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Abstract: Crimean–Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a β-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with sub-nanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggests that GP38 should be evaluated as a vaccine antigen and its structure provides a foundation to investigate functions of this protein in the viral life cycle.

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Abstract: Under limiting sulfur availability, bacteria can assimilate sulfur from alkanesulfonates. Bacteria utilize ATP-binding cassette (ABC) transporters to internalise them for further processing to release sulfur. In gram-negative bacteria the TauABC and SsuABC ensure internalization, although, these two systems have common substrates, the former has been characterized as a taurine specific system. TauA and SsuA are substrate-binding proteins (SBPs) that bind and bring the alkanesulfonates to the ABC importer for transport. Here, we have determined the crystal structure of TauA and have characterized its thermodynamic binding parameters by isothermal titration calorimetry in complex with taurine and different alkanesulfonates. Our structures revealed that the coordination of the alkanesulfonates is conserved, with the exception of Asp205 that is absent from SsuA, but the thermodynamic parameters revealed a very high enthalpic penalty cost for binding of the other alkanesulfonates relative to taurine. Our molecular dynamic simulations indicated that the different levels of hydration of the binding site contributed to the selectivity for taurine over the other alkanesulfonates. Such selectivity mechanism is very likely to be employed by other SBPs of ABC transporters.

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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.

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Abstract: A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial activity, while reducing cardiac ion channel activity. The binding mode and overall design hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although some analogs retained both biochemical activity and whole-cell antibacterial activity, we were unable to significantly improve the activity of the series and analogs retained activity against the cardiac ion channels, therefore we stopped optimization efforts.

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Abstract: Long-wavelength macromolecular crystallography（MX）has been proposed for a long time as a tool for phasing novel macromolecular crystal structures without additional heavy atom labelling. Making use of anomalous diffraction from atoms natively present in the crystal, such as sulphur and phosphorus, has become increasingly popular over the past years. Nevertheless, the full potential of this technique, has not been fully exploited due to lack of dedicated experimental setups able to easily access wavelengths longer than 2 Å. Since the wavelengths for the absorption edges of sulphur and phosphorus are significantly longer, standard beamline setups are not suitable to provide high-quality, high-resolution data, as the experiments are limited by the increasing absorption effects and the diffraction angles for longer wavelengths. Currently only two synchrotron beamlines offer access to optimised sample environments using wavelengths longer than 2.7 Å：BL1A at Photon Factory, Japan and I23 at Diamond Light Source, UK. Here, we describe the challenges and solutions implemented at the in-vacuum long-wavelength MX beamline I23 and present first results.