The structure of the bacterial oxidoreductase enzyme DsbA in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of DsbA enzymes

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the comple

Untersuchungen zur Bedeutung des präpartalen Progesteronentzugs in Hinblick auf die Steuerung der Geburt und die Ablösung der Nachgeburt beim Rind

Beim Rind ist der präpartale Abfall der maternalen Progesteron (P4)-Konzentration Voraussetzung für den Eintritt der Geburt. Der P4-Abfall spiegelt beim Rind primär den Funktionsverlust des Trächtigkeitsgelbkörpers wider. Über die Bedeutung der plazentaren P4-Produktion lagen bisher praktisch keine Informa¬tionen vor. Die Rinder¬plazenta trägt in der Endphase der Gravidität zwar nur minimal zu den maternalen P4-Spiegeln bei, jedoch bildet sie hohe lokale P4-Konzentrationen im Bereich der feto-maternalen Kontakt¬zone. Diese könnten, vermittelt über P4-Rezeptoren (PR) in den maternalen Karunkeln, ein wesentlicher Faktor für die Differenzie¬rung und Funktion der Plazentome sein. Es wurde die Arbeits¬hypothese ent¬wickelt, dass der Entzug hoher lokaler P4-Konzentrationen ein wesentliches Signal für die Vorbereitung eines termin¬gerechten Nachgeburtsabganges darstellt. Entsprechend könnte ein unvollständiges Sistieren der plazentaren P4-Produktion vor der Geburt eine wesentliche Ursache für das Auftreten idiopathischer Nachge-burts¬verhaltungen sein. Zur Überprüfung dieser Hypo¬these wurden drei gravide Kühe am 270. und 271. Graviditätstag mit dem Antigestagen Aglepriston (Ap) behandelt (Gruppe D272+Ap). Dieser PR-Blocker ermöglicht die Ausschaltung aller rezeptor¬vermittelten P4-Wirkungen unabhängig von der P4-Quelle. Als Kontrollen dienten Tiere mit termin¬gerechter spontaner Geburt und termingerechtem Nachgeburts¬abgang (Gruppe Normalgeburt, n = 4, Trächtigkeitsdauer: 280,5 ± 1,7 Tage) sowie unbehan¬delte Tiere mit Schnitt¬entbindung am Tag 272 (Gruppe D272-Ap, n = 3). Die Kühe wurden klinisch überwacht und der Geburtsverlauf dokumentiert. Von allen Tieren wurden Blutproben in regelmäßigen Abständen sowie Plazentome unmittelbar im Anschluss an die Geburt (Gruppen D272+Ap und Normalgeburt) bzw. während der Schnittentbindungen (Gruppe D272-Ap) entnommen. In den Blutproben wurden die Konzentrationen von Progesteron und Östrogenen mittels radioimmuno¬logischer Verfahren und die 13, 14-Di¬hydro-15-Keto-PGF2α (PGFM)-Konzentrationen mittels ELISA gemessen. Als Parameter für die präpartale Umstrukturierung der Plazentome wurde der prozen¬tuale Anteil der Trophoblast-riesenzellen (TGC) an den Trophoblastzellen und die Reduktion des Karunkelepithels erfasst. Weiterhin wurde die Expression von Cyclooxygenase II (Cox II), PR und Glucocorticoidrezeptor (GR) auf Protein- und mRNA-Ebene beurteilt. Die Aglepristonbehandlung führte bei allen drei Kühen zu einer vorzeitigen Terminie¬rung der Gravidität. Erste Geburtsanzeichen traten 46,3 ± 6,0 Stunden nach Be¬handlungsbeginn auf. Es kam zur vollständigen Öffnung der Zervix, eine adäquate Wehentätigkeit setzte jedoch innerhalb der folgenden zwei Stunden nicht ein. So wurde ein manueller Auszug der Kälber durchgeführt. Neben der Öffnung der Zervix wurde durch die Antigestagenbehandlung die Laktogenese induziert. Entge¬gen der eigenen Hypothese wiesen alle drei Kühe der Gruppe D272+Ap, wie die Tiere der Gruppe D272-Ap, eine komplette Nachgeburts¬ver¬haltung auf und die Kälber beider Gruppen waren gleichermaßen prämatur. Die histo¬morphologischen Unter¬suchungen bestätigten, dass durch die Aglepriston¬behand¬lung die präpartale „Plazentareifung“ nicht induziert wurde. So waren, im Gegensatz zur Normalgeburtsgruppe, in den unreifen Plazentomen der Gruppen D272+Ap und D272−Ap weder ein Rück¬gang des relativen Anteils der TGC noch eine Reduktion des Karunkelepithels nach¬weisbar. Überraschenderweise wurde durch die Antigestagenbehandlung die Luteolyse induziert, erkennbar an einem steilen Abfall der P4-Werte vor bzw. während des Auszugs der Kälber. Korrespondierend mit dem Abfall der P4-Konzen¬tra¬tionen wurde bei den Tieren der Gruppe D272+Ap bereits präpartal ein schwacher Anstieg der PGFM-Werte beobachtet. Da bei diesen Tieren, im Gegensatz zur Normal¬geburtsgruppe, keine Aufregulation der plazentaren Cox II-Expression nach¬weisbar war, ist anzunehmen, dass die Antigestagen-induzierte Luteolyse indirekt durch Prostaglandine extraplazentaren, vermutlich endometrialen, Ursprungs aus¬gelöst wurde. Zum Zeitpunkt des Auszugs waren die PGFM-Plasma¬konzentrationen im Vergleich zur Normalgeburtsgruppe jedoch relativ gering (2,14 ± 1,40 ng/ml vs. 8,70 ± 2,20 ng/ml). Somit erklärt vermutlich ein Mangel an uterotonem PGF2α die Wehen¬schwäche bei den Aglepriston-behandelten Tieren. Die Östrogen¬synthese im Trophoblasten sowie die GR- bzw. PR-Expression in den Plazentomen wurden durch das Antigestagen nicht beeinflusst. Insgesamt lassen die Ergebnisse dieser Arbeit darauf schließen, dass nur ein relativ geringer Anteil der geburtsassoziierten Veränderungen direkt durch den präpartalen Progesteronentzug ausgelöst wird, nämlich die Öffnung der Zervix und das Einsetzen