BPS2026 – A targetable cryptic pocket in Ebola VP35 persists under physiological crowding mimics

Cryptic pockets are transient structural features in proteins that can be exploited for therapeutic development, but their behavior in physiologically relevant environments remains unclear. In the interferon inhibitory domain (IID) of Ebola viral protein 35 (eVP35), a cryptic pocket is allosterically linked to double-stranded RNA (dsRNA) binding, a critical step in immune evasion. The balance between open and closed states of this pocket directly tunes RNA affinity, raising the question of whether cell-like macromolecular crowding alters its dynamics. To address this, we applied cysteine thiol-labeling and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to examine the pocket under crowded conditions. Surprisingly, both approaches revealed that the pocket’s accessibility and dynamics are preserved even in highly crowded environments. These findings suggest that the VP35 cryptic pocket is not only structurally robust but also remains dynamically available in conditions that mimic the intracellular milieu, underscoring its potential as a tractable therapeutic target.