An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics, and smFRET Identifies an Aggregation Mechanism
An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics, and smFRET Identifies an Aggregation Mechanism
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DOI：
10.1016/j.jmb.2019.02.009
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发表时间：
2019-03-29
影响因子：
5.6
通讯作者：
Dalby, Paul A.
中科院分区：
文献类型：
--
作者：
Codina, Nuria;Hilton, David;Dalby, Paul A.
蛋白质聚集是许多疾病的根本原因，也限制了许多天然和工程蛋白质在生物技术中的用途。需要更好地理解和表征易聚集状态，以指导防止聚集的蛋白质工程、配方和药物靶向策略。虽然几种最终聚集状态（尤其是淀粉样蛋白）已在结构上得到表征，但人们对引发聚集的天然结构构象异构体知之甚少。我们使用小角 X 射线散射 (SAXS)、原子分子动力学模拟、单分子 Forster 共振能量转移和易聚集区域预测的新颖组合来表征天然人源化 Fab A33 抗体片段中与实验聚集动力学相关的结构变化。 SAXS 显示，在 pH 5.5 及以下时，天然态回转半径 R-g 增加了 2.2% 至 4.1%，并伴随着加速聚集。我们采用尖端方法，将 SAXS 数据拟合到相同条件下的完整 MD 模拟中，并将天然状态下的构象变化定位到轻链 (C-L) 的恒定域。使用两个双标记 Fab 的单分子 Forster 共振能量转移测量独立证实了这种 C-L 位移。还发现这些构象变化会增加预测的 APR 的溶剂暴露，这表明它们促进聚集的可能机制。我们的研究结果提供了一种方法，通过该方法可以通过实验轻松确定易于聚集的构象状态，从而有可能用于指导蛋白质工程或配体结合策略，以稳定蛋白质以防止聚集。 (C) 2019 作者。由爱思唯尔有限公司出版
Protein aggregation is the underlying cause of many diseases, and also limits the usefulness of many natural and engineered proteins in biotechnology. Better mechanistic understanding and characterization of aggregation prone states is needed to guide protein engineering, formulation, and drug-targeting strategies that prevent aggregation. While several final aggregated states-notably amyloids-have been characterized structurally, very little is known about the native structural conformers that initiate aggregation. We used a novel combination of small-angle x-ray scattering (SAXS), atomistic molecular dynamic simulations, single-molecule Forster resonance energy transfer, and aggregation-prone region predictions, to characterize structural changes in a native humanized Fab A33 antibody fragment, that correlated with the experimental aggregation kinetics. SAXS revealed increases in the native state radius of gyration, R-g, of 2.2% to 4.1%, at pH 5.5 and below, concomitant with accelerated aggregation. In a cutting-edge approach, we fitted the SAXS data to full MD simulations from the same conditions and located the conformational changes in the native state to the constant domain of the light chain (C-L). This C-L displacement was independently confirmed using single-molecule Forster resonance energy transfer measurements with two dual-labeled Fabs. These conformational changes were also found to increase the solvent exposure of a predicted APR, suggesting a likely mechanism through which they promote aggregation. Our findings provide a means by which aggregation-prone conformational states can be readily determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation. (C) 2019 The Author(s). Published by Elsevier Ltd.