The RNA Recognition Motif (RRM) domain of the Ewing sarcoma (EWS) protein plays a pivotal role in RNA binding and gene regulation, being crucial for its function. However, its structural dynamics are yet to be revealed. Herein, we performed 5.5 μs cumulative molecular dynamics (MD) simulations to investigate the unfolding pathways of the EWS-RRM domain in urea and DMSO across 300–500 K. The unfolding process was characterized by using free-energy landscape (FEL) analysis, hydrogen-bond occupancy, and Gaussian Mixture Model (GMM) clustering. At lower temperatures (300–350 K), the RRM largely retained its native conformation, while extensive unfolding occurred between 400 and 450 K. Results revealed multiple conformational ensembles: native (N), native-like intermediate (I_N), intermediate (I), and unfolded (U) states, underlying the unfolding pathway of RRM. In urea at 400 K, a long-lived I-state dominated, with transient N and I_N-populations, whereas in DMSO, the I_N-state appeared more stable, that transitioned into tightly packed I-states, reflecting a stepwise unfolding via compact intermediates. At 450 K, the protein reached the U-state in both solvents, though unfolding occurred more readily in urea. This study highlights the solvent-dependent unfolding mechanisms and heterogeneous I-states of EWS-RRM, providing insight into its stability, misfolding, and potential relevance to Ewing sarcoma pathogenesis.

Glioblastoma (GBM) is a highly aggressive brain tumor with a poor prognosis, creating an urgent need for the identification of new therapeutic targets. Lon