School of Chemistry, University of Nottingham, Nottingham, UK.
Department of Molecular and Cell Biology, University of Leicester, Leicester, UK.
Proteomics. 2021 Nov;21(21-22):e2000288. doi: 10.1002/pmic.202000288. Epub 2021 Jun 6.
Protein-ligand interactions are central to protein activity and cell functionality. Improved knowledge of these relationships greatly benefits our understanding of key biological processes and aids in rational drug design towards the treatment of clinically relevant diseases. Carbene footprinting is a recently developed mass spectrometry-based chemical labelling technique that provides valuable information relating to protein-ligand interactions, such as the mapping of binding sites and associated conformational change. Here, we show the application of carbene footprinting to the interaction between eIF4A helicase and a natural product inhibitor, hippuristanol, found in the coral Isis hippuris. Upon addition of hippuristanol we identified reduced carbene labelling (masking) in regions of eIF4A previously implicated in ligand binding. Additionally, we detected hippuristanol-associated increased carbene labelling (unmasking) around the flexible hinge region of eIF4A, indicating ligand-induced conformational change. This work represents further development of the carbene footprinting technique and demonstrates its potential in characterising medicinally relevant protein-ligand interactions.
蛋白质-配体相互作用是蛋白质活性和细胞功能的核心。对这些关系的深入了解极大地促进了我们对关键生物过程的理解，并有助于合理设计药物来治疗临床上相关的疾病。卡宾足迹法是一种最近开发的基于质谱的化学标记技术，可提供有关蛋白质-配体相互作用的有价值信息，例如结合位点的映射和相关构象变化。在这里，我们展示了卡宾足迹法在 eIF4A 解旋酶与天然产物抑制剂 hippuristanol 之间相互作用的应用，该抑制剂存在于珊瑚 Isis hippuris 中。当加入 hippuristanol 时，我们发现先前涉及配体结合的 eIF4A 区域的卡宾标记（掩蔽）减少。此外，我们在 eIF4A 的柔性铰链区域检测到 hippuristanol 相关的卡宾标记增加（去掩蔽），表明配体诱导的构象变化。这项工作代表了卡宾足迹法技术的进一步发展，并展示了其在表征具有医学相关性的蛋白质-配体相互作用方面的潜力。
