protein-crystallization-for-x-ray-crystallography

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signalling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signalling is not well understood. Here, we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16-18-basepair footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signalling model in which the CARDs on MDA5-RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry.

MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA.

Melanom Differenzierung-assoziierte Gen-5 (MDA5) erkennt viralen doppelstr&auml;ngige RNA im Zytoplasma. RNA-Bindung induziert MDA5 den Signalgebung Adapter MAVS durch Interaktionen zwischen den Caspase Recruitment Dom&auml;nen (Karten) der zwei Proteine aktivieren. Der molekulare Mechanismus der MDA5 Signalisierung ist nicht gut verstanden. Hier zeigen wir, dass MDA5 kooperativ kurze RNA-Liganden als ein Dimer mit einer Stellfl&auml;che von 16-18-Basepair bindet. Eine Kristallstruktur der Dom&auml;ne Helicase-Insert MDA5 veranschaulicht eine Evolutions-Verh&auml;ltnis mit der reitenden Hef-Helikasen. In x-ray L&ouml;sung Strukturen die Karten in Unliganded MDA5 sind flexibel und RNA bindet auf der einen Seite von einem asymmetrischen MDA5 Dimer, &Uuml;berbr&uuml;ckung der zwei Untereinheiten. Auf l&auml;ngeren RNA montieren in und Karte gel&ouml;scht MDA5 Konstrukte in ATP-Sensitive-Borsten. Wir schlagen ein Signalgebung Modell, in dem die Karten auf MDA5-RNA-Filamente die Versammlung der MAVS Filamente mit der gleichen Polymer Geometrie nucleate.

MDA5 bildet kooperativ Dimerisierung und ATP-Sensitive-Borsten auf doppelstr&auml;ngige RNA binden.

Toll-like receptor 9 (TLR9) recognizes microbial DNA in endolysosomal compartments. The ectodomain of TLR9 must be proteolytically cleaved by endosomal proteases to produce the active receptor capable of inducing an innate immune signal. We show that the cleaved TLR9 ectodomain is a monomer in solution and that DNA ligands with phosphodiester backbones induce TLR9 dimerization in a sequence-independent manner. Ligands with phosphorothioate (PS) backbones induce the formation of large TLR9-DNA aggregates, possibly due to the propensity of PS ligands to self-associate. DNA curvature-inducing proteins including high-mobility group box 1 and histones H2A and H2B significantly enhance TLR9 binding, suggesting that TLR9 preferentially recognizes curved DNA backbones. Our work sheds light on the molecular mechanism of TLR9 activation by endogenous protein-nucleic acid complexes, which are associated with autoimmune diseases including systemic lupus erythematosus.

DNA binding to proteolytically activated TLR9 is sequence-independent and enhanced by DNA curvature.

Toll-Like-Rezeptor 9 (TLR9) erkennt mikrobiellen DNA in Endolysosomal F&auml;chern. Die APPs&alpha; von TLR9 muss spaltet von Endosomen Proteasen, den aktiven Rezeptor f&auml;hig zeigen, da&szlig; eine angeborene immun-Signal zu produzieren zerspaltet werden. Wir zeigen, dass die gespalten TLR9-APPs&alpha; einem Monomer in L&ouml;sung und, dass DNA-Liganden mit Phosphodiester--Backbones TLR9 Dimerisierung Sequenz-unabh&auml;ngig bewegen. Liganden mit Phosphorothioat (PS)-Backbones induzieren die Bildung von gro&szlig;en TLR9-DNA-Aggregate, m&ouml;glicherweise aufgrund der Neigung der PS-Liganden zu self-associate. DNA Kr&uuml;mmung-induzierende Proteine einschlie&szlig;lich hoch-Mobilit&auml;t Gruppe Feld 1 und Histone H2A und H2B signifikant erh&ouml;hen TLR9-Bindung, die darauf hindeutet, dass TLR9 bevorzugt gebogenen R&uuml;ckgrat der DNA erkennt. Unsere Arbeit wirft Licht auf die molekularen Mechanismen der TLR9 Aktivierung durch endogene Protein-Nukleins&auml;ure S&auml;ure-komplexe, die verbunden sind mit Autoimmunkrankheiten wie Lupus erythematodes.

DNA-Bindung an spaltet aktivierte TLR9 ist unabh&auml;ngig von Sequenz und angereichert mit DNA-Kr&uuml;mmung.

