Conformational landscapes resolved by ion mobility mass spectrometry reveal mechanisms of polyubiquitin-controlled phase separation.

Polyubiquitin chains regulate phase separation of the ubiquitin-binding shuttle protein ubiquilin-2 (UBQLN2) in a manner that depends on the position of the isopeptide linkage and the length of the polyubiquitin chain. However, conformational heterogeneity of the non-covalent complexes formed between these proteins has rendered the molecular mechanisms underlying this regulation elusive. Here, we have used ion mobility mass spectrometry (IM-MS) to disentangle conformational features of the non-covalent complexes formed by UBQLN2 with different polyubiquitin chains. We demonstrate that the length of the polyubiquitin chain binding to UBQLN2 has a large effect on the conformational distribution of the complex, with increasing chain lengths (up to four ubiquitin subunits) allowing access to more extended conformations of the complex, and shorter chains promoting compaction. This length-dependent modulation of conformational landscapes provides the evidence for distinct mechanisms of phase separation regulation encoded by ubiquitin chain length. We also compare polyubiquitin chains linked by lysine residue (K)48 and by K63, both alone and in complex with UBQLN2. By understanding how K48- and K63-linked tetraubiquitin chains behave in the gas phase, which differs to solution, we propose molecular mechanisms for their regulation of UBQLN2 phase separation. This work, enabled by the unique ability of IM-MS to resolve conformational features of highly heterogeneous biomolecular systems, represents a conceptual breakthrough in understanding polyubiquitin-controlled phase separation mechanisms.