Proline-rich domains (PRDs) are among the most prevalent signaling modules of eukaryotes but often unexplored by biophysical techniques as their heterologous recombinant expression poses significant difficulties. Using a “divide-and-conquer” approach, we present a detailed investigation of a PRD (166 residues; ∼30% prolines) belonging to a human protein ALIX, a versatile adaptor protein involved in essential cellular processes including ESCRT-mediated membrane remodeling, cell adhesion, and apoptosis. In solution, the N-terminal fragment of ALIX-PRD is dynamically disordered. It contains three tandem sequentially similar proline-rich motifs that compete for a single binding site on its signaling partner, TSG101-UEV, as evidenced by heteronuclear NMR spectroscopy. Global fitting of relaxation dispersion data, measured as a function of TSG101-UEV concentration, allowed precise quantitation of these interactions. In contrast to the soluble N-terminal portion, the C-terminal tyrosine-rich fragment of ALIX-PRD forms amyloid fibrils and viscous gels validated using dye-binding assays with amyloid-specific probes, congo red and thioflavin T (ThT), and visualized by transmission electron microscopy. Remarkably, fibrils dissolve at low temperatures (2 to 6 °C) or upon hyperphosphorylation with Src kinase. Aggregation kinetics monitored by ThT fluorescence shows that charge repulsion dictates phosphorylation-mediated fibril dissolution and that the hydrophobic effect drives fibril formation. These data illuminate the mechanistic interplay between interactions of ALIX-PRD with TSG101-UEV and polymerization of ALIX-PRD and its central role in regulating ALIX function. This study also demonstrates the broad functional repertoires of PRDs and uncovers the impact of posttranslational modifications in the modulation of reversible amyloids.

富含脯氨酸的结构域（PRD）是真核生物最普遍的信号模块之一，但由于其异源重组表达带来了巨大的困难，因此生物物理技术常常未对其进行探索。使用“分而治之”的方法，我们对属于人类蛋白 ALIX 的 PRD（166 个残基；~30% 脯氨酸）进行了详细研究，ALIX 是一种多功能接头蛋白，参与重要的细胞过程，包括 ESCRT 介导的膜重塑、细胞粘附和细胞凋亡。在解决方案中，ALIX-PRD 的 N 端片段是动态无序的。它包含三个串联的顺序相似的富含脯氨酸的基序，这些基序竞争其信号伙伴 TSG101-UEV 上的单个结合位点，如异核 NMR 光谱所证明的那样。根据 TSG101-UEV 浓度的函数测量弛豫色散数据的全局拟合，可以对这些相互作用进行精确定量。与可溶性 N 端部分相反，ALIX-PRD 的 C 端富含酪氨酸的片段形成淀粉样原纤维和粘性凝胶，并使用淀粉样蛋白特异性探针、刚果红和硫黄素 T (ThT) 进行染料结合测定来验证，并且通过透射电子显微镜观察。值得注意的是，原纤维在低温（2 至 6 °C）下或在 Src 激酶过度磷酸化后溶解。 ThT 荧光监测的聚集动力学表明，电荷排斥决定了磷酸化介导的原纤维溶解，并且疏水效应驱动原纤维形成。这些数据阐明了 ALIX-PRD 与 TSG101-UEV 相互作用和 ALIX-PRD 聚合之间的机制相互作用及其在调节 ALIX 功能中的核心作用。 这项研究还证明了 PRD 的广泛功能，并揭示了翻译后修饰对可逆淀粉样蛋白调节的影响。