The endocytic sorting signal on the low-density lipoprotein receptor for clathrin-mediated internalization is the sequence FDNPVY in the receptor's cytosolic tail. We have used a combination of surface plasmon resonance and crosslinking with a photoactivated peptide probe to demonstrate the interaction between FDNPVY-containing peptides and the mu2 chain of purified AP-2 clathrin adaptors (the complexes responsible for plasma membrane sorting). We show that recognition of the FDNPVY signal is mediated by a binding site in the mu2-subunit that is distinct from the site for the more general YppÃ˜ sorting signal, another tyrosine-based sequence also recognized by mu2-adaptin. These results suggest the possibility that low-density lipoprotein receptor uptake may be modulated specifically and independently of other proteins in the clathrin pathway.

The mu2 subunit of the clathrin adaptor AP-2 binds to FDNPVY and YppÃ˜ sorting signals at distinct sites.

Die MU2 Untereinheit des Clathrin-Adapter AP-2 bindet an FDNPVY und Ypp&#xD8; sortierenden Signale an bestimmten Stellen.

Papillomaviruses propagate in differentiating skin cells, and certain types are responsible for the onset of cervical cancer. We have combined image reconstructions from electron cryomicroscopy (cryoEM) of bovine papillomavirus at 9 A resolution with coordinates from the crystal structure of small virus-like particles of the human papillomavirus type 16 L1 protein to generate an atomic model of the virion. The overall fit of the L1 model into the cryoEM map is excellent, but residues 402-446 in the 'C-terminal arm' must be rebuilt. We propose a detailed model for the structure of this arm, based on two constraints: the presence of an intermolecular disulfide bond linking residues 175 and 428, and the clear identification of a feature in the image reconstruction corresponding to an alpha-helix near the C-terminus of L1. We have confirmed the presence of the disulfide bond by mass spectrometry. Our 'invading arm' model shows that papilloma- and polyomaviruses have a conserved capsid architecture. Most of the rebuilt C-terminal arm is exposed on the viral surface; it is likely to have a role in infection and in immunogenicity.

Atomic model of the papillomavirus capsid.

Atomic-Modell des Papillomavirus Kapsid.

Dengue virus is an emerging global health threat. Its major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. A crystal structure of the soluble ectodomain of E from dengue virus type 2 reveals a hydrophobic pocket lined by residues that influence the pH threshold for fusion. The pocket, which accepts a hydrophobic ligand, opens and closes through a conformational shift in a beta-hairpin at the interface between two domains. These features point to a structural pathway for the fusion-activating transition and suggest a strategy for finding small-molecule inhibitors of dengue and other flaviviruses.

A ligand-binding pocket in the dengue virus envelope glycoprotein.

Ein Ligand-Bindungstasche in dem Dengue-Virus-Glykoprotein.

A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 A. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two linked halves, transmembrane segments 1-5 and 6-10, clamped together by the gamma-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation.

X-ray structure of a protein-conducting channel.

X-ray Struktur eines Proteins f&#xFC;hrenden Kanals.

Dengue virus enters a host cell when the viral envelope glycoprotein, E, binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host-cell membranes. A three-dimensional structure of the soluble E ectodomain (sE) in its trimeric, postfusion state reveals striking differences from the dimeric, prefusion form. The elongated trimer bears three 'fusion loops' at one end, to insert into the host-cell membrane. Their structure allows us to model directly how these fusion loops interact with a lipid bilayer. The protein folds back on itself, directing its carboxy terminus towards the fusion loops. We propose a fusion mechanism driven by essentially irreversible conformational changes in E and facilitated by fusion-loop insertion into the outer bilayer leaflet. Specific features of the folded-back structure suggest strategies for inhibiting flavivirus entry.

Structure of the dengue virus envelope protein after membrane fusion.

Aufbau des Dengue-Virus-H&#xFC;llprotein nach Membranfusion.

