Crowding does not suppress the opening of a cryptic pocket

Cryptic pockets are transient structural features that provide new opportunities for therapeutic intervention, yet their dynamic properties in physiologically relevant environments remain poorly understood. We have identified one such pocket in the interferon inhibitory domain (IID) of Ebola viral protein 35 (eVP35) and demonstrated that it allosterically regulates double-stranded RNA (dsRNA) binding, a crucial step in immune evasion. However, cellular environments, which are rich in dense macromolecular structures, may impose spatial constraints on VP35, potentially affecting the accessibility and function of its cryptic pocket. In this study, we examined the pocket’s behavior under cell-like crowded conditions using cysteine thiol-labeling experiments and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Strikingly, the cryptic pocket’s dynamics and accessibility were unchanged in crowded environments. This robustness indicates the pocket remains structurally and dynamically accessible in crowded cell-like conditions, supporting its potential as a druggable target in vivo.