der Laktation. Dagegen erfordern andere wesentliche geburtsassoziierte Vorgänge, wie eine adäquate Wehentätigkeit und die Ablö¬sung der Plazenta, offensichtlich primär Signale aus dem fetalen Kompartiment. Die eigenständige Bedeutung der plazentaren P4¬-Produktion bleibt unklar.In cattle the prepartal decline in maternal progesterone (P4) levels is a prerequisite for the onset of parturition. This P4 withdrawal predominantly reflects the loss of luteal function, and virtually no information on the importance of placental P4 production is available. Despite its minimal contribution to maternal P4 levels in late gestation the bovine placenta is capable of producing high P4 levels locally at the feto-maternal interface, which – mediated by progesterone receptors (PR) previously detected in the maternal caruncles – could be an essential factor in placental differentiation and function. Thus, the hypothesis was put forward that a well-timed and complete withdrawal of high local P4 concentrations is a crucial signal for the timely release of the placenta. According to this concept, an incomplete cessation of placental P4 production during the initiation of parturition could be an important factor in the etiology of placental retention. To test for this hypothesis, three cows were treated with the antiprogestin aglepristone (Ap) on days 270 and 271 of gestation to abolish receptor mediated effects of P4 irrespective of its origin (group D272+Ap). As controls, four cows giving spontaneous birth at normal term (280.5 ± 1.7 days, group NT) with timely release of fetal membranes and three cows undergoing cesarean section on day 272 (group D272-Ap) were included into the study. The cows were monitored clinically, and the progress of birth was registered. From all animals, blood samples were taken regularly during the experimental period, and placentomes were removed per vaginam immediately after birth (groups D272+Ap and NT) or during cesarean section (group D272-Ap). In blood samples, con-centrations of P4 and estrogens were measured by radioimmunological methods, and for the measurement of 13, 14-dihydro-15-keto prostaglandin F2α (PGFM)-concentrations a commercial ELISA kit was used. To characterize the prepartal remodeling of placentomal microarchitecture, the percentage of trophoblast giant cells (TGC) relative to the total number of trophoblast cells and the reduction of caruncular epithelium were determined. Moreover, the expression of cyclo-oxygenase II (Cox II), PR and glucocorticoid receptor (GR) was investigated at protein and mRNA level. The application of aglepristone significantly reduced gestational length. First signs of impending parturition occurred 46.3 ± 6.0 hours after the start of treatment, and vaginal exploration confirmed that the cervix was fully open during this time. However, no progress in the expulsion of the calves could be observed during the following two hours, obviously due to insufficient myometrial activity. Consequently the calves were extracted. Besides a complete opening of the cervix, antiprogestin treatment induced the onset of lactation. Inconsistent with the working hypothesis, in all cows of the treatment group a severe retention of fetal membranes was observed. Similar cases of retained fetal membrane were also observed in all D272-Ap cows, and calves of both groups were slightly premature to a similar extent. Consistent with clinical observations histological investigations of placentomes showed that antiprogestin treatment did not induce placental maturation, whereas for the placentomes of NT cows the prepartal decline in TGC numbers and the reduction of caruncular epithelium was confirmed. Surprisingly, antiprogestin treatment induced luteolysis, as a significant decline of progesterone concentrations started before or during the expulsion of the calf. Con¬comitant with the decline in P4 concentrations in D272+Ap cows an increase of PGFM levels became detectable. The fact that, different from NT animals, no up-regu¬lation of placental Cox II was found suggests that the antiprogestin induced luteolysis indirectly stimulated prostaglandin pro¬duction from an extraplacental source, presumably the intercaruncular endometrium. However, at parturition PGFM levels in D272+Ap cows were clearly lower in comparison to NT cows (2.14 ± 1.40 ng/ml vs. 8.70 ± 2.20 ng/ml), which suggests that insufficient myometrial activity observed in antiprogestin treated cows was related to a reduced availability of uterotonic PGF2α. Placental estrogen production and the expression of PR or GR were not affected by antiprogestin treatment. In conclusion, the results demonstrate that only a minor part of the processes related to bovine parturition is directly dependent on P4 withdrawal, in particular the opening of the cervix and the onset of lactation. Moreover, they suggest that other important processes such as adequate myometrial activity and timely release of the placenta are predominantly dependent on signals from the fetal compartment. The importance of placental P4 production in cattle remains unknown

