Hydrogen-Deuterium Exchange Identifies the Structural Basis of Phase Separation of the Chromosome Passenger Complex.

The centromere is the locus that directs accurate chromosome segregation, and a master regulatory complex, the chromosome passenger complex (CPC) is enriched in a region of the mitotic centromere termed the inner centromere. In mitosis, the CPC monitors the connections of the centromere to the mitotic spindle, acting to destabilize incorrect attachments but permitting correct attachments to persist. The CPC contains the Aurora-B kinase and three regulatory subunits: INCENP, Survivin and Borealin. Recent findings support a role for CPC phase separation in its localization and function in mitotic error correction (Trivedi et al., 2019, Nat. Cell Biol. 21:1127-1137). Liquid-liquid phase separation is emerging as a central regulatory principle of cell biology. This form of phase separation is often driven by multivalency of adhesive domains and/or linear motifs. The sequences of these domains are often found in unstructured regions where they are enriched in uncharged polar side chains, charged amino acids, or aromatic residues; additionally, structural plasticity allows them to generate multiple, weakly-adhesive, inter- and intramolecular interactions. Currently there are no techniques to globally map the motifs on proteins that drive the multivalent interactions. In this context, we now employ hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) to study dynamics of CPC phase separation. We have identified four regions within the regulatory CPC subunits that upon phase separation exhibit differential HX (three slower and one faster). Importantly, three of these regions map to structured regions of proteins while one maps to the expected intrinsically disordered regions. Each of the four regions is enriched with charged amino acids. Mutation of key residues in one of the differential HX regions within the INCENP subunit diminish droplet size and quantity, suggesting that we have successfully mapped a key surface for the phase separation properties of the CPC. In broader context we have shown for the first time how HXMS coupled with structural data and bioinformatic analysis can be applied to study molecular basics of protein liquid-liquid phase separation.