# Heterogeneous Folding Intermediates Govern the Conformational Pathway of the RNA Recognition Motif Domain of the Ewing Sarcoma Protein

## TL;DR

This study uses molecular dynamics simulations to explore how the RNA Recognition Motif domain of the Ewing sarcoma protein unfolds in different solvents and temperatures.

## Contribution

The study reveals solvent-dependent unfolding mechanisms and heterogeneous intermediate states of the EWS-RRM domain.

## Key findings

- The EWS-RRM domain unfolds through multiple conformational ensembles including native, native-like intermediate, intermediate, and unfolded states.
- Unfolding is more extensive at higher temperatures (400–450 K) and occurs more readily in urea compared to DMSO.
- Solvent-dependent differences in intermediate stability suggest heterogeneous unfolding pathways relevant to Ewing sarcoma pathogenesis.

## Abstract

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 (IN), 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 IN-populations, whereas in DMSO, the IN-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.