Search

Second-generation type III-B rotaxane dendrons, equipped with succinimide and acetylene functional groups, were synthesized successfully and characterized by NMR spectroscopy and mass spectrometry. A cell viability study of a dendron with a normal cell line of L929 fibroblast cells revealed no obvious cytotoxicity at a range of 5 to 100 μM. The nontoxic properties of the sophisticated rotaxane dendron building blocks provided a choice of bio-compatible macromolecular machines that could be potentially developed into polymeric materials.

Cryo-electron microscopy (cryo-EM) together with single particle analysis (SPA) has emerged as a powerful tool to elucidate the 3D structure of macromolecular machines (MMMs) at atomic resolution. In this thesis, three projects are presented where cryo-EM was successfully employed as part of an integrative structural biology approach. The results of these projects provided insights into the molecular interactions and dynamics of the MMMs under investigation. All structural findings are discussed together with the corroborating results from collaboration partners. (i) The λN-mediated transcription antitermination complex (λN-TAC) represents a well- studied example of a viral protein that hijacks the bacterial transcription apparatus. Here, λN induces transcriptional anti-termination in the context of specific stop signals within the RNA, leading to the transcription of viral genes downstream of the stop signal. The presented results shed light onto the mechanism by which the intrinsically disordered protein λN influences a large elongating complex consisting of RNA polymerase, several regulatory Nus protein factors, DNA, and RNA. A multi-pronged strategy is revealed by which λN traverses through the λN-TAC, establishing several favorable interactions to promote anti-termination. (ii) Neuronal development is a tightly controlled process during which translational regulation at high spatiotemporal precision is required. Ex vivo derived ribosomal fractions from the mouse neocortex were purified at different time points during development. Within them, Ebp1 was found as a highly enriched ribosomal regulatory factor. Structural elucidation located Ebp1 at the nascent chain peptide tunnel exit, where it is involved in the translation of several neuron-specific proteins. Thereby, Ebp1 was discovered to be an important regulator of gene expression during neuronal development. (iii) Mammalian cells counteract viral infection by the activity of numerous antiviral factors. One such antiviral factor, SAMHD1, decreases the NTP pool available for viral replication. Lentiviruses like HIV or SIV have evolved the accessory proteins Vpx/Vpr to counteract such antiviral factors. Vpx/Vpr both recruit SAMHD1 as a neo-substrate to the E3 ubiquitin ligase CRL4DCAF1, leading to its ubiquitination and subsequent degradation. In this study, the Vpr- SAMHD1 binding interface was defined, allowing the formulation of a diverging mechanism for the Vpx/Vpr family. Furthermore, insights into the dynamics of the CRL4DCAF1 are provided. Moreover, preliminary work regarding the structural and biochemical characterization of Cdt2, a master regulator of genome stability is presented. These studies combined have shown the power of an integrative structural biology approach that includes cryo-EM/SPA analyses to gain structural insights on a molecular level., Die Kryo-Elektronenmikroskopie (Kryo-EM) in Verbindung mit der Einzelpartikelanalyse (EPA) ist eine etablierte mächtige Methode zur Strukturaufklärung von Makromolekularen Maschinen (MMM). In der vorliegenden Doktorarbeit werden drei verschiedene Projekte beschrieben, bei denen Kryo-EM als Teil eines integrativen Strukturbiologie-Ansatzes erfolgreich angewendet wurde. Die Ergebnisse dieser Projekte geben Aufschluss über molekulare Interaktionen und die Dynamik der verschiedenen MMMs. Die strukturellen Ergebnisse werden zusammen mit unterstützenden Resultaten von Kooperationspartner diskutiert. (i) Der λN-gesteuerte transkriptionelle Anti-Terminationskomplex (TAK) ist ein gut erforschtes Beispiel für die virale Übernahme des bakteriellen Transkriptionsapparates. Dabei induziert λN transkriptionelle Anti-Termination im Kontext von bestimmten Stop-Signalen innerhalb der RNA. Das führt zur Transkription von viralen Genen strangabwärts des Stop- Signals. Die hier präsentierten Ergebnisse deuten auf den Mechanismus hin, mit dem das intrinsisch ungeordnete Protein λN den großen Elongationskomplex bestehend aus RNA- Polymerase, verschiedenen Nus-Proteinen, DNA und RNA beeinflusst. In einer mehrgleisigen Strategie schlängelt sich λN durch den λN-TAK und geht dabei mehrere vorteilhafte Interaktionen ein, um die Anti-Termination einzuleiten. (ii) Die neuronale Entwicklung ist ein engmaschig kontrollierter Prozess, der eine hohe raumzeitliche Präzision der Translationskontrolle benötigt. Zu verschiedenen Zeitpunkten während der Entwicklung des Neocortex wurden ribosomale Fraktionen ex vivo aufgereinigt. Deren Analyse zeigte einer großen Menge von Ebp1, einem regulatorischen Faktor des Ribosoms. Dank der Strukturaufklärung wurde Ebp1 an dem ribosomalen Ausgangstunnel lokalisiert, wo es in die Proteinbiosynthese von verschiedenen neuron-spezifischen Proteinen involviert ist. Durch diese Ergebnisse wurde Ebp1 als ein wichtiger Regulator der Genexpression während der Neuronalentwicklung identifiziert. (iii) Säugetierzellen erwehren sich gegen virale Angriffe durch eine Vielzahl antiviraler Faktoren. Einer dieser antiviralen Faktoren, SAMHD1, dezimiert den für die Virusreplikation nötigen NTP-Pool. Lentiviren wie HIV oder SIV haben die akzessorischen Proteine Vpr/Vpx entwickelt, um diesen antiviralen Faktoren entgegenzuwirken. Vpx/Vpr rekrutieren unter anderem SAMHD1 als neo-Substrat zur E3 Ubiquitin Ligase CRL4DCAF1, was zur Ubiquitinierung und dem anschließenden Abbau von SAMHD1 führt. In dieser Studie, wurde die SAMHD1- Vpr Interaktion definiert, was Einblicke in den divergenten Mechanismus der Vpx/Vpr-Familie erlaubt. Außerdem wurden neue Erkenntnisse in die Dynamik des CRL4DCAF1-Komplexes gewonnen. Des Weiteren werden vorläufige Ergebnisse einer strukturellen und biochemischen Charakterisierung von Cdt2, einem Masterregulator für Genomstabilität, gezeigt. Zusammengenommen stellen diese Studien die Leistungsfähigkeit eines integrativen Strukturbiologie-Ansatzes dar, bei dem Kryo-EM / EPA Einblicke auf molekularer Ebene gewährt.

