Crystallographic Analysis of Pathological Crystals, Periplasmic Domain of Ligand-free CitA Sensor Kinase and PDI-related Chaperones

Die vorliegende Arbeit berichtet über die Erarbeitung von Verfahren, um nicht-merohedrisch verzwillingte Proteinkristalle zu erklären. Aüserdem wird die Strukturanalyse verschiedener Proteine behandelt, die Signaltransfer vermitteln.Nicht-merohedrische Verzwilligung tritt bei der Kristallstrukturanalyse äüserst selten auf. Für nicht-merohedrisch verzwillingte Kleinmolekülkristalle ist die Auswertung der Daten ihrer Röntgenanalyse schon seit einigen Jahren Routine. Bestehende Methoden für Kleinmoleküle wurden dahingehend erweitert, dass zwei verzwillingte Testproteinstrukturen, nämlich Rinder-Insulin (BI-Zwilling) und Glukoseisomerase (GI-Drilling), gelöst werden konnten. Bei der Auswertung wurde das Verfahren anomaler Streuung bei Einfrequenzstrahlung auf Daten angewandt, die an einer Drehanode gemessen wurden.Die bakterielle Antwort auf externe Stimulation wird durch mehrere lebensnotwendige Signaltransfersysteme vermittelt. In Prokaryoten werden diese Systeme Zweikomponentensysteme oder Histidin-Protein-Kinasen genannt. Zweikomponentensysteme bieten sich als Ziele zur Entwicklung neuer Medikamente an, da sie in Säugetiersystemen und/oder im Tierreich noch nicht identifiziert wurden. In der vorliegenden Studie wurde die Kristallstruktur der merohedrisch verzwillingten, ligandenfreien, periplasmischen Domäne des Zitratsensors, CitA, von Klebsiella pneumoniae, bestimmt. Klebsiella pneumoniae ist die Hauptursache von Lungenentzündungn, nosokomialer Infektionen, Blutvergiftungen und Infektionen der Harnröhre. Der Vergleich der Strukturen ligandenfreier und ligandengebundener CitA-Domänen warf ein Licht auf einige mechanistische Aspekte des Signaltransfersystemes des Zitratsensors.Wind, ein Produkt des Windbeutel-Genes und Mitglied der Familie der PDI-verwandten Chaperone, ist eines von mehreren Genen, die notwendig für die dorso--ventrale Polarisierung in der Entwicklung des Embryos von Drosophila melanogaster sind. Dorso-ventrale Polarisierung erfordert die Kommunikation zwischen somatischen Zellen, die aus Folikelzellen abgeleitet sind, und Eizellen, die aus der Keimbahn abgeleitet sind. Sie erfolgt durch eine Kaskade an Signaltransferschritten, die Gurken-EGFR-Signalweg genannt wird. Eine zuvor bestimmte Struktur von Wind schlägt einen Homodimer vor, der eine Dimerisierungsschnittstelle entlang der N-terminalen b-Domäne aufweist. Diese hat die charakteristische Faltung von Thioredoxin. Pipe, ein Produkt von pipe, wurde als potentieller Wechselwirkungspartner des Wind-Dimers identifiziert. Eine mögliche Substratbindungsstelle auf Wind wurde beschrieben. Kristallstrukturen mehrerer nicht-funktioneller Mutationen von Wind sowie ein Wind-Peptide-Komplex deuten an, dass die Wind-Pipe-Wechselwirkung vorwiegend auf aromatischem und/oder hydrophobem Verhalten der Substratbindestelle und Erhaltung der Dimerisierungsschnittstelle basiert. Die Kristallstruktur von ERp29, einem engen Verwandten von Wind, offenbart eine ähnliche dreidimensionale Architektur und Erhaltung der Dimerisierungsschnittstelle

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder–order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates

Crystallization and preliminary crystallographic analysis of the global nitrogen regulator AmtR from Corynebacterium glutamicum

AmtR is a rare example of a member of the TetR family of bacterial transcription regulators that is not regulated by a small-molecule effector but by interaction with a protein named GlnK. Wild-type and SeMet-substituted AmtR have been produced and crystallized and preliminary electron-density maps have been obtained to 3.0 Å resolution

Sesquiterpene Lactones from Elephantopus scaber

The whole plant of Elephantopus scaber afforded the known deoxyelephantopin and isodeoxy-elephantopin, and a new germacranolide sesquiterpene lactone named scabertopin, whose structure and stereo-chemistry were determined by spectroscopic methods and single-crystal X-ray analysis