Plasmids used in this study.

<p>*Cassettes: <i>aadA</i>  =  spectinomycin resistance, <i>tetM</i>  =  tetracycline resistance, <i>aphA-3</i>  =  kanamycin resistance, <i>ermC</i>  =  erythromycin resistance.</p><p>Plasmids used in this study.</p

Bacterial strains and plasmids used in this study.

<p>*Nomenclature is derived from serological typing scheme for capsule polysaccharide (serogroup B):porin B variant (2B):porin A variant (P1.2,5):lipooligosaccharide immunotype (L2). Cassettes: <i>aadA</i>  =  spectinomycin resistance, <i>tetM</i>  =  tetracycline resistance, <i>aphA-3</i>  =  kanamycin resistance, <i>ermC</i>  =  erythromycin resistance.</p><p>+ ND =  not done.</p><p>Bacterial strains and plasmids used in this study.</p

Neisserial LptA::His_x6 transfers PEA to lipid A of <i>E. coli</i> LPS.

<p>Lipid A profiles of LPS extracted from <i>E. coli</i> strains JCB571 expressing <i>Ec</i>DsbA (CKEC272) (Panel A), <i>E. coli</i> JCB571 expressing LptA::His_x6 (CKEC543) (Panel B) and JCB571 expressing LptA::His_x6 and <i>Ec</i>DsbA (CKEC564) (Panel C) as determined by MALDI-TOF MS. <i>bis</i>-Phosphorylated hexaacylated lipid A (m/z = 1796), the mono-phosphorylated derivative (<i>m/z</i> = 1716), and the heptaacylated version due to the addition of a palmitic acyl residue (<i>m/z</i> = 2034) were detected in all strains. <i>bis</i>-Phosphorylated tetraacylated lipid A (<i>m/z</i> = 1360) was found abundantly in the MALDI spectra of all three strains, which was likely produced from <i>bis</i>-phosphorylated hexaacylated lipid A (<i>m/z</i> = 1796) during the ionization step on MALDI. The lipid A preparations from CKEC543 expressing LptA (Panel B) and CKEC564 co-expressing LptA and <i>Ec</i>DsbA (Panel C) also contained ions consistent with one PEA added to the <i>bis</i>-phosphorylated structure (such as <i>m/z</i> 1919; i.e. 1796+123) and the heptaacylated structure (such as <i>m/z</i> = 2157, i.e. 2034+123).</p

LptA::His_x6 stability is dependent upon oxidoreductase activity in <i>E. coli</i>.

