red , reduced form of the nmMsrA; nmMsrA MetSO , C51S nmMsrA in complex with Ac-Met-S-SO-NHMe; nmMsrA CysOH , C198S nmMsrA in which C51 is in the sulfenic acid state; nmMsrA ox , oxidized (Cys51-Cys198) form of nmMsrA.
The electron transport chains of Neisseria meningitidis
2007
Neisseria meningitidis contains c-type and b-type cytochromes. Oxidation of c-type and b-type cytochromes by oxygen was observed in both cells grown under aerobic and denitrifying conditions, whereas oxidation of cytochromes by nitrite was only seen in cells grown under denitrifying conditions, and the predominant oxidizable cytochromes were b-type. These are likely to be associated with the oxidation of a bhaem containing nitric oxide reductase. Nitrite inhibits the oxidation of cytochromes by oxygen in a nitrite reductase-independent manner, indicating that nitrite may inhibit oxidase activity directly, as well as via the intermediate of denitrification, nitric oxide. Cytochromes c4 and c2 are major electron donors to the cbb 3 oxidase. Both strains deficient in cytochrome c4 and c2 exhibits a growth defect under high level of oxygen. These growth defects are linked to their decreased oxygen consumption rate. The growth defect and the decreased oxygen consumption rate indicated that cytochrome c4 dominates electron transfer to cbb 3 oxidase. Cytochrome c5 is an important electron donor to AniA nitrite reductase. A strain deficient in cytochrome c5 exhibits a growth defect under microaerobic conditions in the presence of nitrite. The mutant can not reduce nitrite to nitric oxide although AniA is expressed normally. A growth defect during high cell density culture under aerobic conditions suggests that cytochrome c5 is also an electron donor to ccb 3 oxidase but to a lesser extents than c4 or c2. Lipid-modified azurin (Laz) was heterologously expressed and purified. The purified protein contains copper ion and can be oxidized or reduced. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. Laz can be oxidized by membrane extract in the presence of oxygen or nitrite. This is likely to be due to the activity of cbb 3 oxidase and AniA nitrite reductase, respectively, in membrane extract. However the oxidation rate is very slow. A strain deficient in laz exhibits a growth defect during high cell density culture, similarly to that of c5 mutant. This suggests that Laz might be an electron donor to cbb 3 oxidase. III Acknowledgements It has been a wonderful time for me working in the Moir lab through these four years. I have learned many things and gained many valuable experiences. I would like to express my gratitude to every member of my lab. Most of all, I would like to thank my supervisor Dr. James Moir for all his support and guidance throughout my study.
Oxidative Protein Folding Is Driven by the Electron Transport System
Cell, 1999
An analogous system exists in the endoplasmic reticulum (ER) of yeast. This includes the well-studied protein disulfide isomerase, which catalyzes both the isomerizathe recently discovered Ero1 protein, which is responsi-University of Michigan ble for maintaining the redox status of protein disulfide Ann Arbor, Michigan 48109-1048 isomerase (Frand and Kaiser, 1998;. The Ero1 protein may thus play a somewhat similar role to the prokaryotic DsbB protein.
Progesterone 5-reductase (5-POR) catalyzes the reduction of progesterone to 5-pregnane-3,20-dione and is the first stereospecific enzyme in the putative biosynthetic pathway of Digitalis cardenolides. Selenomethionine-derivatized 5-POR from D. lanata was successfully overproduced and crystallized. The crystals belong to space group P4 3 2 1 2, with unit-cell parameters a = 71.73, c = 186.64 Å . A MAD data set collected at 2.7 Å resolution allowed the identification of six out of eight possible Se-atom positions. A first inspection of the MAD-phased electron-density map shows that 5-POR is a Rossmann-type reductase and the quality of the map is such that it is anticipated that a complete atomic model of 5-POR will readily be built. crystallization communications 188 Egerer-Sieber et al. Progesterone 5-reductase Acta Cryst. (2006). F62, 186-188 Figure 3