Dengue virus is an emerging global health threat. The major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. Antibodies that bind but fail to neutralize noncognate serotypes enhance infection. We have determined the crystal structure of a soluble fragment of the envelope glycoprotein E from dengue virus type 3. The structure closely resembles those of E proteins from dengue type 2 and tick-borne encephalitis viruses. Serotype-specific neutralization escape mutants in dengue virus E proteins are all located on a surface of domain III, which has been implicated in receptor binding. While antibodies against epitopes in domain I are nonneutralizing in dengue virus, there are neutralizing antibodies that recognize serotype-conserved epitopes in domain II. The mechanism of neutralization for these antibodies is probably inhibition of membrane fusion. Our structure shows that neighboring glycans on the viral surface are spaced widely enough (at least 32 A) that they can interact with multiple carbohydrate recognition domains on oligomeric lectins such as DC-SIGN, ensuring maximum affinity for these putative receptors.

Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein.

Variable Oberfl&#xE4;chen-Epitope in der Kristallstruktur des Dengue-Virus Typ 3 H&#xFC;llglycoprotein.

West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.

Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes.

West-Nil-Virus, ein Mitglied der Gattung Flavivirus verursacht Fieber, das auf eine lebensbedrohliche Enzephalitis fortschreiten kann. Das gro&szlig;e Umschlag Glykoprotein, E, dieser Viren vermittelt virale Anhaftung und Eintrag von Membrane Fusion. Wir haben die Kristallstruktur des l&ouml;slichen Fragmentes des West-Nil-Virus E. bestimmt. Die Struktur nimmt die gleiche allgemeine Klappe wie bei der E-Proteine von Dengue-Fieber und FSME-Viren. Die Konformation II-Dom&auml;ne unterscheidet sich in anderen Prefusion E-Strukturen, jedoch und gleicht die Beschaffenheit der Dom&auml;ne II in Postfusion E Strukturen. Die Epitope des West-Nil-Virus-spezifische Antik&ouml;rper-Karte zu einer Region der Dom&auml;ne III, die auf die virale Oberfl&auml;che ausgesetzt ist und hat verwickelt worden, in Rezeptor zu neutralisieren. Im Gegensatz dazu zeigen wir, dass bestimmte rekombinanten therapeutischen Antik&ouml;rper, die Kreuz-West Nile und Dengue-Viren zu neutralisieren, ein Peptid aus Dom&auml;ne binden ich, die nur w&auml;hrend der Membrane Fusion &Uuml;bergang ausgesetzt ist. Durch die Offenbarung der Informationen &uuml;ber die molekulare Landschaft von der Oberfl&auml;che des West-Nil-Virus, helfen unsere Struktur das Design von antiviralen Impfstoffen und Therapeutika.

Kristallstruktur von West-Nil-Virus Umschlag Glykoprotein enth&uuml;llt virale Oberfl&auml;che Epitopes.

The immune response against viral infection relies on the early production of cytokines that induce an antiviral state and trigger the activation of immune cells. This response is initiated by the recognition of virus-associated molecular patterns such as dsRNA, a viral replication intermediate recognized by TLR3 and certain RNA helicases. Infection with West Nile virus (WNV) can lead to lethal encephalitis in susceptible individuals and constitutes an emerging health threat. In this study, we report that WNV envelope protein (WNV-E) specifically blocks the production of antiviral and proinflammatory cytokines induced by dsRNA in murine macrophages. This immunosuppressive effect was not dependent on TLR3 or its adaptor molecule Trif. Instead, our experiments show that WNV-E acts at the level of receptor-interacting protein 1. Our results also indicate that WNV-E requires a certain glycosylation pattern, specifically that of dipteran cells, to inhibit dsRNA-induced cytokine production. In conclusion, these data show that the major structural protein of WNV impairs the innate immune response and suggest that WNV exploits differential vector/host E glycosylation profiles to evade antiviral mechanisms.

West Nile virus envelope protein inhibits dsRNA-induced innate immune responses.

