Department of Chemistry, University at Albany, State University of New York, Albany, New York.
Protein Sci. 2020 Feb;29(2):572-588. doi: 10.1002/pro.3792. Epub 2019 Dec 18.
The molecular chaperone αA-crystallin, mainly localized in the human ocular lens, is believed to protect the lens from opacification and cataract, by suppressing the aggregation of the other lens proteins. The present study provides structural and thermodynamic insights into the ability of human αA-crystallin (HAA) to bind to its partially unfolded clients in the lens, using a small peptide, melittin from bee venom, as a model client. We characterized the thermodynamic parameters of the binding process between melittin and HAA through isothermal titration calorimetry (ITC), and found the binding to be endothermic and entropy-driven. We identified the amino acids in melittin important for binding to HAA by saturation-transfer difference (STD) nuclear magnetic resonance (NMR) experiments, and analysis of NMR line broadening upon titration of melittin with HAA. Our results suggest that hydrophobic residues Ile17 and Ile20 on the C-terminal region of melittin are in close contact with HAA in the melittin-HAA complex. Information obtained from NMR experiments was used to generate structural models of the melittin-HAA complex by molecular docking with high-ambiguity driven docking (HADDOCK). Structural models of the melittin-HAA complex reveal important principles underlying the interaction of HAA with its clients.
分子伴侣αA-晶体蛋白主要定位于人眼晶状体中，据信通过抑制其他晶状体蛋白的聚集来保护晶状体不发生混浊和白内障。本研究使用来自蜂毒的小肽蜂毒素作为模型客户，提供了关于人αA-晶体蛋白（HAA）与晶状体中部分展开的客户结合能力的结构和热力学见解。我们通过等温滴定量热法（ITC）表征了蜂毒素与 HAA 之间结合过程的热力学参数，并发现结合是吸热和熵驱动的。我们通过饱和转移差（STD）核磁共振（NMR）实验鉴定了蜂毒素中与 HAA 结合重要的氨基酸，并分析了蜂毒素与 HAA 滴定时的 NMR 线宽。我们的结果表明，蜂毒素 C 末端区域的疏水性残基 Ile17 和 Ile20 与蜂毒素-HAA 复合物中的 HAA 密切接触。从 NMR 实验获得的信息用于通过高不确定性驱动对接（HADDOCK）进行分子对接生成蜂毒素-HAA 复合物的结构模型。蜂毒素-HAA 复合物的结构模型揭示了 HAA 与其客户相互作用的重要原则。
