High-resolution structure determination of the CylR2 homodimer using paramagnetic relaxation enhancement and structure-based prediction of molecular alignment

Structure determination of homooligomeric proteins by NMR spectroscopy is difficult due to the lack of chemical shift perturbation data, which is very effective in restricting the binding interface in heterooligomeric systems, and the difficulty of obtaining a sufficient number of intermonomer distance restraints. Here we solved the high-resolution solution structure of the 15.4 kDa homodimer CylR2, the regulator of cytolysin production from Enterococcus faecalis, which deviates by 1.1 Å from the previously determined X-ray structure. We studied the influence of different experimental information such as long-range distances derived from paramagnetic relaxation enhancement, residual dipolar couplings, symmetry restraints and intermonomer Nuclear Overhauser Effect restraints on the accuracy of the derived structure. In addition, we show that it is useful to combine experimental information with methods of ab initio docking when the available experimental data are not sufficient to obtain convergence to the correct homodimeric structure. In particular, intermonomer distances may not be required when residual dipolar couplings are compared to values predicted on the basis of the charge distribution and the shape of ab initio docking solutions

Untersuchungen mittels Protein NMR an zwei Systemen mit Einfluss auf bakterielle Pathogenität

Die vorliegende Dissertation beinhaltet neue Strukturdaten über bakterielle Proteine, die an der Transkriptionsregulation und Proteinsekretion beteiligt sind, sowie einen Beitrag zur Verbesserung der Strukturaufklärung von homodimeren Proteinen mittels NMR-Spektroskopie. Ein Hauptaugenmerk liegt auf der Untersuchung von zwei homodimeren bakteriellen Proteinen: Dem Transkriptionsfaktor CylR2 aus Enterococcus faecalis und dem Chaperon CesT aus enteropathogenem Escherichia coli. Enterococcus faecalis hat sich zu einer Hauptursache von Infektionen in Krankenhäusern aufgrund von Antibiotikaresistenzen entwickelt, wobei die schwere der Infektion mit einem von Enterococcus faecalis sekretierten Protein, dem Cytolysin, zusammenhängt. Die Produktion dieses Cytolysins wird von einem zwei-Komponenten CylR1/CylR2 System über einen autoinduzierten Quorum-Sensing Mechanismus reguliert. In dieser Arbeit wird gezeigt, dass das regulatorische Protein CylR2 ein rigides Dimer formt. Die Kristall- und NMR-Struktur sind im wesentlichen identisch. Jedes Monomer besteht aus einem fünf Helix-Bündel, das ein Helix-Turn-Helix DNA-Bindingsmotiv enthält und durch ein zweisträngiges antiparalleles beta-Faltblatt verlängert ist. Die Lösung der NMR-Struktur erfolgt mit Hilfe einer neuartigen Strukturbestimmungsmethode für homodimere Proteine. Um die beiden Untereinheiten als rigide Körper aneinander zu docken, werden experimentelle residuale dipolaren Kopplungen der Rückgrat-Amide und intermolekulare, langreichweitige Distanzen, die über paramagnetische Relaxationsverstärkung ermittelt werden, verwendet. Eine Modellstruktur für CylR2 im Komplex mit seiner spezifischen palindromischen DNA innerhalb des Cytolysinpromotors wird basierend auf der Veränderung der chemischen Verschiebungen von CylR2 bei DNA-Bindung erstellt. Diese Ergebnisse deuten auf eine Rolle von CylR2 als Repressor der Cytolysintranskription hin. Enteropathogenes Escherichia coli ist eine Hauptursache von Durchfallerkrankungen, die durch Sekretion bakterieller Proteine über ein Type III Sekretionssystem in die menschliche Wirtszelle ausgelöst werden. CesT ist an dem Sekretionsmechanismus beteiligt indem es Effektorproteine vor der Sekretion spezifisch im bakteriellen Cytoplasma bindet und dadurch den Effektor in einem sekretionsbereiten Zustand hält. Die genaue Funktion des Chaperons und damit der Mechanismus der bakteriellen Type III Proteinsekretion ist noch ungeklärt. Mittels dipolarer Kopplungen wird gezeigt, dass CesT in Lösung, anders als im Kristall, eine den homologen Proteinen ähnliche Struktur ausbildet. Viele hydrophobe Interaktionen werden für die Erkennung von Effektoren durch CesT als wichtig identifiziert und abschliessend wird ein Modell für die Erkennung des Chaperon/Effektor Komplexes durch das Type III Sekretionssystem vorgeschlagen.The present work provides new structural information about bacterial proteins involved in transcription regulation and protein secretion, as well as a contribution to the improvement of structure determination of homodimeric proteins by NMR spectroscopy. A main focus is placed on two homodimeric bacterial proteins: the transcription factor CylR2 from Enterococcus faecalis and the chaperone CesT from enteropathogenic Escherichia coli. Enterococcus faecalis has emerged as a leading agent of hospital-acquired antibiotic-resistant infections and the severity of its infections has been linked to a secreted protein called cytolysin. Production of this enterococcal cytolysin is regulated by the two-component CylR1/CylR2 system through an autoinduction quorum-sensing mechanism. Here, the regulatory protein CylR2 is found to form a rigid dimer with an essentially identical crystal and solution NMR structure. Each monomer contains a helix-turn-helix DNA-binding motif as part of a five helix-bundle, which is extended by an antiparallel beta-sheet. The determination of the solution NMR structure involves the development of a novel method for homodimeric proteins. This method applies rigid-body docking driven by backbone amide residual dipolar couplings and intermolecular long-range distances from paramagnetic relaxation enhancement. A model structure for CylR2 in complex with its specific palindromic DNA within the cytolysin promotor region is derived based on NMR chemical shift perturbation experiments. These results suggest that CylR2 acts as a repressor of cytolysin transcription. The other studied homodimer, the chaperone CesT, originates from enteropathogenic Escherichia coli which is a chief cause of diarrhea and involves secretion of bacterial proteins via a type three secretion system into the human host cell. CesT participates in the secretion mechanism by specifically binding effector proteins in the bacterial cytoplasm prior to their secretion; the effector is thereby kept in a secretion-competent form. However, the exact function of the chaperone is still open and remains a key question for understanding the bacterial type three protein secretion mechanism. A novel application of NMR dipolar couplings is used to elucidate the structure of CesT in solution. In this way CesT is shown to form a structure similar to its homologues. Many hydrophobic interactions are identified to be important for the complex formation with at least two of its effectors. Finally, a model is suggested for the recognition of the chaperone/effector complex by the type three secretion system

