京都大学 / 新制・課程博士 / 博士(工学) / 甲第24638号 / 工博第5144号 / 新制||工||1982(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 浜地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. The four cluster biogenesis systems that have been discovered require at least one ATPase, but generally the function of nucleotide hydrolysis is understudied. In the cytosolic iron sulfur cluster assembly (CIA) system, responsible for delivering [Fe4-S4] cluster cofactors for cytosolic and nuclear enzymes, the assembly scaffold comprises two homologous ATPases, called Nbp35 and Cfd1 in Saccharomyces cerevisiae. Genetic studies have discovered that the ATPase sites are required for scaffold function in vivo, but in vitro studies have failed to reveal why. The ATPase sites of the Nbp35 and Cfd1 contain a conserved P-loop nucleotide-binding protein fold with a deviant Walker A motif. Known metal trafficking P-loop NTPases’ metallochaperone mechanisms rely on both nucleotide binding and hydrolysis to properly assemble and deliver metal cargo. Furthermore, P-loop NTPases with a deviant Walker A motif commonly serve as central regulatory switches whose hydrolysis activity is modulated by small molecule cargos and/or protein partners. Therefore, it is proposed that the role of Nbp35-Cfd1’s ATPase sites is to direct Fe-S cluster movement by regulating protein and metal cargo interactions. The goal of this thesis is to better understand the scaffold reaction cycle by investigating the metallochaperone mechanism through Nbp35-Cfd1’s protein communications with its ATPase sites. To do this, the identification of at least one nucleotide-dependent partner protein must first be discovered. Herein, in vitro methods have been developed to uncover the scaffold’s ATPase site regulation of protein interactions. We describe a qualitative affinity copurification assay and a quantitative analysis for evaluating the dissociation constant and the kcat and Km values for ATP hydrolysis for the scaffold–partner protein complex. Additionally, the execution of these ATPase assays in an anaerobic environment can be applied to study nucleotide hydrolases involved in metallocluster biogenesis. These in vitro methods are applied to Nbp35-Cfd1 and it is discovered that ATP binding and hydrolysis regulates Nbp35-Cfd1 binding with two CIA factors: Dre2, a reductase proposed to assist in Fe-S cluster assembly, and Nar1, an adaptor between the early and late CIA factors. Although reconstitution of the scaffold’s Fe-S clusters results in a two-fold increase in its ATPase activity, the Dre2 and Nar1 ATP hydrolysis stimulation is dampened, demonstrating that both the Fe-S cargo and partner proteins regulate the scaffold’s ATPase reaction cycle. Next, the domains required for binding and ATPase stimulation were identified for Nbp35-Cfd1 with its partner proteins Dre2 and Nar1. The C-terminal Fe-S binding domain of Dre2 is sufficient for ATPase stimulation, while the Nar1 requires both its N- and C-terminal Fe-S binding domains to activate Nbp35-Cfd1’s ATP hydrolysis. The N-terminal Fe-S binding domain of Nbp35 is dispensable for binding and ATPase stimulation of both Dre2 and Nar1. The CIA targeting complex protein Cia1, which binds to Nar1, competes off Nbp35-Cfd1, indicating a shared binding domain. This data both validates and refines the current working model of the CIA system. To test whether the communication between the ATPase and Fe-S cluster binding domains of the CIA scaffold functions in an analogous manner across multiple species, a preliminary analysis was completed for whether Chaetomium thermophilum and Homo sapien Nbp35-Cfd1 exhibit similar ATPase characteristics and partner protein interaction as their S. cerevisiae ortholog. Human and fungal Nbp35-Cfd1 exhibit ATP binding and demonstrate nucleotide-dependent interactions with Dre2 and Nar1, suggesting that these interactions in a similar manner to effectively communicate in the CIA pathway. Overall, our study uncovers striking similarities between the CIA pathway and other systems which exploit a deviant Walker A NTPase to coordinate complex, multiprotein processes. Identification of the scaffold’s partner proteins significantly advances our understanding as to why the Nbp35/MRP-type Fe-S cluster biogenesis proteins are nucleotide hydrolases. This work provides some mechanistic insight into the functions of these proteins and provides a roadmap for how to investigate this large and widely distributed family and other P-loop NTPase metallochaperones. / 2024-09-19T00:00:00Z

DNA is constantly damaged by a variety of exogenous and endogenous sources. To maintain the integrity of the genome, different DNA repair mechanisms have evolved, which deal with different kinds of DNA damage. One of the DNA repair pathways, Nucleotide Excision Repair (NER), is highly conserved throughout the three kingdoms of life and deals mainly with lesions arising in the DNA duplex after exposure to UV-light. The NER pathway in archaea is more closely related to that of eukarya, although the overall process is not yet well understood. This thesis describes the isolation and characterisation of one of the repair factors, XPD, from the crenarchaeon Sulfolobus acidocaldarius (SacXPD). SacXPD was first identified due to its homology with the eukaryal XPD protein. In eukarya XPD is the 5a' -> 3a' helicase involved in opening the DNA duplex around a damaged site. In eukarya, XPD is part of a 10-subunit complex, where it fulfils important structural roles and takes part in NER, transcription initiation from RNA polymerase II promoters and cell cycle regulation. The archaeal protein on the contrary is a monomer and a 5a' -> 3a' SF2 DNA helicase as its eukaryal counterpart. Its cellular functions, however, are unclear. Upon purification of SacXPD, it was discovered that the protein binds an ironsulphur cluster (FeS), which is essential for its helicase activity, but not for any other enzymatic functions, such as the ATP hydrolysing activity. The FeS cluster domain was not only identified in archaeal XPD, but also in eukaryal XPD and other related eukaryal helicases, such as FancJ. The presence of the FeS cluster was confirmed in the eukaryotic XPD homologue Rad3 from Saccharomyces cerevisiae. Mutagenesis studies were used to investigate a possible function of the FeS cluster, which may be used to engage ssDNA during the duplex unwinding process. This would actively distort the ss/ ds DNA junction. In addition, the resulting bending of the clamped single DNA strand could be used to avoid reannealing. The consequences of some human mutations introduced into the SacXPD gene were investigated and could contribute to our understanding of the development of human diseases.