Die Immunantwort gegen virale Infektion st&uuml;tzt sich auf die fr&uuml;hen Produktion von Zytokinen, die induzieren eines antiviralen Zustand und die Aktivierung von Immunzellen ausl&ouml;sen. Diese Reaktion wird durch die Anerkennung der Virus-assoziierte Molekulare Muster wie sequenzspezifischen, eine Virusreplikation Mittelstufe von TLR3 und bestimmten RNA-Helikasen erkannt initiiert. Infektion mit West-Nil-Virus (WNV) kann auf eine t&ouml;dliche Enzephalitis bei anf&auml;lligen Menschen f&uuml;hren und stellt eine neue Gefahr f&uuml;r die Gesundheit. In dieser Studie berichten wir, dass WNV Umschlag Protein (WNV-E) speziell die Produktion von antivirale und Proinflammatory Cytokines von sequenzspezifischen in murinen Makrophagen induziert blockiert. Diese immunsuppressive Wirkung war nicht abh&auml;ngig von TLR3 oder seine Adapter-Molek&uuml;l Trif. Stattdessen zeigen unseren Experimenten, dass WNV-E auf der Ebene der Rezeptor-Interaktion Protein 1 fungiert. Unsere Ergebnisse zeigen auch, dass WNV-E ein bestimmtes Muster Glycosylation, speziell die der Dipteran Zellen induziert durch sequenzspezifischen Zytokin-Produktion hemmen erfordert. Abschlie&szlig;end zeigen diese Daten, dass die gro&szlig;en strukturellen Proteins des WNV beeintr&auml;chtigt der angeborenen Immunantwort und schlagen vor, dass WNV differenzielle Vektor/Host E Glycosylation profile um antivirale Mechanismen entziehen nutzt.

West-Nil-Virus Umschlag Protein hemmt angeborenen Immunantwort induziert durch sequenzspezifischen.

How influenza virus is locked out of the cell.

Wie Influenza-Virus aus der Zelle gesperrt ist.

Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the "pH sensor" that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

Crystal structure of dengue virus type 1 envelope protein in the postfusion conformation and its implications for membrane fusion.

Dengue-Virus st&uuml;tzt sich auf eine Konformations&auml;nderungen Ver&auml;nderung sein Umschlag Protein, E, zur Sicherung der viralen Lipidmembran mit der Endosomen-Membran und dadurch das virale Genom in das Zytosol zu liefern. Wir haben die Kristallstruktur einer l&ouml;slichen Fragment E (sE) des Dengue-Virustyp 1 (DEN-1) ermittelt. Das Protein ist in der postfusion Konformation, obwohl es keine Lipidmembran oder Reinigungsmittel ausgesetzt war. An der Dom&auml;ne-Dom&auml;ne-III-Schnittstelle, 4 polar R&uuml;ckst&auml;nde bilden einen engen Cluster, die nicht vorhanden ist in anderen Flaviviral postfusion Strukturen. Zwei dieser R&uuml;ckst&auml;nde, His-282 und His-317, sind in Flaviviren konserviert und sind Teil der &quot;pH-Wert-Sensor&quot;, der dem Fusogenic Konformations&auml;nderungen Wandel der E, an den niedrigeren pH-Wert von dem Endosom ausl&ouml;st. In der Fusion-Schleife nimmt Phe-108 eine unterschiedliche Konformation bilden zus&auml;tzliche Trimer Kontakte und f&uuml;llen die sch&uuml;sself&ouml;rmige W&ouml;lbung an der Spitze der DEN-2 sE Trimer beobachtet.

Kristallstruktur von Dengue-Virus Typ 1 Umschlag Protein in der postfusion Konformation und seine Bedeutung f&uuml;r die Membrane Fusion.

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn(60). Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn(70)/Asn(104) flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn(60) reduces adhesion, N-glycans at Asn(70)/Asn(104) of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.

N-glycosylation at the SynCAM (synaptic cell adhesion molecule) immunoglobulin interface modulates synaptic adhesion.

