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Characterization of Cytochrome C Synthetase Ccsba and Latex Clearing Protein

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Characterization of Cytochrome C Synthetase Ccsba and Latex Clearing Protein Characterization of Cytochrome C Synthetase CcsBA and Latex Clearing Protein Inaugural Dissertation zur Erlangung der Doktorgrades der Fakultät für Chemie und Pharmazie der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Lorena Ilcu aus Botosani, Rumänien 2018 Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan Weber Dekan: Prof. Dr. Oliver Einsle Referent: Prof. Dr. Oliver Einsle Korreferentin: Prof. Dr. Susana Andrade Promotionsvorsitzender: Prof. Dr. Hans-Georg Koch Datum der mündlichen Prüfung: 5.12.2018 Diese Arbeit wurde am Institut für Biochemie der Albert-Ludwigs-Universität Freiburg im Breisgau unter der Leitung von Prof. Dr. Oliver Einsle erstellt. Summary The c-type cytochromes are a wide group of metalloproteins, characterized by the covalent attachment of the heme group to the surrounding polypeptide. Their assembly requires assistance from dedicated systems to bring together and to catalyse the thioether bond formation between the vinyl groups of the heme group and two cysteine residues found in a signature motif, most common CXXCH. At least three maturation pathways were developed during evolution [1]. System II or cytochrome c synthesis (Ccs), found in several bacterial species and in chloroplasts, comprises two proteins responsible for the reduction of the heme binding motif on the apocytochrome c and CcsBA complex, dedicated for cytochrome c production. CcsA displays a high conserved region, referred as the WWD motif, and together with two flanking histidine residues orients and positions the heme. The first part of the current study was focused on screening different homologs of CcsBA (or just CcsA) by heterologous production, purification and crystallization in order to elucidate the three-dimensional structure via X-ray crystallography. The CcsBA/CcsA homologs were isolated with a b-type heme found in an oxidized state (Fe3+), indicated by UV/Vis spectral analysis. CcsBA fusion protein from Bacteroides thetaiotaomicron in complex with n-Dodecyl β-d-maltoside (DDM) was the only homolog successfully crystallized. However, the crystal optimization by intensive screening of different conditions did not improve the quality of the diffraction experiments. The second part of this thesis focuses on the structural characterization by X-ray crystallography of latex clearing protein (Lcp) from Streptomyces sp. strain K30, a b-type cytochrome involved in rubber biodegradation. The crystallized structure of Lcp was solved by exploiting the anomalous signal of the heme iron. The three-dimensional model revealed a globular protein, with the core of the protein structurally related to the members of the globin family. Two different structural conformations were observed depending on the nature of the sixth axial ligand of the iron heme. In the close state, a lysine residue from the protein’s backbone unusually binds the iron atom in the distal site, suggesting a role in the enzymatic reaction, while in the open state the same position is occupied by an imidazole molecule, emerged from the crystal growth condition. Additional electron paramagnetic resonance experiments on LcpK30 were conducted to investigate intermediate states of the heme group. V VI Zusammenfassung C-Typ Cytochrome sind eine vielfältige Gruppe der Metalloproteine. Sie zeichnen sich durch eine kovalente Bindung der Häm Gruppe an das umgebende Polypeptid aus. Die Bildung dieser Thioetherbindung benötigt spezielle Enzyme, welche die Reaktion der Vinylgruppen des Häms mit zwei Cysteinresten aus einem Hämbindemotiv, meistens CXXCH, katalysieren. Mindestens drei verschiedene Systeme für die c-Typ Cytochromreifung wurden durch die Evolution entwickelt. System II oder cytochrome c synthesis (Ccs), zu finden in diversen Bakterien und Chloroplasten, besteht aus zwei Proteinen, zuständig für die Reduktion des Hämbindemotivs des Apocytochroms, und dem CcsBA-Komplex, welcher die Bindung des Häms an das Cytochrom und somit die Cytochrom c Reifung ermöglicht. CcsA enthält einen hochkonservierten Bereich, das sogenannte WWD-Motiv, gemeinsam mit zwei flankierenden Histidinen ist dieser Bereich für die Orientierung und Positionierung der Häm-Gruppe verantwortlich. Der erste Teil dieser Arbeit beschäftigt sich mit der Untersuchung verschiedener Homologe von CcsBA (oder nur CcsA) durch heterologe Produktion in E. coli, Reinigung und Kristallisation um mittels Röntgendiffraktometrie die dreidimensionale Struktur von CcsBA aufzuklären. In den isolierten CcsBA/CcsA-Homologen wurde ein oxidiertes Häm b (Fe3+) gefunden, nachgewiesen durch UV/vis-Spekroskopie. CcsBA aus Bacteroides thetaiotaomicron konnte als einziges Homolog erfolgreich kristallisiert