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Bestimmung Des Interaktionsbereiches Sowie Der Struktur Des Bestimmung des Interaktionsbereiches sowie der Struktur des Adrenodoxins im Komplex mit der Adrenodoxin-Reduktase mit Hilfe der paramagnetischen NMR-Spektroskopie Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät III Chemie, Pharmazie, Bio- und Werkstoffwissenschaften der Universität des Saarlandes von Frau Dipl. Chem. Berna Mersinli Saarbrücken 2010 Tag des Kolloquiums: 12. Oktober 2010 Dekan: Prof. Dr. Stefan Diebels Berichterstatter: Prof. Dr. Rita Bernhardt Prof. Dr. Marcellus Ubbink Vorsitz: Prof. Dr. Uli Müller Akademischer Mitarbeiter: Dr. Gert-Wieland Kohring Inhaltsverzeichnis Abkürzungsverzeichnis 4 Zusammenfassung 7 Summary 8 1 Einleitung 10 1.1 Cytochrome P450 . 10 1.2 Ferredoxin-NADPH Reduktasen . 16 1.3 Ferredoxine . 19 1.4 Interaktion des Adx mit AdR und CYP11A1 . 24 1.5 NMR-Spektroskopie . 26 1.6 Ziel der Arbeit . 34 2 Material und Methoden 36 2.1 Materialien . 36 2.2 Molekularbiologische Methoden . 39 2.3 Heterologe Proteinexpression und Reinigung . 42 2.4 Biochemische und biophysikalische Methoden . 46 2.5 NMR-Untersuchungen . 52 3 Ergebnisse 60 3.1 Herstellung der AdR-Mutanten . 61 3.2 Charakterisierung der AdR-Mutanten . 72 3.3 Paramagnetische NMR-Experimente . 84 4 Diskussion und Ausblick 110 4.1 Heterologe Expression und Reinigung der AdR-Formen . 111 4.2 Charakterisierung der AdR-Mutanten . 118 4.3 Paramagnetische NMR-Messungen . 123 3 Inhaltsverzeichnis 5 Veröffentlichungen 143 Literaturverzeichnis 143 Anhang 164 4 Abkürzungsverzeichnis Abkürzungen A450 Absorption bei einer Wellenlänge von 450 nm Adx Adrenodoxin AdxWT Adrenodoxin Wildtyp (1-128) Adx(4-108) verkürzte Form des Adx, fehlende Aminosäuren 1-3 und 109-128 15N2H-AdxWT deuteriertes, 15N-markiertes Adrenodoxin(1-128) 15N2H-Adx(4-108) deuteriertes, 15N-markiertes Adrenodoxin(4-108) AdR Adrenodoxin-Reduktase AdRWT Adrenodoxin-Reduktase Wildtyp bp Basenpaar CLaNP-5 Lanthanoid-ligierende NMR-Sonde-5 (Caged Lanthanide NMR Probe-5 ) CO Carbonmonooxid CSA Chemische-Verschiebungsanisotropie (Chemical Shift Anisotropy) CSP Chemische-Verschiebungsänderung (Chemical Shift Perturbation) CYP Cytochrom P450 CYP11A1 Cytochrom P45011A1, auch P450scc genannt Da Dalton DNA Desoxyribonucleinsäure DRMS Distanz-Effektivwert (Distance Root Mean Square) DTE Dithioerythritol DTT Dithiothreitol DTNB Dithiobisnitrobenzoesäure EDC 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimid EDTA Ethylendiamintetraacetat FAD Flavinadenindinukleotid (oxidierte Form) Gd Gadolinium HEPES 4-(2-Hydroxyethyl)-piperazin-1-propansulfonsäure HPLC Hochleistungsflüssigkeitschromatographie (High Performance Liquid Chromatography) 5 Inhaltsverzeichnis IPTG Isopropyl-β-D-thiogalactosid KA Assoziationskonstante ka Assoziationsrate KD Dissoziationskonstante kd Dissoziationsrate KM Michaelis-Menten-Konstante K Kelvin KPP Kaliumphosphatpuffer LCMS Flüssigkeitschromatographie mit Massenspektrometrie (Liquid Chromatography-Mass Spectrometry) Ln Lanthanoid Lu Lutetium NADPH Nikotinamidadenindinucleotidphosphat (reduzierte Form) NaPP Natriumphosphatpuffer NHS N-Hydroxysuccinimid NMR Kernspinresonanzspektroskopie (Nuclear Magnetic Resonance) OD