W&auml;hlen Sie Adh&auml;sionsmolek&uuml;le vor- und postsynaptisches Membranen verbinden und entwickelnde Synapsen zu organisieren. Die Verordnung dieser Trans-synaptische Interaktionen ist eine wichtige Frage der neurobiologischen. Wir haben bereits gezeigt, dass die synaptische ZELLADH&Auml;SIONSMOLEK&Uuml;LE (SynCAMs) 1 und 2 in Homo- und Heterophilic Interaktionen engagieren und die synaptischen Spalt &uuml;berbr&uuml;cken, pr&auml;synaptische Klemmen genau. Hier zeigen wir, dass standortspezifische N-Glycosylation wirkt sich auf die Struktur und Funktion der Klebstoff SynCAM Interaktionen. Durch kristallographische Analyse von SynCAM 2 haben wir die selbstklebende Oberfl&auml;che seine Ig1 Dom&auml;ne ein N-Glykan in R&uuml;ckstand Asn(60) identifiziert. Strukturelle Modellierung des entsprechenden SynCAM 1 Ig1 Dom&auml;ne zufolge seiner Glycosylation Standorte Asn(70)/Asn(104) flankieren die Bindung-Schnittstelle in diesem Bereich. Mass spectrometric und teilhabende Studien best&auml;tigen und die &Auml;nderung dieser drei Sites zu charakterisieren. Diese standortspezifische N-Glykane beeinflusst SynCAM Adh&auml;sion noch differenzielle handeln. Obwohl Glykosylierung von SynCAM 2 bei Asn(60) Haftung reduziert, erh&ouml;hen N-Glykane am Asn(70)/Asn(104) des SynCAM 1 seine Interaktionen. Die &Auml;nderung der SynCAM 1 mit Sialins&auml;ure S&auml;uren tr&auml;gt zur St&auml;rkung seiner Bindung Glykan-abh&auml;ngigen. Funktional, N-Glykosylierung f&ouml;rdert die Trans-synaptischen Interaktionen von SynCAM 1 und f&uuml;r Synapse Induktion ist erforderlich. Diese Ergebnisse zeigen, dass N-Glycosylation SynCAM Proteine differentiell betrifft ihre Bindung-Schnittstelle und implizieren post-translationale Modifikation als Mechanismus zur Trans-synaptische Adh&auml;sion zu regulieren.

N-Glycosylation an der SynCAM (synaptische Zelladh&auml;sionsmolek&uuml;l)-Immunoglobulin-Schnittstelle moduliert synaptische Adh&auml;sion.

Profilins promote actin polymerization by exchanging ADP for ATP on monomeric actin and delivering ATP-actin to growing filament barbed ends. Apicomplexan protozoa such as Toxoplasma gondii invade host cells using an actin-dependent gliding motility. Toll-like receptor (TLR) 11 generates an innate immune response upon sensing T. gondii profilin (TgPRF). The crystal structure of TgPRF reveals a parasite-specific surface motif consisting of an acidic loop, followed by a long Î²-hairpin. A series of structure-based profilin mutants show that TLR11 recognition of the acidic loop is responsible for most of the interleukin (IL)-12 secretion response to TgPRF in peritoneal macrophages. Deletion of both the acidic loop and the Î²-hairpin completely abrogates IL-12 secretion. Insertion of the T. gondii acidic loop and Î²-hairpin into yeast profilin is sufficient to generate TLR11-dependent signaling. Substitution of the acidic loop in TgPRF with the homologous loop from the apicomplexan parasite Cryptosporidium parvum does not affect TLR11-dependent IL-12 secretion, while substitution with the acidic loop from Plasmodium falciparum results in reduced but significant IL-12 secretion. We conclude that the parasite-specific motif in TgPRF is the key molecular pattern recognized by TLR11. Unlike other profilins, TgPRF slows nucleotide exchange on monomeric rabbit actin and binds rabbit actin weakly. The putative TgPRF actin-binding surface includes the Î²-hairpin and diverges widely from the actin-binding surfaces of vertebrate profilins.

Structure-based analysis of Toxoplasma gondii profilin: a parasite-specific motif is required for recognition by Toll-like receptor 11.

