Structural and biochemical analysis of selected nucleic acid-protein complexes

Authors: Christina Schmidt (Universität Hamburg)
Co-authored by industrial partner: No

Type: Thesis

State: Published (Approved)
Published: October 2019

Abstract: In the course of this work, structural and biochemical information about a transcription factor from the pathogenic Mycobacterium avium ssp. paratuberculosis and of an L-RNA aptamer, in complex with the orexigenic Peptide ghrelin, was obtained. In the first part of the thesis the peroxide sensitive, transcription factor Ferric Uptake Regulator (FurA) from Mycobacterium avium ssp. paratuberculosis (MAP), the causative agent for paratuberculosis in ruminants and especially in livestock, was studied. This disease is often fatal and responsible for huge economic losses for the livestock industry worldwide. Here, first structural information about MAP FurA is presented. For the first time, the open and the closed confirmation of MAP FurA were observed, where the open form is putatively able to bind DNA to regulate gene expression. The open form is the metalized, unoxidized MAP FurA. It was shown, that MAP FurA is able to bind manganese and iron, as well as zinc. This might indicate, that upon binding of different metal ions, a fine-tuning mechanism of regulation is possible, depending on the current cytosolic composition. The apo-protein and the oxidized form are in the closed form, indicating that these forms are not able to bind DNA. When peroxide stress is occurring, the iron gets oxidized and dissociated from the protein resulting in a conformational change. Crystallization trials were performed and first initial microcrystals were obtained. Further optimization is needed to obtain structural information about the metal binding and DNA interaction. The second part of this thesis provides structural insights into the Ghrelin•NOX-B11 complex and presents several methods that can be used for phase retrieval for nucleic Acid containing crystals. In the 1990s the development of SELEX (Systematic Evolution of Ligands by Exponential enrichment) led to the development of aptamers, a novel group of DNA or RNA molecules that bind to their cognate targets with high affinity via their 3D structure. Spiegelmers were developed by the NOXXON Pharma AG and are a Special class of aptamers that consist of unnatural L-nucleotides and are hence not susceptible to endo- and exo-nucleases. NOX-B11 is a Spiegelmer that binds the active octanoyl-ghrelin with high affinity and effectiveness has been demonstrated in vitro and in vivo. Ghrelin is an orexigenic peptide that is associated with various physiological processes, mostly in the regulation of food metabolism and hunger. It is therefore an important drug target. The active form of ghrelin, which binds its receptor (growth hormone secretagogue receptor (GHSR)1a), has a posttranslational, and for mammals unique, modification, namely a fatty acid side chain bound at Ser3. Here, first structural information About complex formation was obtained. It was shown, that the L-RNA and the complex are highly stable. In situ DLS studies revealed a conformational change of the Spiegelmer upon ghrelin addition and stability of the L-RNA and the complex was monitored over 40 days. Furthermore, SAXS measurements demonstrated the conformational change as well, as the L-RNA transitions from an elongated molecule to a more compact complex with ghrelin. Extensive crystallization trials were performed and it was possible to obtain X-ray diffraction suitable crystals. X-ray diffraction data were collected from a single crystal to a resolution of 2.65 Å and the space group was determined to be C2. Since no structural information about the Spiegelmer, nor ghrelin, was known, several Phase retrieval methods were applied. The phase problem of crystallography is still the main bottleneck of structure determination, as only the amplitudes of a reflection can be measured and the phases are not observed. Native SAD is a comparatively new Approach for phase retrieval, using the intrinsic atoms (sulfur for proteins, phosphorous for nucleic acids) as anomalous scattering atoms.

Journal Keywords: Biochemistry; Structural Biology; Molecular Biology; X-ray diffraction

Subject Areas: Biology and Bio-materials

Instruments: I23-Long wavelength MX