Launching Spiking Ligands into a Protein Protein Interface: A Promising Strategy To Destabilize and Break Interface Formation in a tRNA Modifying Enzyme
Launching Spiking Ligands into a Protein Protein Interface: A Promising Strategy To Destabilize and Break Interface Formation in a tRNA Modifying Enzyme
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DOI：
10.1021/cb400020b
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发表时间：
2013-06-01
影响因子：
4
通讯作者：
Klebe, Gerhard
中科院分区：
文献类型：
--
作者：
Immekus, Florian;Barandun, Luzi Jakob;Klebe, Gerhard
除了蛋白质功能的竞争性活性位点抑制外，寡核苷酸酶中的小分子对蛋白质-蛋白质相互作用的干扰为创新治疗开辟了新的前景。tRNA鸟嘌呤转糖基酶（TGT）是治疗志贺氏菌病的潜在靶点，仅以同二聚体形式具有活性。因此，通过小分子破坏二聚体界面提供了一种新的抑制模式。该酶的一个特殊特征是活性位点与二聚体边缘界面之间的距离短。这表明设计。的扩大的活性位点抑制剂装饰刚性，针型取代基刺入潜在的热点的相互作用界面。已经合成了具有连接乙炔基型取代基的配体，并通过K-d测量、晶体学、非共价质谱和计算机模拟对其进行了表征。与先前确定的晶体结构与非扩展的活性位点抑制剂，一个很好的环螺旋基序，涉及在几个接触整个二聚体界面，福尔斯除了，并建议增强灵活性，一旦钉配体绑定。质谱法表明溶液中复合的TGT同二聚体显著不稳定，但未完全破坏。由于环-螺旋基序的定向相互作用显然不能决定二聚体的稳定性，因此建议由几个芳香族氨基酸组成的结构保守的疏水补丁作为相互作用热点。该补丁的残基位于结构高度保守的螺旋-转角-螺旋基序上，其不受结合的加标配体的影响。然而，它被屏蔽从溶剂访问的环螺旋基序，成为扰动后，结合的尖峰配体，这作为一个可能的解释减少界面稳定性。
Apart from competitive active-site inhibition of protein function, perturbance of protein protein interactions by small molecules in oligodornain enzymes opens new perspectives for innovative therapeutics tRNA guanine transglycosylase (TGT), a potential target to treat shigellosis, is active only as the homodimer. Consequently, disruption of the dimer interface by small molecules provides a novel inhibition mode. A special feature of this enzyme is the short, distance between active site and rim of the dimer,interface. This suggests design. of expanded active site inhibitors decorated with rigid, needle type substituents to spike into potential hot spots of the interaction interface. Ligands with attached ethinyl-type substituents have been synthesized and characterized by K-d measurements, crystallography, noncovalent mass spectrometry, and computer simulations. In contrast to previously determined crystal structures with nonextended active site inhibitors, a well loop helix motif, involved in several contacts across the dimer interface, falls apart and suggests enhanced flexibility once the spiking ligands are bound. Mass spectrometry indicates significant destabilization but not full disruption of the cornplexed TGT homodimer in solution. As directed interactions of the loop-helix motif obviously do not determine dimer stability, a structurally conserved hydrophobic patch composed of several aromatic amino acids is suggested as interaction hot spot The residues of this patch reside on a structurally highly conserved helix-turn-helix motif, which remains unaffected by the bound spiking ligands. Nevertheless, it is shielded from solvent access by the loop helix motif that becomes perturbed upon binding of the spiking ligands, which serves as a possible explanation for reduced interface stability.