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

The Role of PAS Kinase in PASsing the Glucose Signal

PAS kinase is an evolutionarily conserved nutrient responsive protein kinase that regulates glucose homeostasis. Mammalian PAS kinase is activated by glucose in pancreatic beta cells, and knockout mice are protected from obesity, liver triglyceride accumulation, and insulin resistance when fed a high-fat diet. Yeast PAS kinase is regulated by both carbon source and cell integrity stress and stimulates the partitioning of glucose toward structural carbohydrate biosynthesis. In our current model for PAS kinase regulation, a small molecule metabolite binds the sensory PAS domain and activates the enzyme. Although bona fide PAS kinase substrates are scarce, in vitro substrate searches provide putative targets for exploration

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

Engineering Genetically Encoded Nanosensors for Real-Time In Vivo Measurements of Citrate Concentrations

Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation

Structures of enveloped virions determined by cryogenic electron microscopy and tomography : Advances in Virus Research

Enveloped viruses enclose their genomes inside a lipid bilayer which is decorated by membrane proteins that mediate virus entry. These viruses display a wide range of sizes, morphologies and symmetries. Spherical viruses are often isometric and their envelope proteins follow icosahedral symmetry. Filamentous and pleomorphic viruses lack such global symmetry but their surface proteins may display locally ordered assemblies. Determining the structures of enveloped viruses, including the envelope proteins and their protein-protein interactions on the viral surface, is of paramount importance. These structures can reveal how the virions are assembled and released by budding from the infected host cell, how the progeny virions infect new cells by membrane fusion, and how antibodies bind surface epitopes to block infection. In this chapter, we discuss the uses of cryogenic electron microscopy (cryo-EM) in elucidating structures of enveloped virions. Starting from a detailed outline of data collection and processing strategies, we highlight how cryo-EM has been successfully utilized to provide unique insights into enveloped virus entry, assembly, and neutralization.Peer reviewe

Structural Basis for c-di-GMP-Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis

X-ray crystallographic structural analyses of the bacterial transmembrane receptor LapD identify conserved molecular mechanisms that control biofilm formation in response to changes in the intracellular levels of the second messenger c-di-GMP

Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets

In bacteria, adaptation to changing environmental conditions is often mediated by two-component signal transduction systems. In the prototypical case, a specific stimulus is sensed by a membrane-bound histidine kinase and triggers autophosphorylation of a histidine residue. Subsequently, the phosphoryl group is transferred to an aspartate residue of the cognate response regulator, which then becomes active and mediates a specific response, usually by activating and/or repressing a set of target genes. In this review, we summarize the current knowledge on two-component signal transduction in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for the large-scale biotechnological production of amino acids and can also be applied for the synthesis of a wide variety of other products, such as organic acids, biofuels, or proteins. Therefore, C. glutamicum has become an important model organism in industrial biotechnology and in systems biology. The type strain ATCC 13032 possesses 13 two-component systems and the role of five has been elucidated in recent years. They are involved in citrate utilization (CitAB), osmoregulation and cell wall homeostasis (MtrAB), adaptation to phosphate starvation (PhoSR), adaptation to copper stress (CopSR), and heme homeostasis (HrrSA). As C. glutamicum does not only face changing conditions in its natural environment, but also during cultivation in industrial bioreactors of up to 500 m3 volume, adaptability can also be crucial for good performance in biotechnological production processes. Detailed knowledge on two-component signal transduction and regulatory networks therefore will contribute to both the application and the systemic understanding of C. glutamicum and related species

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

Membrane-assisted mineral trioxide aggregate apical plug for management of traumatized immature anterior teeth: Clinical case reports

Apexification using calcium hydroxide has many disadvantages, such as, it needs more time for the treatment, chance for fracture of the tooth, and incomplete calcification of the bridge. There are many alternative treatments introduced, which have gained popularity, such as, forming an apical plug using mineral trioxide aggregate (MTA), for excellent results. In cases of wide open apices, it is difficult to limit the restoration to the working length, as such situations lead to the apical extrusion of the material into the periapical region, which prevents further healing. Such conditions can be best treated with the use of a resorbable collagen membrane, which limits the restoration till the working length and prevents the extrusion of the material beyond the apex. The present case reports highlight the non-surgical management of immature teeth by using a membrane as a barrier, with an MTA apical plug, followed by crown rehabilitation

Similarities in the structure of the transcriptional repressor AmtR in two different space groups suggest a model for the interaction with GlnK

AmtR belongs to the TetR family of transcription regulators and is a global nitrogen regulator that is induced under nitrogen-starvation conditions in Corynebacterium glutamicum. AmtR regulates the expression of transporters and enzymes for the assimilation of ammonium and alternative nitrogen sources, for example urea, amino acids etc. The recognition of operator DNA by homodimeric AmtR is not regulated by small-molecule effectors as in other TetR-family members but by a trimeric adenylylated P(II)-type signal transduction protein named GlnK. The crystal structure of ligand-free AmtR (AmtR(orth)) has been solved at a resolution of 2.1 Å in space group P2(1)2(1)2. Comparison of its quaternary assembly with the previously solved native AmtR structure (PDB entry 5dy1) in a trigonal crystal system (AmtR(tri)) not only shows how a solvent-content reduction triggers a space-group switch but also suggests a model for how dimeric AmtR might stoichiometrically interact with trimeric adenylylated GlnK