Type IV pili are responsible for a diverse range of functions, including twitching motility and cell adhesion. Assembly of the pilus fiber is driven by a cytoplasmic ATPase: it interacts with an inner membrane complex of biogenesis proteins which, in turn, bind to nascent pilin subunits and mediate fiber assembly. Here we report the structural characterization of the PilF TFP assembly ATPase from Thermus thermophilus. The crystal structure of a recombinant C-terminal fragment of PilF revealed bound, unhydrolysed ATP, although the full length complex was enzymatically active. 3D reconstructions were carried out by single particle cryoelectron microscopy for full length apoprotein PilF and in complex with AMPPNP. The structure forms an hourglass-like shape, with the ATPase domains in one half and the N1 domains in the second half which, we propose, interact with the other pilus biogenesis components. Molecular models for both forms were generated: binding of AMPPNP causes an upward shift of the N1 domains towards the ATPase domains of ~8 Å. We advocate a model in which ATP hydrolysis is linked to displacement of the N1 domains which is associated with lifting pilin subunits out of the inner membrane, and provide the activation energy needed to form the pilus fiber.

International audience; We report here cryoelectron microscopy reconstructions of type IV pili (T4P) from two important human pathogens, Pseudomonas aeruginosa and Neisseria gonorrhoeae, at ∼ 8 and 5 Å resolution, respectively. The two structures reveal distinct arrangements of the pilin globular domains on the pilus surfaces, which impart different helical parameters, but similar packing of the conserved N-terminal α helices, α1, in the filament core. In contrast to the continuous α helix seen in the X-ray crystal structures of the P. aeruginosa and N. gonorrhoeae pilin subunits, α1 in the pilus filaments has a melted segment located between conserved helix-breaking residues Gly14 and Pro22, as seen for the Neisseria meningitidis T4P. Using mutagenesis we show that Pro22 is critical for pilus assembly, as are Thr2 and Glu5, which are positioned to interact in the hydrophobic filament core. These structures provide a framework for understanding T4P assembly, function, and biophysical properties.