In der vorliegenden Arbeit wurde der ATP-gekoppelte uphill Elektronentransfer von reduziertem RACo auf Kobalt(II)-CoFeSP untersucht. Dazu wurden zunächst die Bedingungen der rekombinanten Genexpression in Escherichia coli und die Reinigungsstrategie der Proteine verbessert, um einen Cofaktorgehalt beider Proteine von annähernd 100 % zu erreichen. Anschließend wurden die Reaktionsbedingungen des Elektronentransfers optimiert, um eine tiefergehende Analyse zu ermöglichen. Die Ergebnisse dieser Arbeit deuten darauf hin, dass durch die Bindung von ATP ein bidirektionaler Elektronentransfer induziert wird. Der Elektronentransfer konnte mit nicht-hydrolysierbaren ATP-Analoga und mit ADP induziert werden. Weder für die nicht-hydrolysierbaren ATP-Analoga noch für ADP konnten anschließend Hydrolyseprodukte nachgewiesen werden. Zusätzlich konnte für die limitierende Rate der ATP-Hydrolyse ein mehr als 100-fach kleinerer Wert bestimmt werden als für den Elektronentransfer. Beide Ergebnisse zeigen, dass der Elektronentransfer unabhängig von der ATP-Hydrolyse ist. Kobalt(I)-CoFeSP kann jedoch auch ein Elektron auf oxidiertes RACo übertragen, was auf einen bidirektionalen Elektronentransfer hindeutet. Diese These wurde mit der Beobachtung untermauert, dass sich durch Zugabe von ADP und der Erhöhung der ADP-Konzentration die Anzahl der transferierten Elektronen pro CoFeSP zunimmt und sich somit die Lage des entstehenden Gleichgewichts verschieben lässt. Auf dieser Datengrundlage konnten drei mögliche Modelle für den Reaktionsmechanismus erstellt werden, von welchen ein Modell als am wahrscheinlichsten erscheint. In diesem Reaktionsmechanismus gleichen sich die Redox-Potentiale beider Redox-Zentren durch die ATP-Bindung an. Dies ermöglicht den Elektronentransfer vom [2Fe2S]-Cluster von RACo auf das Kobalt-Ion des Cobalamins. Die Rückreaktion wird durch eine erneute Reduktion des [2Fe2S]-Clusters verhindert und durch die anschließende ATP-Hydrolyse dissoziiert der Komplex. / In the present work, ATP-coupled uphill electron transfer from reduced RACo to cobalt(II)-CoFeSP was investigated. For this purpose, the conditions of recombinant gene expression in Escherichia coli and the purification strategy of the proteins were improved to achieve a cofactor content of both proteins close to 100%. Subsequently, the electron transfer reaction conditions were optimized to enable a more in-depth analysis. The results of this work indicate that a bidirectional electron transfer is induced by the binding of ATP. Electron transfer could be induced with non-hydrolysable ATP analogues and with ADP. Neither for the nonhydrolyzable ATP analogues nor for ADP hydrolysis products could subsequently be detected. In addition, a value more than 100-fold smaller could be determined for the limiting rate of ATP hydrolysis than for electron transfer. Both results indicate that electron transfer is independent of ATP hydrolysis. However, cobalt(I)-CoFeSP can also transfer an electron to oxidized RACo, suggesting bidirectional electron transfer. This hypothesis was supported with the observation that adding ADP and increasing the ADP concentration increases the number of transferred electrons per CoFeSP by shifting the position of the emerging equilibrium. Based on these data, three possible models for the reaction mechanism are suggested, of which one model appears to be the most plausible. In this reaction mechanism, the redox potentials of both redox centers equalize due to ATP binding. This allows electron transfer from the [2Fe2S] cluster of RACo to the cobalt ion of cobalamin. The back reaction is prevented by a further reduction of the [2Fe2S] cluster, and subsequent ATP hydrolysis dissociates the complex