Intrinsically disordered proteins (IDPs) often undergo liquid-liquid phase separation (LLPS) to organize cellular processes. LLPS drives the formation of biomolecular condensates with distinct functional roles. How LLPS influences the specific interactions of IDPs remains poorly understood. Here, we investigate tau, a microtubule-associated IDP implicated in neurodegenerative diseases, as a model system to explore LLPS-dependent client binding.
Using a native mass spectrometry (nMS)-compatible droplet assay, we induced tau LLPS by reducing ammonium acetate concentration and monitored structural changes across conditions. nMS and ion mobility revealed that LLPS conditions promote tau compaction, while mass photometry detected transient oligomerization at intermediate salt concentrations. Our assay induces droplet formation via intermolecular electrostatic interactions, while the same interactions intramolecularly induce tau compaction.
We next examined how LLPS alters interactions of tau with known binding partners. The polyphenol EGCG bound tau exclusively under LLPS conditions, demonstrating LLPS-specific small-molecule binding. Similarly, the molecular chaperone domain BRICHOS interacted with tau only in droplets. AlphaFold predictions and molecular dynamics simulations identified a BRICHOS-binding site in the proline-rich region of tau that becomes more accessible under LLPS conditions. Remarkably, this region overlaps with a known tubulin-binding site, suggesting potential competition.
Mass photometry and fluorescence microscopy confirmed that LLPS enhances tau–tubulin complex formation, facilitating microtubule assembly. However, BRICHOS competes with tubulin for tau binding, reducing complex formation and inhibiting microtubule growth from tau–tubulin droplets. These findings reveal how LLPS enables tau to interact with binding partners and mediates tau function as a microtubule-associated protein.
Our work demonstrates how combining nMS, mass photometry, and structural modeling provides a powerful strategy to dissect LLPS-specific interactions in IDPs. This approach uncovers the mechanistic principles of phase separation and its role in modulating protein function.