Nichtkompetitive Inhibition der tRNA-Guanin Transglycosylase durch Störung der essentiellen Protein-Protein-Interaktion

Protein-Protein-Kontakte stellen eine reiche Vielfalt an Interaktionsmöglichkeiten der Polypeptide mit regulatorischen Funktionen innerhalb lebender Organismen dar. Das homodimere Enzym tRNA-Guanin Transglykosylase(TGT) katalysiert z. B. eine Basenaustauschreaktion, die essentiell für die Pathogeni...

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Bibliographic Details
Main Author: Jakobi, Stephan
Contributors: Klebe, Gerhard (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2013
Online Access:PDF Full Text
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Table of Contents: Protein-Protein-Interactions are commonly known as part of a regulatory network within cells and organisms. The homodimer tRNA-guanine transglycosylase (TGT) catalyzes a base exchange reaction and is therefore crucial to the pathogenicity of Shigella bacteria. By placing a ligand directly in the contact region of the two enzyme partners, the complex is supposed to collapse. In this thesis, the contact area was first analyzed in detail by molecular dynamics simulations. As a major finding a hot-spot-network was identified, consisting of Trp326, Tyr330, His333, and Phe92’ respectively. These four amino acids are in contact with each other by stacking and also interact with acceptor functionalities on the opposite side of the other dimer partner. Furthermore, mutagenesis studies were able to underline this result by inducing monomerization of the permanent and obligate homodimer of TGT. Structure solution by X-ray crystallography showed the monomers to tend to assemble crystallographic dimers in the same manner as the wildtype forms its homodimers (space group C2). Interestingly enough, the structure of each monomer is stable. Also when a disulfide bridge via the introduced cysteine 330 (C330-C330’) is formed and induces a completely different packing in space group P6(5)22, only a small part of the interface is found to be flexible. In some conformations, loop 46 to 52 opens possible pockets in which putative ligands could be placed. First attempts to focus on protein-protein-interface-inhibitors started in silico with docking and molecular dynamics simulations and in vitro by using the tethering approach combined with mass spectrometry. Further experiments might succeed in a way to new PPI-Inhibitors of the homodimeric enzyme TGT.