NMR and bioinformatic approaches to understanding how intrinsically disordered proteins modulate biomolecular condensates

The role of biomolecular condensates in regulating biological function and the importance of dynamic interactions involving intrinsically disordered protein regions (IDRs) in their assembly are increasingly appreciated. Elucidating the critical interactions that govern condensation via phase separation is challenging due to the lack of applicability of standard structural biological tools to study these highly dynamic large-scale associated states and the lack of applicability of standard bioinformatic tools to study low complexity sequences that are not easily alignable. Using the C-terminal IDR (607 to 709) of CAPRIN1, an RNA-binding protein found in cytoplasmic biomolecular condensates, we have applied NMR methods developed by Lewis Kay to obtain site-specific information on key interactions that control phase separation and on the modulation of phase separation by post-translational modifications and ATP. We have developed, with Alan Moses, a bioinformatics approach for IDRs that does not require alignments and that identifies conserved molecular features (such as biophysical properties), termed “evolutionary signatures.” Groups of IDRs with similar evolutionary signatures are highly enriched for functional annotations and phenotypes, including clusters that are strongly linked to particular biomolecular condensates. We can utilize a feature-based approach to understand the types of interactions within specific condensates and how disease mutations that perturb conserved features may drive pathology. We also developed a new phase separation predictive tool based on sequence statistics for different physicochemical interactions within the folded protein database that provides insights into the key interactions underlying condensates. These experimental and computational methods should enable deeper understanding of how IDRs contribute to biological regulation via biomolecular condensates.