Perturbance of enzyme function by blocking dimer interface formation: Novel route to specific antibiotics

• Antragsteller: Professor Dr. Gerhard Klebe
• Fachliche Zuordnung: Pharmakologie
• Förderung: Förderung von 2010 bis 2014
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 29078704

The tRNA modifying enzyme, tRNA-guanine transglycosylase (Tgt), constitutes a putative target for new selective antibiotics against Shigella bacteria. Based on several crystal structures of Tgt in complex with tRNA the formation of a Tgt homodimer was suggested. Non-covalent nanoESI mass spectrometry that can study protein complexes under non-degrading conditions confirms the dimeric oligomerisation state in solution and 2:1 binding stoichiometry with its Substrate tRNA. Point mutations were introduced to destabilize the protein-protein dimer interface. Enzyme kinetics reveal a reduced catalytic activity of these mutated variants supposedly related to the destabilization of the dimer as further evidenced by both, non-covalent mass spectrometry and X-ray crystallography. Extended active site inhibitors penetrating into the dimer interface region perturb the interaction geometry of the protein-protein contact. These inhibitors will be further developed into sole interface binders preventing dimer formation and thus blocking protein function. Alternatively, virtual and experimental screening will be applied to discover novel interface binders. A tethering approach connecting appropriate disulfides with Cys residues engineered into surface-exposed positions will be performed to identify weak binders in the interface region. These will then be optimized to result in potent interface binders.

• DFG-Verfahren: Forschungsgruppen
• Teilprojekt zu: FOR 806: Interfering with intracellular protein-protein interactions - probing protein functions with small molecules