Profilins f&ouml;rdern Aktin-Polymerisation durch den Austausch von ADP f&uuml;r ATP auf Monomer Aktin und Bereitstellung von ATP-Actin Filament Stacheldraht enden w&auml;chst. Apicomplexan Protozoen wie Toxoplasma Gondii dringen in Wirtszellen mit ein Aktin-abh&auml;ngige gleiten-Motilit&auml;t. Toll-Like-Rezeptor (TLR) 11 generiert eine angeborene Immunantwort auf Sensorik, T. Gondii profilin (TgPRF). Die Kristallstruktur von TgPRF zeigt ein Parasit-spezifische Oberfl&auml;che-Motiv, bestehend aus einer sauren Schleife, gefolgt von einem langen &beta;-Haarnadel. Eine Reihe von Struktur-basierte profilin Mutanten zeigen, dass TLR11 Anerkennung der sauren Schleife f&uuml;r die meisten der Interleukin (IL)-12 Sekretion Reaktion auf TgPRF im peritonealen Makrophagen verantwortlich ist. L&ouml;schen von der sauren-Schleife und die &beta;-Haarnadel hebt vollst&auml;ndig Sekretion von IL-12. Einf&uuml;gen von T. Gondii sauren Schleife und &beta;-Haarnadel in Hefe profilin ist ausreichend, um TLR11-abh&auml;ngige Signalisierung zu generieren. Ersetzung der sauren Schleife in TgPRF mit der homologen Schleife aus den Apicomplexan Parasiten Cryptosporidium Parvum betroffen TLR11-abh&auml;ngige IL-12-Sekretion, w&auml;hrend der Substitution durch die sauren Schleife von Plasmodium Falciparum f&uuml;hrt zu reduzierten, aber signifikante IL-12-Sekretion nicht. Wir schlie&szlig;en, dass das Parasit-spezifische-Motiv in TgPRF die wichtigsten Molekulare Muster von TLR11 erkannt. Im Gegensatz zu anderen Profilins TgPRF verlangsamt Nukleotid-B&ouml;rse auf Monomer Kaninchen Aktin und bindet Kaninchen Aktin schwach. Die vermeintliche TgPRF-Aktin-Bindung-Oberfl&auml;che enth&auml;lt die &beta;-Haarnadel und weicht stark von den Aktin-Bindung-Oberfl&auml;chen der vertebrate Profilins.

Struktur-basierte Analyse von Toxoplasma Gondii profilin: ein Parasit-spezifische-Motiv ist erforderlich f&uuml;r die Anerkennung von Toll-Like-Rezeptor 11.

Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory-secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75Â Ã… resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinant AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2's netrin-like fold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.

Ancylostoma ceylanicum excretory-secretory protein 2 adopts a netrin-like fold and defines a novel family of nematode proteins.

Hakenw&uuml;rmer sind menschliche Parasiten, die verheerende Auswirkungen auf die globale Gesundheit, besonders in den unterentwickelten L&auml;ndern haben. Ancylostoma Ceylanicum bef&auml;llt Mensch und Tier, so dass es eine n&uuml;tzliche Modellorganismus Pathogenese der Krankheit zu studieren. A. Ceylanicum Exkretorische sekretorischen Protein 2 (AceES-2), ein h&ouml;chst immunreaktivem Molek&uuml;l abgesondert von Erwachsenen W&uuml;rmer auf dem Gel&auml;nde des intestinalen Anlage ist teilweise Schutzma&szlig;nahmen als Schleimhaut-Immunisierung gegen Hakenwurm An&auml;mie verabreicht. Die Kristallstruktur von AceES-2 bestimmt bei 1,75 &Aring; Aufl&ouml;sung zeigt, die es A Netrin-wie Falte &auml;hnlich annimmt, die im Gewebe-Hemmer von Matrix-Metalloproteinasen (TIMPs) und in Erg&auml;nzung Faktoren C3 und C5 gefunden. Jedoch beeintr&auml;chtigt rekombinante AceES-2 nicht signifikant die 10 am h&auml;ufigsten vorkommenden menschlichen Matrix Metalloproteinasen oder Erg&auml;nzung-vermittelte Zellenzerst&ouml;rung. Das Vorhandensein einer stark sauren Oberfl&auml;che auf AceES-2 legt nahe, dass es als ein Zytokin-K&ouml;der-Rezeptor fungieren kann. Mehrere kleine Fadenw&uuml;rmer Proteine, die als TIMPs annotiert wurden oder Netrin-Dom&auml;ne-haltige Proteine zeigen Sequenz-Homologie in strukturell wichtigen Regionen der AceES-2 Netrin-wie Falten. Unseren Ergebnissen zufolge zusammen, AceES-2 eine neuartige Familie der Fadenw&uuml;rmer Netrin-&auml;hnliche Proteine, definiert das funktionieren wird, auf um dem Host Immunantwort zur Hakenwurm und andere Parasiten zu modulieren.

Ancylostoma Ceylanicum Exkretorische sekretorischen Protein 2 nimmt eine Falte Netrin-&auml;hnliche und definiert eine neue Familie der Fadenw&uuml;rmer Proteine.

