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Cryptic pockets are of growing interest as potential drug targets, particularly to control protein-nucleic acid interactions that often occur via flat surfaces. However, it remains unclear whether cryptic pockets contribute to protein function or if they are merely happenstantial features that can easily be evolved away to achieve drug resistance. Here, we explore whether a cryptic pocket in the Interferon Inhibitory Domain (IID) of viral protein 35 (VP35) of Zaire ebolavirus aids its ability to bind double-stranded RNA (dsRNA). We use simulations and experiments to study the relationship between cryptic pocket opening and dsRNA binding of the IIDs of two other filoviruses, Reston and Marburg. These homologs have nearly identical structures but block different interferon pathways due to different affinities for blunt ends and backbone of the dsRNA. Simulations and thiol-labeling experiments demonstrate that the homologs have varying probabilities of pocket opening. Subsequent dsRNA-binding assays suggest that closed conformations preferentially bind dsRNA blunt ends while open conformations prefer binding the backbone. Point mutations that modulate pocket opening proteins further confirm this preference. These results demonstrate the open cryptic pocket has a function, suggesting cryptic pockets are under selective pressure and may be difficult to evolve away to achieve drug resistance.
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This content will become publicly available on November 17, 2026
Crowding does not suppress the opening of a cryptic pocket
Cryptic pockets are transient structural features that could provide new opportunities for drug design. However, their utility would be greatly diminished if the probability of pocket opening were universally suppressed in cells due to generic crowding effects like excluded volume. To address this concern, we study the effect of generic crowders on the opening of a cryptic pocket in the interferon inhibitory domain (IID) of Ebola viral protein 35 (eVP35) that allosterically regulates double-stranded RNA (dsRNA) binding. We find that crowding has negligible effect on dsRNA binding or the probability of cryptic pocket opening, as measured by both thiol labeling and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The probability that a specific pocket opens may still be altered by specific environments. However, our results establish that crowding effects that are common to a variety of cellular environments do not pose a universal barrier to targeting cryptic pockets. Therefore, the potential of targeting cryptic pockets continues to present exciting opportunities.
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- Award ID(s):
- 2218156
- PAR ID:
- 10710821
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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