<p>Standardised whole cell lysates were separated by SDS-PAGE. A Western immunoblot was developed using anti-His tag antibody to detect the presence of LptA::His_x6 in the cellular extracts. Lanes were: Lane 1, ColorPlus pre-stained protein molecular weight marker (New England Biolabs); Lane 2: <i>E. coli</i> JCB571 expressing <i>Ec</i>DsbA (CKEC272); Lane 3: <i>E. coli</i> JCB571 carrying pTrc99A (CKEC288); Lane 4: <i>E. coli</i> JCB571 expressing LptA::His_x6 (CKEC543); Lane 5: <i>E. coli</i> JCB571 expressing LptA::His_x6 and <i>Ec</i>DsbA (CKEC564); Lane 6: CKEC564 treated with DTT and alkylated with AMS; and Lane 7: CKEC564 alkylated with AMS. Molecular weights (kDa) are indicated on the left.</p

Oxidation status of LptA::His_x6 in oxidoreductase mutants of <i>N. meningitidis</i>.

<p>Standardised cell lysates were separated by SDS-PAGE, followed by transfer to a membrane and western immunoblot using anti-His_x6 HRP conjugate antibody to detect the presence of LptA::His_x6. Panel A. Lane 1, protein molecular weight standard (New England Biolabs, Cat-2-212); Lane 2: NMBΔ<i>dsbA1/dsbA2</i> expressing LptA::His_x6 from pCMK1001 (CKNM221) untreated; Lane 3: CKNM221 treated with DTT and alkylated with AMS; Lane 4: CKNM221 alkylated with AMS, Lane 5: NMBΔ<i>dsbA3</i> expressing LptA::His_x6 (CKNM222) untreated; Lane 6: CKNM222 treated with DTT and alkylated with AMS; Lane 7: CKNM222 alkylated with AMS. Panel B. Lane 1, protein molecular weight standard (New England Biolabs, Cat-2-212); Lane 2: NMB expressing LptA::His_x6 (CKNM216) untreated; Lane 3: CKNM216 treated with DTT and alkylated with AMS; Lane 4: CKNM216 alkylated with AMS; Lane 5: NMBΔ<i>dsbA1</i>/<i>NmdsbA2/dsbA3</i> expressing LptA::His_x6 (CKNM755); Lane 6: CKNM755 treated with DTT and alkylated; Lane 7: CKNM755 alkylated with AMS.</p

Lipid A substitution profiles of meningococcal oxidoreductase mutants.

<p>Lipid A profiles of LOS extracted from <i>N. meningitidis</i> strain NMB (Panel A), NMBΔ<i>lptA::aadA</i> (Panel B), NMBΔ<i>NmdsbA1</i>/<i>NmdsbA2</i> (Panel C), NMBΔ<i>NmdsbA3</i> (Panel D) and NMBΔ<i>dsbA1/dsbA2/dsbA3</i> (Panel E) as determined by MALDI-TOF MS. <i>bis</i>-Phosphorylated hexaacylated lipid A (<i>m/z</i> = 1712), the <i>mono</i>-phosphorylated (<i>m/z</i> = 1632) and the <i>tri</i>-phosphorylated derivative (<i>m/z</i> = 1792) were detected in all strains. Strain NMB and the oxidoreductase mutants all expressed the mono-phosphorylated, <i>bis</i>-phosphorylated and <i>tri</i>-phosphorylated hexaacylated lipid A with a single PEA addition (<i>m/z</i> = 1755, <i>m/z</i> = 1835 and <i>m/z</i> = 1915). Consistent with the loss of LptA activity, NMBΔ<i>lptA::aadA</i> lacked these ions.</p

Structural and biochemical characterisation of the oxidoreductase NmDsbA3 from Neisseria meningitidis

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyses the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Whilst most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whilst NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3 &Aring; resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidised by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.<br /