An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening
An allosteric pocket for inhibition of bacterial Enzyme I identified by NMR-based fragment screening
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DOI：
10.1016/j.yjsbx.2020.100034
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发表时间：
2020-01-01
影响因子：
2.9
通讯作者：
Venditti, Vincenzo
中科院分区：
文献类型：
--
作者：
Nguyen, Trang T.;Venditti, Vincenzo
酶I（EI）是激活细菌磷酸转移酶系统的关键酶，在多种代谢途径的调控中发挥重要作用，在多个水平上控制细菌细胞的生物学。EI在不同类型细菌中的保守性和普遍性使得该酶成为抗菌研究的潜在靶标。在这里，我们使用NMR为基础的片段筛选，以确定新的抑制剂EI。我们确定了三个分子片段，变构抑制EI催化的磷酰基转移反应，通过与酶在表面口袋位于超过10埃远离底物结合位点的相互作用。有趣的是，虽然这三个分子共享相同的结合口袋，我们观察到，两个发现的EI配体作为竞争性抑制剂，而第三个配体作为混合抑制剂。通过NMR和分子动力学模拟表征EI-抑制剂复合物揭示了干扰活性位点折叠的关键相互作用，并为所鉴定的分子片段的不同抑制活性提供了结构基础。特别是，我们表明，抑制剂和V292的侧链之间的接触是至关重要的不稳定结合的底物EI。相反，混合抑制是由抑制剂和α-螺旋2之间的额外接触引起的，其以变构方式扰乱活性位点结构和周转。我们希望我们的研究结果为开发第二代变构抑制剂提供基础，并提出新的分子策略来对抗耐药感染。
Enzyme I (EI), which is the key enzyme to activate the bacterial phosphotransferase system, plays an important role in the regulation of several metabolic pathways and controls the biology of bacterial cells at multiple levels. The conservation and ubiquity of EI among different types of bacteria makes the enzyme a potential target for antimicrobial research. Here, we use NMR-based fragment screening to identify novel inhibitors of EI. We identify three molecular fragments that allosterically inhibit the phosphoryl transfer reaction catalyzed by EI by interacting with the enzyme at a surface pocket located more than 10 angstrom away from the substrate binding site. Interestingly, although the three molecules share the same binding pocket, we observe that two of the discovered EI ligands act as competitive inhibitors while the third ligand acts as a mixed inhibitor. Characterization of the EI-inhibitor complexes by NMR and Molecular Dynamics simulations reveals key interactions that perturb the fold of the active site and provides structural foundation for the different inhibitory activity of the identified molecular fragments. In particular, we show that contacts between the inhibitor and the side-chain of V292 are crucial to destabilize binding of the substrate to EI. In contrast, mixed inhibition is caused by additional contacts between the inhibitor and alpha-helix 2 that perturb the active site structure and turnover in an allosteric manner. We expect our results to provide the basis for the development of second generation allosteric inhibitors of increased potency and to suggest novel molecular strategies to combat drug-resistant infections.