werden. Intensives Screening der Kristallisationsbedingungen brachte jedoch keine Verbesserung bezüglich der Qualität der Diffraktionsexperimente. Der zweite Teil dieser Arbeit beschäftigt sich mit der strukturellen Charakterisierung des Latex clearing protein (Lcp) aus Streptomyces sp. strain K30, einem b-Typ Cytochrom, welches am biologischen Abbau von Kautschuk beteiligt ist, durch Röntgendiffraktometrie. Die Struktur des kristallisierten Lcp konnte unter Nutzung des anomalen Signals des Häm-Eisens gelöst werden. Das dreidimensionale Modell offenbarte eine globuläre Strukture, deren Kern strukturell mit denen der Globine verwandt ist. Zwei verschiedene strukturelle Konformationen konnten beobachtet werden, abhängig von der Art des sechsten Liganden des Häm-Eisens. Im geschlossenen Zustand bindet ein Lysin aus dem Peptidrückgrat, auf ungewöhnliche Weise, distal an das Eisenatom, während im offenen Zustand diese Position von einem Imidazolmolekül eingenommen wird, welches aus der Kristallisationsbedingung stammt. Dies weist auf eine Rolle des Lysins für die enzymatische Reaktion hin. Zusätzlich wurden VII Elektronenspinresonanzexperimente mit LcpK30 durchgeführt, um die Zwischenstufen der Häm-Gruppe zu untersuchen. VIII Table of Contents Characterization of the CcsBA Synthetase from System II of Cytochrome c Assembly .............................................................................................................. 1 1. Introduction .................................................................................................................. 3 1.1 Cytochromes c ............................................................................................................. 3 1.2 Heme synthesis ............................................................................................................ 4 1.3 Apocytochrome c translocation over various membranes ........................................... 7 1.4 Cytochrome c maturation systems ............................................................................... 8 1.5 System IV and V .......................................................................................................... 8 1.6 System III ..................................................................................................................... 9 1.7 System I ..................................................................................................................... 11 1.7.1 Bacterial cytochrome c maturation system ......................................................... 11 1.7.2 Mitochondrial cytochrome c maturation system ................................................. 17 1.8 System II .................................................................................................................... 18 1.8.1 Bacterial cytochrome c synthesis system ............................................................ 18 1.8.2 Chloroplastidial cytochrome c synthesis system ................................................ 22 1.9 Bacterial species with System II, selected for CcsBA or CcsA characterization ...... 24 1.9.1 Aquifex aeolicus .................................................................................................. 24 1.9.2 Bacillus megaterium ........................................................................................... 24 1.9.3 Bacteroides thetaiotaomicron ............................................................................. 25 1.9.4 Geobacter sulfurreducens ................................................................................... 26 1.9.5 Helicobacter hepaticus ....................................................................................... 26 1.9.6 Hydrogenobacter thermophilus .......................................................................... 27 1.9.7 Micrococcus luteus ............................................................................................. 27 1.9.8 Symbiobacterium thermophilum ......................................................................... 28 1.10 Human pathogenicity ............................................................................................... 28 IX 1.11 Evolution of cytochrome c biogenesis systems ....................................................... 29 1.12 X-Ray Crystallography ............................................................................................ 31 1.12.1 Protein Crystallization ...................................................................................... 31 1.12.2 X-Ray diffraction .............................................................................................. 33 1.12.3 Molecular replacement...................................................................................... 36 1.12.4 Single-wavelength anomalous diffraction (SAD) ............................................. 37 2. Scope
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