Debora Lucarelli

Publications (4)11.37 Total impact

• Article: The RecB nuclease domain binds to RecA-DNA filaments: implications for filament loading.

  Debora Lucarelli, Ying A Wang, Vitold E Galkin, Xiong Yu, Dale B Wigley, Edward H Egelman
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  ABSTRACT: The E. coli RecBCD enzyme facilitates the loading of RecA onto single-stranded DNA produced by the combined helicase/nuclease activity of RecBCD. The nuclease domain of RecB protein, RecB(nuc), has been previously shown to bind RecA. Surprisingly, RecB(nuc) also binds to phage and eukaryotic homologs of RecA, leading to the suggestion that RecB(nuc) interacts with the polymerization motif that is present in all three proteins. This mode of interaction could only be with monomeric RecA, as this motif would be buried in filaments. We show that RecB(nuc) binds extensively to the outside of RecA-DNA filaments. Three-dimensional reconstructions suggest that RecB(nuc) binds to the ATP-binding core of RecA, with a displacement of the C-terminal domain of RecA. Solution experiments confirm that the interaction of RecB(nuc) is only with the RecA core. Since the RecA C-terminal domain has been shown to be regulatory, the interaction observed may be part of the loading mechanism where RecB displaces the RecA C-terminal domain and activates a RecA monomer for polymerization.
  Journal of Molecular Biology 07/2009; 391(2):269-74. · 4.00 Impact Factor
• Source

  Available from: PubMed Central

  Article: The Metal-Dependent Regulators FurA and FurB from Mycobacterium Tuberculosis.

  Debora Lucarelli, Michael L Vasil, Wolfram Meyer-Klaucke, Ehmke Pohl
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  ABSTRACT: The ferric uptake regulators (Fur) form a large family of bacterial metal-activated DNA-binding proteins that control a diverse set of genes at the transcriptional level. Mycobacterium tuberculosis, the causative agent of tuberculosis, expresses two members of the Fur family, designated FurA and FurB. Although both belong to the same family, they share only approximately 25% sequence identity and as a consequence, they differ significantly in some of their key biological functions. FurA appears to be a specialized iron-dependent regulator that controls the katG gene, which encodes for a catalase-peroxidase involved in the response of M. tuberculosis to oxidative stress. KatG is also the key mycobacterial enzyme responsible for the activation of the first-line tuberculosis drug Isoniazid. FurB in contrast requires Zn(2+) rather than Fe(2+), to bind to its target sequence in regulated genes, which include those involved in Zn(2+)-homeostasis. Recent biochemical, crystallographic and spectroscopic data have now shed light on the activation and metal discrimination mechanisms in this protein family.
  International Journal of Molecular Sciences 09/2008; 9(8):1548-60. · 2.60 Impact Factor
• Article: Crystal structure and function of the zinc uptake regulator FurB from Mycobacterium tuberculosis.

  Debora Lucarelli, Santina Russo, Elspeth Garman, Anna Milano, Wolfram Meyer-Klaucke, Ehmke Pohl
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  ABSTRACT: Members of the ferric/zinc uptake regulator (Fur/Zur) family are the central metal-dependent regulator proteins in many Gram-negative and -positive bacteria. They are responsible for the control of a wide variety of basic physiological processes and the expression of important virulence factors in human pathogens. Therefore, Fur has gathered significant interest as a potential target for novel antibiotics. Here we report the crystal structure of FurB from Mycobacterium tuberculosis at a resolution of 2.7A, and we present biochemical and spectroscopic data that allow us to propose the functional role of this protein. Although the overall fold of FurB with an N-terminal DNA binding domain and a C-terminal dimerization domain is conserved among the Zur/Fur family, large differences in the spatial arrangement of the two domains with respect to each other can be observed. The biochemical and spectroscopic analysis presented here reveals that M. tuberculosis FurB is Zn(II)-dependent and is likely to control genes involved in the bacterial zinc uptake. The combination of the structural, spectroscopic, and biochemical results enables us to determine the structural basis for functional differences in this important family of bacterial regulators.
  Journal of Biological Chemistry 04/2007; 282(13):9914-22. · 4.77 Impact Factor
• Article: Structural Studies on Ferric Uptake Regulator Proteins from Mycobacterium tuberculosis

