Search

INTRODUCTION The eukaryotic nucleus pro­tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the larg­est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf­fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma­tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal­lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra­tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc­tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf­fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight­forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol­ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)–based prediction to generate an exten­sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac­terized. Benchmarking against previous and unpublished x-ray and cryo–electron micros­copy structures revealed unprecedented accu­racy. We obtained well-resolved cryo–electron tomographic maps of both the constricted and dilated conformational states of the hu­man NPC. Using integrative modeling, we fit­ted the structural models of individual NUPs into the cryo–electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf­fold. We elucidated in great detail how mem­brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en­vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re­solved model covers >90% of the human NPC scaffold. It captures conforma­tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy­namic model of the NPC. Our study exempli­fies how AI-based structure prediction may accelerate the elucidation of subcellular ar­chitecture at atomic resolution. A 70-MDa model of the human nuclear pore complex scaffold architecture. The structural model of the human NPC scaffold is shown for the constricted state as a cut-away view. High-resolution models are color coded according to nucleoporin subcomplex membership. The nuclear envelope is shown as a gray surface.

Die vorliegende Dissertation ist in zwei Thematiken unterteilt: 1.Molekulare Mechanismen der Segregation von Chromosomen: Die Segregation der Chromosomen ist ein zentraler Prozess während der Zellteilung. Dieser wird durch hierarchische Proteinkomplexe reguliert, die Kinetochore. Sie kontrollieren die synchrone Teilung der Schwesterchromatiden in höheren Eukaryoten in einem Prozess, der als spindle assembly checkpoint (SAC) bezeichnet wird. Der 800 kDa große ROD-Zw10-Zwilch (RZZ) Proteinkomplex besitzt eine Schlüsselfunktion in der Formierung des SAC sowie der Anheftung der Mikrotubuli an die Kinetochore. Diese Arbeit beschreibt die erste mittels Kryo-Elektronenmikroskopie bestimmte hochaufgelöste Struktur des RZZ Komplexes. Die Organisation der Bestandteile des Komplexes wurde durch Einbeziehung diverser biochemischer Methoden charakterisiert. Diese Arbeit stellt den ersten Schritt für ein strukturelles Verständnis des SAC dar und ermöglicht hierdurch ein besseres Verständnis der komplexen Signalwege, welche die Zellteilung steuern. 2. Molekulare Grundlagen der Batten-Krankheit: Das Ceroid-lipofuscinosis neuronal-3 (cln3) Gen kodiert für ein Transmembranprotein (Cln3p). Mutationen dieses Proteins bilden die Grundlage für die Batten-Krankheit, die am weitesten verbreitete aller neuronalen Ceroid-Lipofuscinosen (NCL). Diese vererbbaren neuronalen Krankheiten sind durch den Verlust der Sehkraft, myoklonische Anfälle, Verlust der geistigen Fähigkeiten sowie Störungen der motorischen Fertigkeiten charakterisiert. In dieser Arbeit wird die erste erfolgreiche Expression und Reinigung des humanen Cln3p mittels eines Baculovirus-Insektenzellen-Expressionssystems beschrieben. Initiale biochemische Charakterisierungen der posttranslationalen Modifikationen des Proteins wurden durchgeführt. Weiterhin wurden erste Versuche zum Aufklärung der Proteinfunktion und der Struktur des Proteins begonnen., The work presented in this thesis can be divided into two topics: 1. Molecular mechanism of chromosome segregation: Chromosome segregation is a central process in cell division. Hierarchical protein assemblies called kinetochores navigate this process. They monitor the synchronous separation of sister chromatids in higher eukaryotes by a pathway commonly referred to as spindle assembly checkpoint (SAC). The 800 kDa ROD-Zw10-Zwilch (RZZ) complex is a key player both in SAC and formation of kinetochore – microtubule attachments. This work highlights the first high-resolution structure of the RZZ complex determined by cryo-electron microscopy. The organization of constituent proteins in the complex has been understood by interweaving various biochemical methods. This study is essentially the first step in understanding the SAC from a structural perspective and enables to use hints from the structure in understanding the complex signaling process that drives cell division. 2. Molecular basis of the Batten disease: Ceroid – lipofuscinosis neuronal-3 (cln3) gene encodes a trans-membrane protein (Cln3p) mutations in which cause Batten’s disease. Batten disease is the most common of all neuronal ceroid-lipofuscinoses (NCL, genetically inherited neuronal disorders) characterized by loss of vision, myoclonic seizures, loss of cognitive function, motor dysfunction. This work reports the first expression and purification of human Cln3p from the baculovirus driven insect cell expression system. Initial biochemical characterization of the protein is also reported. Efforts to understand the protein function have been initialized and variety of techniques used to understand the structure of the protein.