Marginal Stability of the YB1 Cold‐Shock Domain in Cells Enables Binding of Multiple Nucleic Acids

YB1 is an intrinsically disordered protein with a folded cold shock domain (CSD) required for translation, transcription, and RNA metabolism. This multifunctionality and cancer involvement make it a therapeutically attractive target. YB1-CSD and nucleic acid interaction is essential for function. The CSD is marginally stable in vitro, with unknown implications for its function in the cell. In this study, the folding stability of the CSD in living cells is studied at the physiological levels of nucleic acids. The CSD is highly stabilized (increase in T_M by 10 °C) by a disordered 11 amino acid tail encoded in the C-terminal YB1-domain (CSDex). Still, CSDex remains 50% folded and 50% unfolded inside cells, whereas CSD alone is mostly unfolded. The stability strongly increased upon nucleic acid binding, an effect that is sequence-dependent and stronger for RNA than DNA. The thermodynamic and kinetic analysis revealed an ensemble of native state conformers for CSDex when probed in its native cellular environments. It is proposed that this allows the protein to retain its native fold upon binding to different ligands. The marginal stability can be a trade-off for its high demand to bind efficiently to different ligands, mediating the multifunctionality of YB1.