Abstract
<jats:p>Intrinsically disordered proteins drive liquid–liquid phase separation (LLPS) through dynamic interactions with RNA, known as fuzzy interactions, yet the molecular mechanisms underlying fuzzy complex formation remain poorly understood. Here, we investigated the interactions between a fragment of the wild-type RGG1 region (Y239–G249) of Fused in Sarcoma (FUS) and a five-nucleotide polyuracil (U5), as well as the corresponding complexes containing the amyotrophic lateral sclerosis (ALS)-associated mutation R244C and functional mutations R244A and R244K, using conventional molecular dynamics and Parallel Cascade Selection Molecular dynamics (PaCS-MD) simulations. Analysis of the free-energy landscape revealed that fuzzy interactions are governed primarily by the dynamic rearrangement of residue–base contact patterns rather than by the intrinsic conformational flexibility of the RGG1 fragment or U5. Among these residue–base contacts, nonspecific interactions (represented by van der Waals interactions) involving residue 244 contributed more to maintaining fuzzy interactions than specific interactions (represented by hydrogen bonds). These findings provide a molecular framework for understanding how dynamic intermolecular contacts generate fuzzy protein–RNA interactions and thereby facilitate higher-order assembly into multivalent protein–RNA complexes and biomolecular condensates.</jats:p>