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Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6

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Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6 Research Collection Doctoral Thesis Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6 Author(s): Voigts-Hoffmann, Felix Publication Date: 2012 Permanent Link: https://doi.org/10.3929/ethz-a-007303759 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library ETH Zurich Dissertation No. 20189 Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6 A dissertation submitted to ETH ZÜRICH for the degree of Doctor of Sciences (Dr. sc. ETH Zurich) presented by Felix Voigts-Hoffmann MSc Molecular Biotechnology, Universität Heidelberg born April 11, 1981 citizen of Göttingen, Germany accepted on recommendation of Prof. Dr. Nenad Ban (Examiner) Prof. Dr. Raimund Dutzler (Co-examiner) Prof. Dr. Rudolf Glockshuber (Co-examiner) 2012 blank page ii Summary Ribosomes are large complexes of several ribosomal RNAs and dozens of proteins, which catalyze the synthesis of proteins according to the sequence encoded in messenger RNA. Over the last decade, prokaryotic ribosome structures have provided the basis for a mechanistic understanding of protein synthesis. While the core functional centers are conserved in all kingdoms, eukaryotic ribosomes are much larger than archaeal or bacterial ribosomes. Eukaryotic ribosomal rRNA and proteins contain extensions or insertions to the prokaryotic core, and many eukaryotic proteins do not have prokaryotic counterparts. Furthermore, translation regulation and ribosome biogenesis is much more complex in eukaryotes, and defects in components of the translation machinery are associated with human diseases and cancer. Atomic resolution structures of eukaryotic ribosomes are required to understand the regulatory aspects of translation in higher organisms at a molecular level, and to elucidate the structural differences to the prokaryotic ribosome that are the basis of antibiotic selectivity. T. We thermophila have determined the crystal structure of the large ribosomal subunit of the ciliate in complex with initiation factor 6 and cycloheximide (CHX), an inhibitor of eukaryotic protein synthesis. At a resolution of 3.5 angstrom (Å), the atomic model reveals the structure and localization of the eukaryotic-­‐specific protein moieties and rRNA expansion segments. While the peptidyl transferase center (PTC), the subunit interface, and the surface around the exit tunnel are conserved, rRNA expansion segments and eukaryotic specific protein moieties form a complex network of interactions on the solvent-­‐exposed side of the large subunit, and on the back of stalk regions. Some proteins also reach deeply into the core of large subunit, suggesting that they may have a role in the stabilization of the rRNA. We observe difference density for the glutarimide antibiotic cycloheximide (CHX) at the tRNA exit site and localize mutations of ribosomal proteins that are known to confer CHX resistance, rationalizing existing biochemical data. Furthermore, differences to the prokaryotic ribosome in the polypeptide exit tunnel may have important consequences for the binding of macrolide antibiotics such as erythromycin. Eukaryotic initiation factor 6 is required to prevent joining of the small subunit to 60S precursors during ribosome biogenesis and nuclear export. It has also been linked to cellular signaling pathways and is known to play an important role during development, in human diseases, and in cancer. The structure of the 60S:eIF6 complex resolves an iii ambiguity about the binding site and facilitates further characterization of the cellular function of eIF6. In eukaryotes, but not in archaea, RPL40 is expressed as a ubiquitin fusion. In analogy to the small ribosomal subunit ubiquitin fusion protein rpS27A, it occupies a functionally important position near the elongation factor binding site, indicating that ubiquitin cleavage is prerequisite T. thermophila for ribosome function. The structure of the large ribosomal subunit from complements the recently published structure of the 40S subunit from the same organism, establishing a structural framework for analyses of the eukaryotic-­‐specific aspects of translation. Furthermore, crystals of individual ribosomal subunits provide the opportunity to study complexes with regulatory factors and proteins required for ribosome biogenesis. Subtle structural differences to the bacterial ribosome in the areas surrounding conserved functional centers may be exploited for the design of antibiotics, and inhibitors of eukaryotic translation may hold therapeutic potential for the treatment of fungal infections and cancer. iv Zusammenfassung Ribosomen sind die Proteinfabriken der Zelle und katalysieren die Polymerisation