Ewing sarcoma, an aggressive pediatric bone and soft tissue cancer, is driven by a chromosomal translocation that fuses the low-complexity domain (LCD) of the RNA-binding protein EWS to the DNA-binding domain of the transcription factor FLI1, generating the potent oncogenic driver EWS::FLI1. This fusion protein reprograms transcription, disrupts RNA processing, and engages in aberrant biomolecular condensation, yet the underlying protein mechanisms that enable these activities remain poorly understood. We employed an integrated structural and biophysical strategy that included NMR spectroscopy, paramagnetic relaxation enhancement, mutagenesis, microscopy, and all-atom molecular dynamics simulations, to dissect the molecular interactions and condensate properties of EWS and EWS::FLI1. Our studies reveal that the EWS LCD undergoes extensive self-association mediated by tyrosine-rich segments that promote compact intramolecular conformations and robust intermolecular networks, driving phase separation. These condensates are further modulated by the RNA-binding domains of EWS (RGG repeats and RRM), which engage in distributed, transient contacts with EWS LCD through electrostatic and aromatic interactions and by direct binding of ETS DNA-binding domains, which enhance condensate rigidity through conserved “wing”-mediated contacts. DNA binding to ETS domains antagonizes this process, highlighting a tunable balance between nucleic acid recognition and protein-driven condensation. Together, these findings support a model in which weak, multivalent, and chemically specific interdomain contacts underlie the assembly and regulation of EWS and EWS::FLI1 condensates. By delineating how sequence-encoded features, domain interplay, and competitive binding shape condensate dynamics, this work establishes a molecular framework for understanding the biophysical basis of EWS::FLI1 oncogenic function and provides insight into the broader roles of FET proteins in transcription, RNA metabolism, and genome maintenance.
Libich et al. (Sun,) studied this question.