Dokument

Zusammenfassung:
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 Pathogenität der Shigella Bakterien ist. Durch einen geeigneten Liganden, der an die Kontaktfläche der beiden Monomere bindet, könnte die Ausbildung des aktiven Komplexes unterdrückt werden. In dieser Arbeit wurde deshalb zunächst das Interface mittels Molekulardynamik-Simulationen ausführlich und eingehend charakterisiert. So konnte ein zentraler Hot-Spot, bestehend aus den aromatischen Aminosäuren Trp326, Tyr330, His 333 und Phe92‘, identifiziert werden. Sie stehen am Interface durch Stacking miteinander in Kontakt und bilden zusätzlich ein Netzwerk von Wasserstoffbrückenbindungen zu Akzeptorfunktionalitäten am Dimerpartner aus. Durch umfassende Mutagenesestudien konnte gezeigt werden, dass Veränderungen innerhalb dieses Clusters zur Monomerisierung des sonst permanenten, obligaten Homodimers führen. Die Aufklärung mittels Röntgenstrukturbestimmung ließ, bei stabiler Tertiärstruktur, einen klaren Trend zum Erhalt eines kristallographischen Dimers in analoger Weise zum Wildtypprotein erkennen (RG C2). Durch eine Disulfidbrücke C330-C330‘ konnte die Variante TGT Y330C jedoch auch in einer geänderten geometrischen Anordnung kristallisiert werden (RG P6(5)22). In allen Strukturen zeigte sich ein am Interface beteiligter Loop (AS 46-52) als besonders flexibel, wobei sich nach ersten Analysen durch verschiedene Konformationen Bindetaschen mit der Möglichkeiten zur Interaktion von geeigneten Protein-Protein-Interface-Liganden öffnen. Hier könnte ein Schlüsselmechanismus für einen möglichen Wirkstoff gefunden worden sein. Erste Versuche, einen potentiellen Liganden zu platzieren, bedienten sich in silico der Molekulardynamik und des Dockings, in vitro eines MS-gekoppelten Fragment-Tetherings. Weitere, vertiefende Experimente könnten den Durchbruch für einen neuen Protein-Protein-Interface-Inhibitor am Homodimer der TGT bedeuten.

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