Metal-induced nanoscale clusterization initiates protein liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) of biomolecules is a crucial mechanism in regulating cellular functions through dynamic formation of membrane-less organelles. The assembly of nucleation seeds is a key step that triggers LLPS; however, it is challenging to precisely study its assembly mechanism due to the complexity of the condensation process. Recently, metal ions have been found to play important roles in inducing LLPS. To elucidate the assembling mechanism, a small ubiquitin-like modifier (SUMO) protein was employed as a model protein to study metal-induced condensation. The results indicate that SUMO possesses two weak Cu(II)-binding sites across the protein surface, enabling intermolecular bridging among SUMO molecules. The formation of assembling seeds is confirmed by mass photometry analysis, showing the Cu(II)-induced dynamic clusterization of SUMO at nanoscale. Increasing the Cu(II) binding affinity of SUMO significantly promotes the protein condensation, underscoring the pivotal role of Cu(II) coordination in LLPS. This work provides insights into the protein assembly mechanism through non-specific intermolecular metal coordination.