optische Dichte ox oxidiert PCR Polymerase-Kettenreaktion (Polymerase Chain Reaction) PCS Pseudokontaktverschiebung (Pseudocontact Shift) PDB Proteindatenbank (Protein Data Bank) PMSF Phenylmethylsulfonylfluorid ppm parts per million PRE Paramagnetische-Relaxationsverstärkung (Paramagnetic Relaxation Enhancement) Q-Wert Reinheitsgrad der Proteinlösung der AdR (A270 nm/A450 nm) red reduziert RMS Effektivwert (Root Mean Square) RMSD Standardabweichung (Root Mean Square Deviation) RU Resonanzeinheit (Responce Unit) SPR Oberflächenplasmonresonanz (Surface Plasmon Resonance) T Temperatur 6 Inhaltsverzeichnis Tm Thulium TROSY Transversale Relaxationsoptimierte Spektroskopie (Transverse Relaxation Optimized Spectroscopy) u.a. unter anderem UV/Vis Ultravioletter und visueller Wellenlängenbereich WT Wildtyp Abkürzungen für Aminosäuren A Ala Alanin M Met Methionin C Cys Cystein N Asn Asparagin D Asp Aspartat P Pro Prolin E Glu Glutamat Q Gln Glutamin F Phe Phenylalanin R Arg Arginin G Gly Glycin S Ser Serin H His Histidin T Thr Threonin I Ile Isoleucin V Val Valin K Lys Lysin W Trp Tryptophan L Leu Leucin Y Tyr Tyrosin 7 Zusammenfassung In dieser Arbeit wurden der Interaktionsbereich und die Struktur des Adrenodoxins (Adx) im Komplex mit der Adrenodoxin-Reduktase (AdR) beschrieben. Beide Pro- teine sind wichtige Komponenten der Steroidhormon-Biosynthese. Für die Unter- suchungen wurden AdR-Mutanten (AdR_A: AdR_C364S/S425C/K429C, AdR_B: AdR_C145S/C364S/Q232C/K236C) hergestellt, die jeweils ein exponiertes Cysteinpaar besitzen. Beide Mutanten zeigten gleiche strukturelle und funktionale Eigenschaften wie der AdR-Wildtyp (AdRWT). Der Interaktionsbereich des 15N2H-Adx im Komplex mit AdRWT wurde durch die Bestimmung der Chemischen-Verschiebungsänderung unter Anwendung der Transversalen Relaxationsoptimierten NMR-Spektroskopie (TROSY) bestimmt. CLaNP-5, eine Lanthanoid-ligierende Sonde, wurde über Disulfidbrücken an die AdR gekoppelt und für paramagnetische TROSY-NMR-Untersuchungen der Pseu- dokontaktverschiebungen und Paramagnetischen-Relaxationsverstärkungen eingesetzt. Die dadurch erhaltenen, weitreichenden Distanzinformationen wurden für Docking- Berechnungen des AdR-Adx-Komplexes verwendet. Diese Berechnungen wurden von Herrn Dr. Peter H.J. Keizers (Universität Leiden, Niederlande) durchgeführt. Der Inter- aktionsbereich des Adx in Wechselwirkung mit der AdR konnte damit in dieser Arbeit zum ersten Mal detailliert beschrieben werden, der seinerseits die eingehendere Bestim- mung des AdR-Adx-Komplexes in Lösung ermöglichte. 8 Summary In this work the structure of adrenodoxin (Adx) and its region, which is important for the interaction with adrenodoxin reductase (AdR), will be described. Both pro- teins are important components of the steroid hormone biosynthesis in the adrenal cortex. Therefore, two AdR mutants (AdR_A: AdR_C364S/S425C/K429C, AdR_B: AdR_C145S/C364S/Q232C/K236C) containing two exposed cysteins on the protein surface, respectively, have been prepared and characterized. AdR_A and AdR_B sho- wed the same structural and functional properties as the wild type form of AdR. The exact interaction region of 15N2H-Adx in interaction with AdR wild type has been deter- mined by measuring