  Debora Lucarelli
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  ABSTRACT: Iron is the most abundant trace element in the human body and zinc is the second one. Control of ion homeostasis is of vital importance for mammals and bacteria. Regulation of the ion flux into or out of the cell is a complex and articulated mechanism that still needs to be elucidated. The highly specialized pathogen Mycobacterium tuberculosis has to contend with iron sequestration in order to survive in the human body. Iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage. Paucity of this metal triggers an extensive response to increase iron acquisition whereas an excess of it can be toxic for the cell. The control of intracellular iron concentration is also linked to other important processes including oxidative stress response and the regulation of virulence factors. Recent studies have shown that, in patients affected by TB/HIV exposed to high level of dietary iron, the risk of active pulmonary tuberculosis increases. In M. tuberculosis the ferric uptake regulator A (FurA) is activated by Fe2+ to bind specifically to its target DNA sequence thereby repressing the downstream genes. Zinc is also an important element for all living organisms and serves as a cofactor in all six classes of enzymes and also in several regulatory proteins. The intracellular concentration of this metal must be carefully regulated due its toxicity. Compared with eukaryotes, little is known about zinc homeostasis in bacteria. In the tuberculosis genome several genes coding for zinc proteins have been classified but curiously no zinc regulator has been yet defined. Surprisingly, instead, two Fur genes were identified: Mtb furA and Mtb furB, but no clear structural or functional distinction has been reported. In this thesis a careful and detailed structural and biological description of FurA and FurB proteins is presented. Using a variety of biochemical and biophysical methods - including electrophoretic mobility shift assay (EMSA), site directed mutations, isothermal calorimetry (ITC), microPIXE analysis, extended X-ray absorption fine spectroscopy (EXAFS) and X-ray crystallography - we investigated the metal binding sites together with the nature and the structure of these proteins. The combination of these results enable us to distinguish between structurally and functionally distinct metal binding sites, provide a meticulous description and qualitative and quantitative characterization of them, propose biological roles and present for the first time a 3D picture of a zinc uptake regulator. Eisen ist das häufigste Spurenelement im menschlichen Körper, Zink das zweithäufigste. Die Kontrolle der Ionenhomeostase ist von überlebenswichtiger Bedeutung für alle Organismen. Die Regulierung des Ionenflusses in oder aus der Zelle ist ein komplexer und und steng regulierter Mechanismus, der noch weitestgehend ungeklaert ist. Der hochspezialisierte Krankheitserreger Mycobacterium tuberculosis muss sich mit der Sequestration von Eisen begnügen, um im menschlichen Körper überleben zu können. Der Eisenstoffwechsel wird durch die transktriptionelle Regulation von Genen kontrolliert, die mit der Aufnahme, dem Transport und der Speicherung von Eisen zusammenhängen. Der Mangel dieses Metalls löst eine weitreichende Reaktion hin zur erhöhten Aufnahme von Eisen aus, während ein Überschuss für die Zelle toxisch sein kann. Die Kontrolle der intrazellulären Eisenkonzentration ist mit anderen wichtigen Prozessen wie der Reaktion auf oxidativen Stress und der Regulation von Virulenzfaktoren. Neuere Studien haben gezeigt, dass bei TB/HIV-Patienten die hohe Mengen diätisches Eisen zu sich nehmen das Risiko einer akuten offenen Tuberkulose ansteigt. In M. tuberculosis wird der Eisenaufnahmeregulator A (engl. ferric uptake regulator A: FurA) durch Fe2+ zur spezifischen Bindung an seine DNA-Zielsequenz aktiviert, wodurch nachgeordnete Gene reprimiert werden. Zink ist ein weiteres wichtiges Element für alle lebenden Organismen und dient als Kofaktor in allen sechs Enzymklassen sowie in verschiedenen Regulationsproteinen. Die intrazelluläre Konzentration dieses Metalls muss aufgrund seiner Toxizität sehr genau reguliert werden. Verglichen mit Eukaryonten ist nur sehr wenig über die Zinkhomeostase in Bakterien bekannt. Im Genom von M. tuberculosis wurden mehrere Gene fuer potentiell Zink bindende Proteine identifiziert, allerdings wurde bisher seltsamerweise kein Zinkregulator gefunden. Stattdessen wurden erstaunlicherweise zwei Fur-Gene identifiziert: Mtb furA und Mtb furB, allerdings wurde kein eindeutiger struktureller oder funktioneller Unterschied berichtet. In dieser Arbeit wird eine sorgfältige und detaillierte strukturelle und biologische Beschreibung der FurA- und FurB-Proteine dargestellt. Mit Hilfe einer Reihe von biochemischen und biophysikalischen Methoden, darunter Untersuchungen der elektrophoretischen Mobilitätsveränderungen (engl. electrophoretic mobility shift assay: EMSA), ortsspezifische Mutationen, isothermale Titrations Kalorimetrie (ITC), microPIXE, Röntgenabsorptionsspektroskopie (engl. extended X-ray absorption fine structure: EXAFS) und Röntgenkristallographie, haben wir die Metallbindungsstellen zusammen mit der Struktur und den Eigenschaften dieser Proteine charakterisiert. Die Kombination dieser Ergebnisse erlaubte es zwischen strukturell und funktionell unterschiedlichen Metallbindungsstellen zu unterscheiden, eine aeusserst genaue qualitative und quantitative Charakterisierung von ihnen zu erstellen. sowie eine Erklärung ihrer biologischen Aufgaben und zum ersten Mal eine dreidimensionale Darstellung eines Zinkaufnahmeregulators vorzustellen.