The structure of full-length human TLR5 determined by electron microscopy single-particle image reconstruction at 26Ã… resolution shows that TLR5 forms an asymmetric homodimer via ectodomain interactions. The structure shows that like TLR9, TLR5 dimerizes in the absence of ligand. The asymmetry of the dimer suggests that TLR5 may recognize two flagellin molecules cooperatively to establish an optimal flagellin response threshold. A TLR5 homology model was generated and fitted into the electron microscopy structure. All seven predicted N-linked glycosylation sites are exposed on the molecular surface, away from the dimer interface. Glycosylation at the first five sites was confirmed by tandem mass spectrometry. Two aspartate residues proposed to interact with flagellin (Asp294 and Asp366) are sterically occluded by a glycan at position 342. In contrast, the central region of the ectodomains near the dimer interface is unobstructed by glycans. Ligand binding in this region would be consistent with the ligand binding sites of other TLRs.

Toll-like receptor 5 forms asymmetric dimers in the absence of flagellin.

Die Struktur der in menschlichen TLR5 durch Elektronenmikroskopie Einzel-Teilchen Bildrekonstruktion bei 26&Aring; Aufl&ouml;sung bestimmt zeigt, dass TLR5 eine asymmetrische Homodimer &uuml;ber APPs&alpha; Interaktionen bildet. Die Struktur zeigt, dass wie TLR9, TLR5 in Ermangelung Ligand dimerizes. Die Asymmetrie der Dimer zufolge TLR5 zwei Flagellin-Molek&uuml;le kooperativ herstellen eine optimalen Flagellin-Antwort-Schwelle erkennen kann. Ein TLR5 Homologie-Modell generiert und passte in der Elektronenmikroskopie-Struktur. Alle sieben vorhergesagten N-chromosomale Glycosylation Sites werden auf der molekularen Oberfl&auml;che, Weg von der Dimer-Schnittstelle verf&uuml;gbar gemacht. Glykosylierung an den ersten f&uuml;nf Standorten wurde durch Tandem-Massenspektrometrie best&auml;tigt. Zwei Aspartat-R&uuml;ckst&auml;nde vorgeschlagen Interaktion mit Flagellin (Asp294 und Asp366) sind durch eine Glykan an Position 342 sterisch verschlossen. Im Gegensatz dazu ist die Region, die Ectodomains in der N&auml;he der Dimer-Schnittstelle ungehindert durch Glykane. Ligand-Bindung in dieser Region w&uuml;rde mit der Ligand-Bindungsstellen von anderen TLRs &uuml;bereinstimmen.

Article	Year
MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA. The EMBO journal\| PubMed ID: 22314235	2012
DNA binding to proteolytically activated TLR9 is sequence-independent and enhanced by DNA curvature. The EMBO journal\| PubMed ID: 22258621	2011
The mu2 subunit of the clathrin adaptor AP-2 binds to FDNPVY and YppÃ˜ sorting signals at distinct sites. Traffic (Copenhagen, Denmark)\| PubMed ID: 12121421	2002
Atomic model of the papillomavirus capsid. The EMBO journal\| PubMed ID: 12234916	2002
A ligand-binding pocket in the dengue virus envelope glycoprotein. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 12759475	2003
X-ray structure of a protein-conducting channel. Nature\| PubMed ID: 14661030	2004
Structure of the dengue virus envelope protein after membrane fusion. Nature\| PubMed ID: 14737159	2004
Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein. Journal of virology\| PubMed ID: 15613349	2005
Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes. Journal of virology\| PubMed ID: 16943291	2006
West Nile virus envelope protein inhibits dsRNA-induced innate immune responses. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 18056386	2007
How influenza virus is locked out of the cell. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19033206	2008
Crystal structure of dengue virus type 1 envelope protein in the postfusion conformation and its implications for membrane fusion. Journal of virology\| PubMed ID: 19244332	2009
N-glycosylation at the SynCAM (synaptic cell adhesion molecule) immunoglobulin interface modulates synaptic adhesion. The Journal of biological chemistry\| PubMed ID: 20739279	2010
Structure-based analysis of Toxoplasma gondii profilin: a parasite-specific motif is required for recognition by Toll-like receptor 11. Journal of molecular biology\| PubMed ID: 20851125	2010
Ancylostoma ceylanicum excretory-secretory protein 2 adopts a netrin-like fold and defines a novel family of nematode proteins. Journal of molecular biology\| PubMed ID: 21352830	2011
Toll-like receptor 5 forms asymmetric dimers in the absence of flagellin. Journal of structural biology\| PubMed ID: 22173220	2011

Yorgo Modis

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