Chemical Shift Perturbations (CSP) using the method of Transverse Relaxation Optimized NMR-Spectroscopy (TROSY). Furthermore, to obtain long-range distance information by paramagnetic TROSY-NMR measurements, a Caged Lanthani- de NMR Probe-5 (CLaNP-5) has been ligated to the AdR mutant surface via disulfide bridges, causing Pseudocontact Shifts (PCS) and Paramagnetic Relaxation Enhance- ment (PRE). These restraints (CSP, PCS, and PRE) have been used by Dr. Peter H.J. Keizers (University Leiden, The Netherlands) to perform docking experiments of Adx and AdR. This work describes in detail for the first time the interaction region of Adx and the structure of the AdR-Adx-complex in solution. 9 1 Einleitung 1.1 Cytochrome P450 1.1.1 Allgemeine Aspekte 1958 wiesen Garfinkel und Klingenberg (Garfinkel, 1958; Klingenberg, 1958) in Leber- mikrosomen ein CO bindendes Pigment nach, welches im reduzierten und CO- gebundenen Komplex ein untypisches Absorptionsmaximum bei einer Wellenlänge von 450 nm zeigte (Omura & Sato, 1964a,b). Dieses Pigment konnte als ein b-Typ Hämopro- tein identifiziert werden und erhielt durch diese Eigenschaft seinen Namen Cytochrom P450, wobei das P für Pigment und 450 die entsprechende Wellenlänge steht (Omura & Sato, 1964a,b). Dieses für Cytochrome untypische Absorptionsspektrum wird durch das Thiolat-Anion eines Cysteins induziert, welches die fünfte Ligandenstelle am Ei- sen des Protoporphyrins IX besetzt (Ichikawa & Yamano, 1967; Murakami & Mason, 1967). Durch diese Eigenschaft erfolgt die Klassifizierung der Cytochrom P450 Enzyme als Häm-Thiolat-Proteine. Cytochrome P450 zählen zu den Oxidoreduktasen. Sie katalysieren die reduktive Spal- tung des molekularen Sauerstoffs und zählen somit zur Untergruppe der Oxygenasen, die weiterhin in die Gruppen der Mono-, und Dioxygenasen unterteilt wird. Cytochro- me P450 katalysieren den Einbau eines Sauerstoffatoms in das Substrat, während das zweite zu Wasser umgesetzt wird und zählen somit zu den Monooxygenasen (Mason et al., 1955; Mason, 1957; Hayaishi & Nozaki, 1969). Hayaishi und Nozaki unterteilten die Gruppe der Monooxygenasen in die Gruppen der internen und externen Monooxy- genasen (Hayaishi & Nozaki, 1969). Während interne Monooxygenasen die benötigten Elektronen zur Reduktion des Sauerstoffatoms dem Substrat entnehmen, erhalten die ex- ternen Monooxygenasen diese von einem externen Elektronendonator wie z.B NADPH. Die Einordnung der Cytochrome P450 ist in Abbildung 1.1 aufgeführt. 10 1 Einleitung Abbildung 1.1: Einordnung der Cytochrome P450 in Enzymgruppen. Die Cytochrome P450 werden zu einer Proteinsuperfamilie zusammengefasst, die aus 11294 bekannten Enzymen besteht und die in 977 Familien unterteilt werden kann (Stand November 2009: http://drnelson.utmem.edu/CytochromeP450.html). Cytochrome P450 sind ubiquitäre Hämproteine, die in allen Lebensformen vorkommen und ein weitreichen- des Spektrum an Reaktionen katalysieren. Um die Vielfalt der vorhandenen Cytochrome klassifizieren zu können, führte Nebert 1987 eine systematische Nomenklatur ein (Nebert et al., 1987). In Abbildung 1.2 wird anhand des Beispiels CYP11A1 diese Systematik veranschaulicht. CYP steht für das
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