High-resolution characterization of structural changes involved in prion diseases and dialysis-related amyloidosis

Proteinaggregation ist die Ursache vieler Krankheiten, wie Diabetes Mellitus Typ 2, Parkinsonsche, Alzheimersche und Huntingtonsche Krankheit, spongiforme Encephalopathien, Stauungsinsuffizienz und Dialyse-assoziierte Amyloidose. All diesen Störungen liegt eine Proteinfehlfaltung zugrunde, die zur Bildung von Fibrillen und Ablagerung amyloider Plaques an verschiedenen Stellen im Körper führt.Aufgrund des hohen Molekulargewichts von Fibrillen und der immanenten Heterogenität von Zwischenzuständen im Entstehungsprozess stellt die Untersuchung solcher Aggregate eine besondere Herausforderung für einen Strukturbiologen dar. Die Kernresonanzspektroskopie (nuclear magnetic resonance NMR) bietet jedoch eine einzigartige Möglichkeit, solche Aggregationsprozesse in beliebigen Stadien zu untersuchen vom Monomer bis hin zur fibrillären Form.Unter Verwendung verschiedener NMR-Techniken werden im Rahmen dieser Arbeit strukturelle Veränderungen untersucht, die an Prionenerkrankungen und Dialyse-assoziierter Amyloidose beteiligt sind.Die Ursache von Prionenerkrankungen liegt in der Aggregation des nativen α-helikalen Prionproteins PrPC in seine pathologische β-faltblattreiche Isoform PrPSc. Obwohl der Umwandlungsmechanismus nach wie vor unklar ist, sind bereits mehrere Modelle für PrPSc vorgeschlagen worden. Die meisten beruhen auf der Annahme, dass sich während der Aggregation des Prionproteins Helix 1 in ein β-Faltblatt umfaltet. Im 3. Kapitel der Arbeit wird unter Verwendung verschiedener Stop-Mutanten des humanen Prionproteins gezeigt, dass keine solche Umfaltung auftritt. Darüber hinaus wird gezeigt, dass obwohl Helix 1 die Aggregation des Proteins unterstützt, sie von Proteinase K verdaut wird und somit nicht als β-Faltblatt vorliegt. Untersuchungen der Lösungsmittelinteraktion von PrP-Fibrillen zeigen eine erhöhte Flexibilität der Helix 1 und des β-Faltblattes 2 und führen zur Identifizierung einer kleinen und starren fibrillären Kernstruktur, bestehend aus 4 β-Faltblättern. Wichtig hierbei ist, dass Aminosäuren tief im Inneren dieser Kernstruktur der humPrP Fibrillen gefunden werden, die für die Artenbarriere und den M/V Polymorphismus am Codon 129 verantwortlich sind. Außerdem wird gezeigt, dass die fibrillären Formen einiger Stop-Mutanten des humanen Prionproteins gemeinsame strukturelle Merkmale aufweisen. Schließlich wird aufgrund der vorhandenen Daten ein Model für PrPsc vorgeschlagen.Die zweite Proteinfehlfaltungs-Krankheit, die hier untersucht wird, ist die Dialyse-assoziierte Amyloidose. Die Krankheit wird bei Patienten beobachtet, die unter chronischer Niereninsuffizienz leiden und einer Langzeithämodialyse unterliegen, und wird verursacht durch die Aggregation des beta-2-Microglobulins (hβ2m) der leichteren Kette des Typ I Haupt-Histokompatibilitätskomplexes. Aufgrund des niedrigen Molekulargewichtes (11 kDa) und des gut untersuchten siebensträngigen β-sandwich Faltungsmotivs betrachtet man hβ2m als ein gutes Modell, um Amyloiderkrankungen zu untersuchen. Im 4. Kapitel der Arbeit werden strukturelle Unterschiede zwischen zwei säuredenatuierten Zwischenzuständen hβ2m aufgezeigt, und experimentelle Daten werden vorgestellt die darauf hindeuten, dass die Dynamik der Vorläuferensemble die Morphologie der resultierenden Fibrillen beeinflusst. Darüber hinaus konnten neue Informationen bezüglich der Lösungsmittelinteraktion der amyloiden Aggregate gewonnen werden und flexible Bereiche innerhalb der Fibrillen des hβ2m identifiziert werden.Unabhängig von den oben genannten Projekten wird eine Untersuchung der elektrostatischen Interaktionen von Molekülorientierungen intrinsisch ungefalteter Proteine im Anhang A in Form eines Nachdruckes gezeigt. In der NMR-Spektroskopie werden schwache Molekülorientierungen verwendet, um dipolare Kopplungen