通过核磁共振片段筛选鉴定出抑制细菌酶I的变构口袋

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Trang T. Nguyen , Vincenzo Venditti
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引用次数: 5

摘要

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起着重要作用,在多个水平上控制着细菌细胞的生物学。EI在不同类型细菌中的保守性和普遍性使其成为抗菌研究的潜在靶点。在这里,我们使用基于核磁共振的片段筛选来鉴定新的EI抑制剂。我们发现了三个分子片段,它们通过在距离底物结合位点超过10 Å的表面口袋中与酶相互作用来变质抑制EI催化的磷酸化酰基转移反应。有趣的是,虽然这三个分子具有相同的结合袋,但我们观察到其中两个EI配体作为竞争性抑制剂,而第三个配体作为混合抑制剂。通过核磁共振和分子动力学模拟对ei -抑制剂复合物进行表征,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,我们发现抑制剂和V292侧链之间的接触对于破坏底物与EI的不稳定结合至关重要。相反,混合抑制是由抑制剂和螺旋2之间的额外接触引起的,这种接触以变构方式扰乱了活性位点结构和周转。我们希望我们的研究结果能够为第二代变构抑制剂的开发提供基础,并提出新的分子策略来对抗耐药感染。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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

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 Å 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 ⍺-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.

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来源期刊
Journal of Structural Biology: X
Journal of Structural Biology: X Biochemistry, Genetics and Molecular Biology-Structural Biology
CiteScore
6.50
自引率
0.00%
发文量
20
审稿时长
62 days
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