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

tRNA修饰酶tRNA-鸟嘌呤转糖基酶（Tgt）是一种新的选择性抗生素靶向志贺氏菌。根据Tgt与tRNA配合物的几种晶体结构，提出Tgt同型二聚体的形成。非共价纳米esi质谱法可以在非降解条件下研究蛋白质复合物，证实了其在溶液中的二聚体寡聚化状态，并与其底物tRNA进行了2:1的结合化学计量。引入点突变来破坏蛋白质-蛋白质二聚体界面的稳定性。酶动力学揭示了这些突变变体的催化活性降低，这些突变变体可能与二聚体的不稳定有关，非共价质谱和x射线晶体学进一步证明了这一点。扩展活性位点抑制剂渗透到二聚体界面区域扰乱了蛋白质-蛋白质接触的相互作用几何形状。这些抑制剂将进一步发展成为单界面结合剂，阻止二聚体的形成，从而阻断蛋白质的功能。另外，虚拟和实验筛选将用于发现新的界面粘合剂。将采用系留方法将适当的二硫化物与Cys残基连接到表面暴露位置，以识别界面区域的弱结合物。然后，这些将被优化，以产生有效的界面粘合剂。

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.