beobachten zu können, welche als sensibler Gradmesser für die Struktur und Dynamik eines Biomoleküls gelten. Es wird gezeigt, dass die Orientierung ungefalteter Proteine maßgeblich von elektrostatischen Wechselwirkungen abhängt, mit der Ionenstärke in der Lösung skaliert und mittels eines vereinfachten elektrostatischen Modells vorhergesagt werden kann.Protein aggregation is the cause of several human diseases such as diabetes mellitus type 2, Parkinson s disease, Alzheimer s disease, Huntington s disease, spongiform encephalopathies, congestive heart failure or dialysis-related amyloidosis. All of these disorders result from protein misfolding which leads to fibrillization and deposition of amyloid plaques in different parts of the body.Due to high molecular weight of the amyloid fibrils and intrinsic heterogeneity of the intermediate states, protein aggregation is a very challenging field of study for the structural biologist. However, nuclear magnetic resonance (NMR) provides a unique possibility to investigate aggregation at all stages, from the monomer to the fibrils.In this work, structural changes involved in prion diseases and dialysis-related amyloidosis are investigated with the help of various NMR techniques.Prion diseases are caused by the aggregation of the natively α-helical prion protein PrPC into its pathological β-sheet-rich isoform PrPSc. While the mechanism of conversion remains unclear, several models of PrPSc have been proposed. Most of them originate from the assumption that, upon aggregation of the prion protein, helix 1 is converted into β-sheet. In Chapter 3, using different stop mutants of the human prion protein, it is shown that no such conversion occurs. Moreover, evidence is provided that while helix 1 region promotes aggregation of the protein, it is not resistant to proteinase K digestion and therefore not converted into a β-strand. Investigation of solvent protection of PrP fibrils reveals increased flexibility of helix 1 and β-strand 2 regions and identifies a small, rigid fibrillar core comprising four β-strands. Importantly, residues responsible for the species barrier and the M/V polymorpshism at codon 129 are found to be deeply buried in the core of humPrP fibrils. Furthermore, it is shown that the fibrillar forms of different stop mutants of the human prion protein share common structural features. Finally, based on the available data a model for PrPSC is proposed.The second protein misfolding diseases studied here is dialysis related amyloidosis. The disease is observed in patients with chronic renal failure undergoing long-term hemodialysis and is caused by aggregation of beta-2-microglobulin (hβ2m) - the light chain of the type I major histocompatibility complex. Due to its low molecular weight (11kDa) and a well-defined seven-stranded β-sandwich native fold, hβ2m is considered a very good model for studying all amyloid disorders. In Chapter 4, structural differences between two acid-denatured intermediate states of hβ2m are shown, and experimental data is presented that strongly suggests an effect of the dynamics of the precursor ensembles on the morphology of the resulting fibrils. Furthermore, new information on solvent protection of the amyloid aggregates is provided and flexible regions within the fibrils of hβ2m are identified.Independent of the above-mentioned projects, a study on the effects of electrostatic interactions on molecular alignment of intrinsically unstructured proteins is presented in form of a reprint in Appendix A. Weak molecular alignment is required in NMR for observation of dipolar couplings, which are a sensitive probe of the structure and dynamics of biomolecules. It is demonstrated that alignment of disordered proteins depends critically on electrostatic interactions, is scaled with the ionic strength of the solution, and can be predicted using a simplified electrostatic model