Assignment-free solution NMR method reveals CesT as an unswapped homodimer

The X-ray structure of the homodimeric chaperone CesT is the only structure among the type three secretion system (TTSS) chaperones that shows a domain swap. This swap has potential importance for the mechanism of effector translocation through a TTSS. Here we present two nuclear magnetic resonance strategies exploiting pre-existing structural models and residual dipolar couplings (RDCs), which reveal the unswapped 35.4-kDa dimer to be present in solution. Particularly efficient is the discrimination of a swapped and unswapped structural state performed simultaneously to automatic backbone assignment using only HN-RDCs and carbonyl backbone chemical shifts. This direct approach may prove to be generally useful to rapidly differentiate two structural models

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

07 avril 19271927/04/07 (A28,N9609)

Structure and DNA-binding properties of the cytolysin regulator CylR2 from Enterococcus faecalis

Enterococcus faecalis is one of the major causes for hospital-acquired antibiotic-resistant infections. It produces an exotoxin, called cytolysin, which is lethal for a wide range of Gram-positive bacteria and is toxic to higher organisms. Recently, the regulation of the cytolysin operon was connected to autoinduction by a quorum-sensing mechanism involving the CylR1/CylR2 two-component regulatory system. We report here the crystal structure of CylR2 and its properties in solution as determined by heteronuclear NMR spectroscopy. The structure reveals a rigid dimer containing a helix–turn–helix DNA-binding motif as part of a five-helix bundle that is extended by an antiparallel β-sheet. We show that CylR2 is a DNA-binding protein that binds specifically to a 22 bp fragment of the cytolysin promoter region. NMR chemical shift perturbation experiments identify surfaces involved in DNA binding and are in agreement with a model for the CylR2/DNA complex that attributes binding specificity to a complex network of CylR2/DNA interactions. Our results propose a mechanism where repression is achieved by CylR2 obstruction of the promoter preventing biosynthesis of the cytolysin operon transcript

Nitrite Dismutase Reaction Mechanism: Kinetic and Spectroscopic Investigation of the Interaction between Nitrophorin and Nitrite

Nitrite is an important metabolite in the physiological pathways of NO and other nitrogen oxides in both enzymatic and nonenzymatic reactions. The ferric heme <i>b</i> protein nitrophorin 4 (NP4) is capable of catalyzing nitrite disproportionation at neutral pH, producing NO. Here we attempt to resolve its disproportionation mechanism. Isothermal titration calorimetry of a gallium­(III) derivative of NP4 demonstrates that the heme iron coordinates the first substrate nitrite. Contrary to previous low-temperature EPR measurements, which assigned the NP4-nitrite complex electronic configuration solely to a low-spin (<i>S</i> = 1/2) species, electronic absorption and resonance Raman spectroscopy presented here demonstrate that the NP4-NO₂^– cofactor exists in a high-spin/low-spin equilibrium of 7:3 which is in fast exchange in solution. Spin-state interchange is taken as evidence for dynamic NO₂^– coordination, with the high-spin configuration (<i>S</i> = 5/2) representing the reactive species. Subsequent kinetic measurements reveal that the dismutation reaction proceeds in two discrete steps and identify an {FeNO}⁷ intermediate species. The first reaction step, generating the {FeNO}⁷ intermediate, represents an oxygen atom transfer from the iron bound nitrite to a second nitrite molecule in the protein pocket. In the second step this intermediate reduces a third nitrite substrate yielding two NO molecules. A nearby aspartic acid residue side-chain transiently stores protons required for the reaction, which is crucial for NPs’ function as nitrite dismutase