Neue Ansätze zur Entwicklung von Modulatoren der homodimeren tRNA-Guanin-Transglycosylase
Das Ziel dieser Dissertation war die biophysikalische Charakterisierung einer Dimerstruktur am Beispiel der tRNA-Guanin-Transglycosylase (TGT). Für die intensive Untersuchung des Dimers wurden neben der Röntgenkristallographie auch enzymkinetische Messungen, isotherme Titrationskalorimetrie, native...
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Table of Contents: The aim of this dissertation was the biophysical characterization of the dimer structure of tRNA-guanine transglycosylase (TGT) as a case study. In addition to X-ray crystallography, enzyme kinetic measurements, isothermal titration calorimetry, native mass spectrometry, thermal shift assay and protein-based 19F NMR spectroscopy were used for the comprehensive study of the ligand-induced influence on dimer stability. The Z. mobilis TGT catalyzes the incorporation of a modified nucleobase into the tRNA as a homodimer. This modification is essential in the pathogenesis of the inflammatory bowel dis¬ease dysentery caused by Shigella. For this purpose, TGT replaces the purine base guanine at the wobble position-34 of tRNAsAsn,Asp,His,Tyr with the modified nucleobase preQ1. The lin-benzoguanine scaffold derived from the purine base competitively binds to the active site of the protein. By inserting modifications at the C(2') or C(4') positions, the adjacent ribose-33/uracil-33 and ribose-34/phosphate-35 site pockets can be addressed. Since TGT can catalytically convert the natural tRNA substrate only in a 2:1 ratio (monomer:tRNA) as a homodimer, the stabilization of the quaternary structure in an incompetent arrangement represents an alternative way of inhibition besides the classical blocking of the active site. The contact surface of the two TGT monomers (dimer interface) falls close to the ribose-34/phosphate-35 pocket. By addressing the hydrophobic region of the above-mentioned pocket, the geometry of an adjacent loop-helix motif can be disturbed, reducing the dimer sta¬bility associated with a massive rearrangement of up to 20 amino acids. Three ligands have been crystallographically identified which, when bound in the ribose-34 pocket, induce a "twisted" arrangement of the TGT dimer (twisted dimer), which should prevent catalytic activity due to its rearrangement in a catalytically incompetent geometry. By introducing a fluorine probe inside the dimer, the "twisted" dimer was investigated under native conditions in solution (Chapter 2). The 19F NMR results indicate that the loop-helix motif and especially the amino acid Trp95 have a regulatory function for the dimer arrangement and formation. Therefore, modulation of the malleable motif with suitable ligands appears as an ideal concept to inhibit the dimer protein. Within the ribose-33/uracil-33 pocket, a water network was replaced by binding of C(2')-monosaccharide-modified lin-benzoguanine inhibitors in a charge-neutral manner (Chapter 3). It was also shown that acetonide-protected sugar motifs play a decisive role in the stabiliza¬tion of a [5+5+4] water cluster and thus significantly influencing the binding affinity of the applied ligands. In addition to the binding affinity, the motif used increases the physicochemical proper¬ties of the lin-benzoguanine ligands. Based on an earlier study, the TGT mutant Y330C was introduced. Although the crystal struc¬ture under investigation does not allow the complete structural elucidation of the TGT mono¬mer, it shows an arrangement of the molecules in space group P6522 in a largely unconstrained arrangement lacking restrictions from the packing of the neighboring dimer mate. Interest¬ingly, the β1α1 loop (Thr47 – Lys55) is observed in a previously unobserved conformation. The new arrangement of the loop is not compatible with the formation of the functional dimer interface. Therefore, the determination of the monomer structure remains an unresolved problem. For this purpose, MS-coupled tethering screening was applied at the Cys330 position for the search of optimal crystallization conditions (Chapter 4). In addition to the use of reversible disulfide binders, the concept has been extended by the introduction of irreversible binders. It turned out that acrylamides used have a high potential for the modification of cysteine residues and bind irrespectively of the affinity of the fragment to the target protein. This opens up a new perspective for possibilities of dimer investigation. The functionality of TGT is only guaranteed in the dimeric arrangement. Thus, the regulation of dimer formation seems to be a possibility to inhibit its catalytic capability. A cluster consisting of four aromatic amino acids (Phe92', Trp326, Tyr330, His333) was identified, which is essential for the stability of the dimer interface. By introducing phenylalanine mutants, the hydrogen bonds have been selectively removed, while the aromatic character of the residues is preserved (Chapter 5). The effect on the dimer interface was investigated by X-ray crystallo-graphy, native mass spectrometry, enzyme kinetics and the determination of the melting temperature of the respective mutants. The position His333 seems to be of greater importance, and seemingly its protonation strongly influences the stability of the dimer interface. This finding is also based on the experience that crystals grown at pH 5.5 show a more pronounced change in the structural loop-helix motif. Also, the mutagenesis study shows that the Trp95 residue, which is located in the center of the loop-helix motif, appears to be essential concerning dimer regulation. The reduction of this residue to phenylalanine causes a significant depression of the melting temperature by >15 °C. Furthermore, dimerization rate, structural stability of the loop-helix motif and binding behavior of differently substituted ligands, which are characterized more in detail in chapter 2, are significantly influenced.