Mitglied des Betreuungsausschusses:

High-resolution characterization of structural changes involved in prion diseases and dialysis-related amyloidosis Dissertation zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades "Doctor rerum naturalium&quot

19F-NMR-based dual-site reporter assay for the discovery and distinction of catalytic and allosteric kinase inhibitors

In modern kinase drug discovery, allosteric inhibitors have become a focus of attention due to their potential selectivity, but such compounds are difficult to identify. Here we describe an NMR-based competition assay using 19F-containing reporter molecules, which allows for rapid identification and discrimination between ATP-competitive and allosteric kinase inhibitors. We illustrate the principle of such a dual-site competition assay with the example of catalytic and allosteric ABL1 kinase inhibitors. The assay can also be used to identify and characterize mixed binding modes of well-known drugs, as shown for crizotinib and fingolimod

Contributions of biomolecular NMR to allosteric drug discovery

Three case studies for how NMR can support drug discovery: Bcr-Abl, FPPS, Pak

¹⁹F‑NMR-Based Dual-Site Reporter Assay for the Discovery and Distinction of Catalytic and Allosteric Kinase Inhibitors

In modern kinase drug discovery, allosteric inhibitors have become a focus of attention due to their potential selectivity, but such compounds are difficult to identify. Here we describe an NMR-based competition assay using ¹⁹F-containing reporter molecules, which allows for rapid identification and discrimination between ATP-competitive and allosteric kinase inhibitors. We illustrate the principle of such a dual-site competition assay with the example of catalytic and allosteric ABL1 kinase inhibitors. The assay can also be used to identify and characterize mixed binding modes of well-known drugs, as shown for crizotinib and fingolimod

NMR reveals the allosteric opening and closing of Abelson tyrosine kinase by ATP-site and myristoyl pocket inhibitors

Successful treatment of chronic myelogenous leukemia is based on inhibitors binding to the ATP site of the deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein. Recently, a new type of allosteric inhibitors targeting the Abl myristoyl pocket was shown in preclinical studies to overcome ATP-site inhibitor resistance arising in some patients. Using NMR and small-angle X-ray scattering, we have analyzed the solution conformations of apo Abelson tyrosine kinase (c-Abl) and c-Abl complexes with ATP-site and allosteric inhibitors. Binding of the ATP-site inhibitor imatinib leads to an unexpected open conformation of the multidomain SH3-SH2-kinase c-Abl core, whose relevance is confirmed by cellular assays on Bcr-Abl. The combination of imatinib with the allosteric inhibitor GNF-5 restores the closed, inactivated state. Our data provide detailed insights on the poorly understood combined effect of the two inhibitor types, which is able to overcome drug resistance

Uniform isotope labeling of proteins expressed in insect cells using a custom medium

Production of proteins uniformly labeled with 2H, 13C and 15N isotopes is essential for advanced NMR studies. Here we present an affordable strategy for uniform isotope labeling in the insect cell system. Compared to commercial media the cost of the newly developed media are about 10-fold lower at comparable isotope incorporation. Additionally, there is no restriction on the labeling pattern (only 15N and 13C,15N are available commercially), but 2H, 13C and 15N in every combination can be labeled. The method was evaluated by the NMR studies on expressed proteins with four different uniform labeling patterns

Affordable uniform isotope labeling with 2H, 13C and 15N in insect cells.

A simple and affordable protocol was developed to produce uni-form labeled proteins in insect cells. Incorporation levels of 80% can be achieved for 15N and 13C and yields are comparable to expression in full media. For 2H,15N and 2H,13C,15N labeling, incorporation is slightly lower with 75% and 73%, respectively, and yields are typically two-fold reduced. The media were optimized for simplicity, reproducibility, isotope incorporation and cost. They consist of only five components, which are all commercially available at less than 8% of the costs than labeling media from vendors. High isotope incorporation and low cost are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The approach was applied to several cytosolic and secreted target proteins

Identification of Two Secondary Ligand Binding Sites in 14-3‑3 Proteins Using Fragment Screening

Proteins typically interact with multiple binding partners, and often different parts of their surfaces are employed to establish these protein–protein interactions (PPIs). Members of the class of 14-3-3 adapter proteins bind to several hundred other proteins in the cell. Multiple small molecules for the modulation of 14-3-3 PPIs have been disclosed; however, they all target the conserved phosphopeptide binding channel, so that selectivity is difficult to achieve. Here we report on the discovery of two individual secondary binding sites that have been identified by combining nuclear magnetic resonance-based fragment screening and X-ray crystallography. The two pockets that these fragments occupy are part of at least three physiologically relevant and structurally characterized 14-3-3 PPI interfaces, including those with serotonin <i>N</i>-acetyltransferase and plant transcription factor FT. In addition, the high degree of conservation of the two sites implies their relevance for 14-3-3 PPIs. This first identification of secondary sites on 14-3-3 proteins bound by small molecule ligands might facilitate the development of new chemical tool compounds for more selective PPI modulation