School of Pharmaceutical Sciences, Beijing Advanced Innovation Center for Structural Biology, and Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, P. R. China.
Mol Pharm. 2021 Jan 4;18(1):267-274. doi: 10.1021/acs.molpharmaceut.0c00905. Epub 2020 Dec 12.
Adenosine triphosphate (ATP) is amphiphilic in nature and has the characteristics of a hydrotrope because of the charged triphosphate moiety and the large aromatic ring located on each end of its structure. Previous studies revealed that ATP can effectively maintain the solubility and prevent liquid-liquid phase separation (LLPS) of some biological proteins. In this study, we assessed the impact of ATP on the stability of a model therapeutic IgG1 antibody (MA1) to evaluate its potential application in protein formulation design. In our system, ATP promotes rapid LLPS of MA1 and we demonstrate that the ATP-MA1 static interaction drives phase separation of MA1. The attractive protein-protein interaction increased exclusively in the presence of ATP but not in the presence of other ATP analogues, such as adenosine diphosphate, adenosine monophosphate, and adenine. Through an intrinsic fluorescence quenching study, we revealed that ATP bound to MA1 electrostatically and formed static interactions; furthermore, such static ATP-MA1 interactions significantly altered the surface property of the protein and the protein-protein interactions and subsequently induced LLPS of MA1. This ATP-induced LLPS could be effectively eliminated by Mg, which chelated with ATP and thus negated ATP-MA1 static interaction. Our results revealed the unique molecular mechanism of ATP-induced rapid LLPS of MA1.
三磷酸腺苷（ATP）本质上具有两亲性，由于其带有电荷的三磷酸基团和位于结构两端的大芳香环，因此具有水增溶特性。先前的研究表明，ATP 可以有效地维持一些生物蛋白的溶解度并防止液-液相分离（LLPS）。在本研究中，我们评估了 ATP 对模型治疗性 IgG1 抗体（MA1）稳定性的影响，以评估其在蛋白质配方设计中的潜在应用。在我们的系统中，ATP 促进 MA1 的快速 LLPS，我们证明 ATP-MA1 静态相互作用驱动 MA1 的相分离。仅在存在 ATP 的情况下，吸引力的蛋白质-蛋白质相互作用才会增加，而不存在其他 ATP 类似物（如二磷酸腺苷、一磷酸腺苷和腺嘌呤）。通过本征荧光猝灭研究，我们揭示了 ATP 与 MA1 静电结合并形成静态相互作用；此外，这种静态 ATP-MA1 相互作用显著改变了蛋白质的表面性质和蛋白质-蛋白质相互作用，随后诱导 MA1 的 LLPS。这种由 ATP 诱导的 LLPS 可以通过与 ATP 螯合的 Mg 有效消除，从而否定了 ATP-MA1 的静态相互作用。我们的研究结果揭示了 ATP 诱导 MA1 快速 LLPS 的独特分子机制。
