Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, New South Wales, Australia.
Molecular Horizons, University of Wollongong, Wollongong, New South Wales, Australia.
Protein Sci. 2019 Feb;28(2):365-374. doi: 10.1002/pro.3534. Epub 2018 Dec 20.
Apolipoprotein-D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein-D adopts an eight-stranded β-barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent α-helix. Crystallography studies of recombinant apolipoprotein-D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX-MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X-ray crystallography, making HDX-MS an invaluable orthogonal technique. Here, we report an HDX-MS protocol for apolipoprotein-D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand-free apolipoprotein-D revealed apolipoprotein-D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein-D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N-terminus and C-terminus. Together, our experiments provide insight into apolipoprotein-D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein-D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein-D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein-D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein-D structure from X-ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein-D, to elucidate how proteins change flexibility when binding to small molecules.
载脂蛋白 D 是一种糖基化的四聚体脂联素,可结合并转运小疏水分子,如孕酮和花生四烯酸。与其他脂联素一样,载脂蛋白 D 采用由两个分子内二硫键稳定的八链 β-桶折叠结构,其旁边是一个 α-螺旋。重组载脂蛋白 D 的晶体学研究表明,在与孕酮结合时没有发生主要的构象变化。酰胺氢-氘交换质谱(HDX-MS)通过监测蛋白质骨架中酰胺氢与氘的交换来报告溶液中蛋白质的结构变化。HDX-MS 检测蛋白质构象和结构动力学的变化,以响应配体结合等蛋白质功能,这些变化可能在 X 射线晶体学中无法检测到,因此 HDX-MS 是一种非常有价值的正交技术。在这里,我们报告了一种载脂蛋白 D 的 HDX-MS 方案,该方案使用严格的淬灭条件和更长的氘化时间解决了蛋白质刚性高和胃蛋白酶切割率低的挑战,得到了 85%的序列覆盖率和 50%的氘交换。无配体载脂蛋白 D 的相对分数氘交换表明载脂蛋白 D 是一种高度结构的蛋白质。孕酮结合通过八个肽中的氘交换显著减少来检测到。载脂蛋白 D 动力学的稳定可以解释为配体结合口袋中的正位效应和 N 端和 C 端的变构效应的组合。总之,我们的实验提供了对载脂蛋白 D 结构动力学的深入了解,并绘制了孕酮结合对蛋白质远端部分的传递效应。孕酮结合导致载脂蛋白 D 动力学稳定的现象表明,在脂联素家族中存在一种常见的行为,这可能对载脂蛋白 D 与受体或脂蛋白颗粒的相互作用有影响。结论:我们首次揭示了在阿尔茨海默病中具有保护作用的载脂蛋白 D 与类固醇激素分子结合时如何变得更加有序。这些结果通过纳入有关蛋白质随时间运动变化的信息,极大地扩展了基于 X 射线晶体学研究的载脂蛋白 D 结构的理解。为了实现这些结果,开发了一种改进的方案,适用于类似于载脂蛋白 D 的蛋白质,以阐明当结合小分子时蛋白质如何改变灵活性。