Abstract 1965 Revealing the structural basis for self-association of the RNA binding protein EWS

Ewing sarcoma RNA-binding protein 1 (EWS) is a member of the FET (FUS, EWS, TAF15) family of RNA binding proteins that function in transcription, splicing, and DNA damage repair. Mutations in FET proteins are linked to various neurodegenerative diseases, including frontal temporal dementia and amyotrophic lateral sclerosis, and the FET proteins participate in oncogenic fusions that cause numerous pediatric malignancies. EWS, like all FET family members, harbors extensive intrinsically disordered regions that lack a defined structure and facilitate multivalent and highly dynamic homo- and heterotypic interactions. Through a chromosomal translocation event, the intrinsically disordered N-terminal low complexity domain of EWS (EWSLCD) becomes fused to the FLI1 DNA-binding domain to yield the prototypical FET oncogenic fusion EWS::FLI1, the driving mutation of Ewing sarcoma. The EWSLCD forms transient self-associative interactions that promote biomolecular condensate formation and are critical for oncogenic transformation. Here, we utilize paramagnetic relaxation enhancement (PRE) NMR and molecular dynamics simulations of EWSLCD to reveal self-associative hotspots at tyrosine residues throughout the domain. These residues also drive biomolecular condensate formation, as confirmed by light microscopy and Csat measurements. Additionally, we uncover differential molecular compaction states in distinct regions of the EWSLCD, with the C-terminal region exhibiting a more compact state. These results establish the molecular drivers of EWSLCD self-association and provide insights into how these interactions impact the native functions of EWS and EWS::FLI1. Taken together, this work enhances our understanding of how intrinsically disordered proteins influence cellular processes and control their spatial environment through transient and dynamic interactions. This study was funded in part by the St. Baldrick's Foundation 634706 (DSL), the Welch Foundation AQ-2001-20190330 (DSL) and A-2113-20220331 (JM), NIGMS R01GM140127 (DSL) and R01GM136917 (JM). The Mays Cancer Center Drug Discovery and Structural Biology Shared Resource is supported in part by NCI P30CA05417X4.