The probability of cryptic pocket opening controls functional tradeoffs in filovirus immune evasion.

Cryptic pockets are of growing interest as potential drug targets. However, it remains unclear whether they 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 has a functional role. We use simulations and experiments to study the relationship between cryptic pocket opening and double-stranded RNA (dsRNA) binding of the IIDs of two other filoviruses, Reston and Marburg. These homologs have structures nearly identical to Zaire but block different interferon pathways because Marburg IID only binds the backbone, while Zaire and Reston IIDs bind both backbone and blunt ends of dsRNA. We hypothesized that these differences arise from alterations in their pocket dynamics. Simulations and thiol-labeling experiments demonstrate that Reston has a lower probability of opening the cryptic pocket while Marburg has a higher probability than Zaire. Subsequent dsRNA-binding assays with different length substrates suggest that closed conformations preferentially bind dsRNA blunt ends while open conformations prefer binding the backbone. Further, a point mutation that increases the probability of cryptic pocket opening in Reston shows that the open states prefer backbone binding, while a substitution that lowers the probability of pocket opening in Zaire improves binding to blunt ends. These results demonstrate the cryptic pocket controls a functional tradeoff, suggesting cryptic pockets are under selective pressure and may be difficult to evolve away to achieve drug resistance.