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.
Cytokinetic abscission, the last step of cell division, is regulated by the ESCRT machinery. In response to mitotic errors, ESCRT proteins, namely, ALIX, CHMP4B, and CHMP4C, accumulate in the cytosolic compartments termed “abscission checkpoint bodies” (ACBs) to delay abscission and prevent tumorigenesis. ALIX contributes to the biogenesis and stability of ACBs via an unknown mechanism. We show that ALIX phase separates into nondynamic condensates in vitro and in vivo, mediated by the amyloidogenic portion of its proline-rich domain. ALIX condensates confined CHMP4 paralogs in vitro. These condensates dissolved and reformed upon reversible tyrosine phosphorylation of ALIX, mediated by Src kinase and PTP1B, and sequestration of CHMP4C altered their Src-mediated dissolution. NMR analysis revealed how ALIX triggers the activation of CHMP4 proteins, which is required for successful abscission. These results implicate ALIX’s phase separation in the modulation of ACBs. This study also highlights how posttranslational modifications can control protein phase separation.
Solution Space (SolSpace) Scanning. We have tested a wide variety of IDRs and have found each to have its own unique set of responses to changes in solution conditions, i.e., its own SolSpace ''fingerprint.'' These results underline the idea that the structure and function of an IDR cannot be understood in terms of its sequence alone, but can be fully understood only in the context of its environment. We are now placing labeled IDRs in live cells to test how their structure and behavior can be influenced by perturbations to the cell that change the cell's inner environment.
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