von Aminosäuren in der durch die Boten RNA (Messenger RNA, mRNA) festgelegten Reihenfolge. Sie bestehen aus mehreren ribosomalen RNAs (rRNAs) und Dutzenden von Proteinen. Basierend auf den Kristallstrukturen prokaryotischer Ribosomen konnte im letzten Jahrzehnt ein detailliertes Verständnis der Proteinbiosynthese in diesen Organismen erarbeitet werden. Während die grundlegenden Funktionen in allen Organismen konserviert sind, gibt es signifikante Abweichungen in Bezug auf die Biogenese und Regulierung. Eukaryotische Ribosomen sind deutlich grösser als die Ribosomen von Bakterien oder Archaen und enthalten zusätzliche Proteine sowie Expansionssegmente, welche den konservierten Kern der ribosomalen RNA erweitern. Genetische Abweichungen in der Proteinbiosynthese Maschinerie führen zu schweren Erkrankungen und Entwicklungsdefekten, welche mit einem erhöhten Krebsrisiko einhergehen. Um die Regulationsmechanismen der eukaryotischen Translation zu verstehen, und die Unterschiede zum bakteriellen System zu eruieren, welche die selektive Wirkung von Antibiotika ermöglichen, sind hochaufgelöste Strukturen des eukaryotischen Ribosoms erforderlich. Wir haben die T. Kristallstruktur thermophila eines Komplexes der großen ribosomalen Untereinheit des Wimperntierchens mit dem eukaryotischen Initiationsfaktor 6 bei einer Auflösung von 3.5 Ångstrom (Å) bestimmt. Das atomare Modell zeigt die Struktur und Lokalisierung der rRNA Expansionssegmente und der zusätzlichen Proteinbestandteile. Der Komplex enthält weiterhin das Antibiotikum Cycloheximid (CHX), welches die Proteinbiosynthese bei Eukaryoten blockiert. Das aktive Zentrum und die Grenzfläche zur kleinen ribosomalen Untereinheit sind im Vergleich zum prokaryotischen Ribosom stark konserviert. Die für eukaryotische Ribosomen spezifischen rRNA Expansionssegmente und Proteinbestandteile bilden ein komplexes Netzwerk von Interaktionen auf der gegenüberliegenden, der kleinen Untereinheit abgewandten Seite. Davon ausgenommen ist die Region um den ribosomalen Tunnel, durch den die synthetisierten Polypeptide die große Untereinheit verlassen. Einige eukaryotische Proteinbestandteile reichen weit in den Kern der großen ribosomalen Untereinheit hinein und könnten die Struktur der ribosomalen RNA stabilisieren. v Anhand von Differenz-­‐Elektronendichte Karten können wir die Bindungsstelle von Cycloheximid in der ribosomalen E-­‐Stelle lokalisieren und biochemische Ergebnisse rationalisieren, nach denen Mutationen in ribosomalen Proteinen die Bindung von Cycloheximid beeinträchtigen. Strukturelle Unterschiede zum prokaryotischen Ribosom im Bereich des Tunnels können signifikante Auswirkungen auf die Bindung von Makrolid-­‐Antibiotika haben, zu denen auch Erythromycin gehört. Der eukaryotische Translations-­‐Initiationsfaktor eIF6 verhindert während der Biogenese von Ribosomen die Assoziation von kleiner und großer Untereinheit. Des Weiteren spielt eIF6 eine Rolle bei menschlichen Erkrankungen und Krebs. Die Kristallstruktur des Komplexes klärt eine Unsicherheit in Bezug auf die Bindungsstelle von eIF6 an der großen Untereinheit und ermöglicht weitere Experimente zur Untersuchung einer möglichen Verbindung zwischen eIF6 und zellulären Signaltransduktionswegen. Im Gegensatz zu Archaen ist das ribosomale Protein RPL40 bei Eukaryoten mit einer Ubiquitin Domäne fusioniert. Wie ein Ubiquitin Fusionsprotein der kleinen ribosomalen Untereinheit, rpS27A, nimmt auch RPL40 eine funktionell wichtige Position in der Nähe der ribosomalen Bindungsstelle für Elongationsfaktoren ein. Daher ist die proteolytische Entfernung von Ubiquitin Domänen für die Funktion des eukaryotischen Ribosoms unerlässlich. In Verbindung mit der kürzlich veröffentlichen Kristallstruktur der T. kleinen ribosomalen Untereinheit thermophila bietet die Struktur der großen Ribosomalen Untereinheit von eine Grundlage für das Verständnis eukaryotischer Aspekte der Proteinbiosynthese auf molekularer Ebene. Insbesondere bieten Kristalle der einzelnen Untereinheiten die Möglichkeit, Komplexe mit Regulations-­‐ und Biogenesefaktoren zu untersuchen. Die strukturellen Unterschiede zum prokaryotischen Ribosom können für die Entwicklung selektiver Antibiotika genutzt werden. Darüber hinaus könnten auch Inhibitoren der eukaryotischen Proteinbiosynthese therapeutisch einsetzbar sein, beispielsweise in der Behandlung von Krebserkrankungen oder Pilzinfektionen. vi Acknowledgements Nenad Ban Sebastian Klinge and Marc André Leibundgut Sofia Arpagaus, Tabitha Bucher, Timm Meier, Julius Rabl and Jan Erzberger The Ban group The SLS Team Family and Friends vii blank page viii Table of Contents Summary ..............................................................................................................................................................
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