posted on 2021-02-11, 16:04authored byHarsh Bansia, Pranjal Mahanta, Neela H. Yennawar, Suryanarayanarao Ramakumar
Cryptic pockets are visible in ligand-bound
protein structures
but are occluded in unbound structures. Utilizing these pockets in
fragment-based drug-design provides an attractive option for proteins
not tractable by classical binding sites. However, owing to their
hidden nature, they are difficult to identify. Here, we show that
small glycols find cryptic pockets on a diverse set of proteins. Initial
crystallography experiments serendipitously revealed the ability of
ethylene glycol, a small glycol, to identify a cryptic pocket on the
W6A mutant of the RBSX protein (RBSX-W6A). Explicit-solvent molecular
dynamics (MD) simulations of RBSX-W6A with the exposed state of the
cryptic pocket (ethylene glycol removed) revealed closure of the pocket
reiterating that the exposed state of cryptic pockets in general are
unstable in the absence of ligands. Also, no change in the pocket
was observed for simulations of RBSX-W6A with the occluded state of
the cryptic pocket, suggesting that water molecules are not able to
open the cryptic pocket. “Cryptic-pocket finding” potential
of small glycols was then supported and generalized through additional
crystallography experiments, explicit-cosolvent MD simulations, and
protein data set construction and analysis. The cryptic pocket on
RBSX-W6A was found again upon repeating the crystallography experiments
with another small glycol, propylene glycol. Use of ethylene glycol
as a probe molecule in cosolvent MD simulations led to the enhanced
sampling of the exposed state of experimentally observed cryptic sites
on a test set of two proteins (Niemann-Pick C2, Interleukin-2). Further,
analyses of protein structures with validated cryptic sites showed
that ethylene glycol molecules bind to sites on proteins (Bcl-xL,
G-actin, myosin II, and glutamate receptor 2), which become apparent
upon binding of biologically relevant ligands. Our study thus suggests
potential application of the small glycols in experimental and computational
fragment-based approaches to identify cryptic pockets in apparently
undruggable and/or difficult targets, making these proteins amenable
to drug-design strategies.