Structure-Based Drug Design to Perturb Function of a tRNA-Modifying Enzyme by Active Site and Protein-Protein Interface Inhibition
Drug research increasingly focuses on the interference with protein-protein interface formation as attractive opportunity for therapeutic intervention. The tRNA-modifying enzyme Tgt, a putative drug target to fight Shigellosis, is only functionally active as a homodimer. To better understand the driving forces responsible for assembly and stability of the formed homodimer interface we embarked onto a computational and mutational analysis of the interface-forming residues. We also launched spiking ligands into the interface region to perturb contact formation. We controlled by non-degrading mass spectrometry the actual ratio of monomer-dimer equilibrium in solution and used crystal structure analysis to elucidate the geometrical changes resulting from the induced perturbance. A patch of four aromatic amino acids, embedded into a ring of hydrophobic residues and further stabilized by a network of H-bonds is essential for the dimer contact. Apart from the aromatic hot spot, the interface shows an extended loop-helix motif, which exhibits remarkable flexibility. In the destabilized mutant variants and the complexes with the spiking ligands, the loop-helix motif adopts deviating conformations in the interface region. This motivated us to follow a strategy to raise small molecule binders against this motif to mould the loop geometry in a conformation incompatible with the interface formation.
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