Serendipitous Fatty Acid Binding Reveals the Structural Determinants for Ligand Recognition in Apolipoprotein M

Apolipoprotein M (ApoM) is a 25-kDa HDL-associated apolipoprotein and a member of the lipocalin family of proteins. Mature apoM retains its signal peptide, which serves as a lipid anchor attaching apoM to the lipoproteins, thereby keeping it in the circulation. Studies in mice have suggested apoM to be antiatherogenic, but its physiological function is yet unknown. We have now determined the 1.95 angstrom resolution crystal structure of recombinant human apoM expressed in Escherichia coli and made the unexpected discovery that apoM, although refolded from inclusion bodies, was in complex with fatty acids containing 14, 16 or 18 carbon atoms. ApoM displays the typical lipocalin fold characterised by an eight-stranded antiparallel beta-barrel that encloses an internal ligand-binding pocket. The crystal structures of two different complexes provide a detailed picture of the ligand-binding determinants of apoM. Additional fatty acid- and lipid-binding studies with apoM and the mutants apoM(W47F) and apoM(W100F) showed that sphingosine-1-phosphate is able to displace the bound fatty acids and efficiently quenched the intrinsic fluorescence with an IC50 of 0.90 mu M. Whereas the fatty acids bound in the crystal structure could be a mere consequence of recombinant protein production, the observed binding of sphingosine-l-phosphate might provide a key to a better understanding of the physiological function of apoM. (C) 2009 Elsevier Ltd. All rights reserved

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of YvoA from Bacillus subtilis

YvoA from B. subtilis, a member of the poorly understood HutC family of bacterial transcription factors, has been recombinantly produced, characterized as a homodimer in solution and crystallized in space group C2

regulator TetR

reverses the function of the transcriptio

Pacmanvirus, a New Giant Icosahedral Virus at the Crossroads between Asfarviridae and Faustoviruses

International audienceAfrican swine fever virus, a double-stranded DNA virus that infects pigs, is the only known member of the Asfarviridae family. Nevertheless, during our isolation and sequencing of the complete genome of faustovirus, followed by the description of kaumoebavirus, carried out over the past 2 years, we observed the emergence of previously unknown related viruses within this group of viruses. Here we describe the isolation of pacmanvirus, a fourth member in this group, which is capable of infecting Acanthamoeba castellanii. Pacmanvirus A23 has a linear compact genome of 395,405 bp, with a 33.62% G + C content. The pacmanvirus genome harbors 465 genes, with a high coding density. An analysis of reciprocal best hits shows that 31 genes are conserved between African swine fever virus, pacmanvirus, faustovirus, and kaumoebavirus. Moreover, the major capsid protein locus of pacmanvirus appears to be different from those of kaumoebavirus and faustovirus. Overall, comparative and genomic analyses reveal the emergence of a new group or cluster of viruses encompassing African swine fever virus, faustovirus, pacmanvirus, and kaumoebavirus. IMPORTANCE Pacmanvirus is a newly discovered icosahedral double-stranded DNA virus that was isolated from an environmental sample by amoeba coculture. We describe herein its structure and replicative cycle, along with genomic analysis and genomic comparisons with previously known viruses. This virus represents the third virus, after faustovirus and kaumoebavirus, that is most closely related to classical representatives of the Asfarviridae family. These results highlight the emergence of previously unknown double-stranded DNA viruses which delineate and extend the diversity of a group around the asfarvirus members

Structural Basis of Zika Virus Specific Neutralization in Subsequent Flavivirus Infections

Zika virus (ZIKV), a mosquito-borne human flavivirus that causes microcephaly and other neurological disorders, has been a recent focus for the development of flavivirus vaccines and therapeutics. We report here a 4.0 Å resolution structure of the mature ZIKV in complex with ADI-30056, a ZIKV-specific human monoclonal antibody (hMAb) isolated from a ZIKV infected donor with a prior dengue virus infection. The structure shows that the hMAb interactions span across the E protein dimers on the virus surface, inhibiting conformational changes required for the formation of infectious fusogenic trimers similar to the hMAb, ZIKV-117. Structure-based functional analysis, and structure and sequence comparisons, identified ZIKV residues essential for neutralization and crucial for the evolution of highly potent E protein crosslinking Abs in ZIKV. Thus, this epitope, ZIKV’s “Achilles heel”, defined by the contacts between ZIKV and ADI-30056, could be a suitable target for the design of therapeutic antibodies