Imagine an alien species sees a single photo of a human. They would have no idea how our arms and legs move, or that our mouths can open and close. So it is with protein crystal structures: even multiple static images often fail to show possible conformations. Pockets open and close in unexpected places, and these can be critical for drug discovery. But how do you find these “cryptic” pockets? Harsh Bansia, Suryanarayanarao Ramakumar, and collaborators at Indian Institute of Science, Bengaluru and Pennsylvania State University provide a new approach in J. Chem. Inf. Mod.
The researchers were studying a bacterial xylanase called RBSX and had mutated a tryptophan residue to an alanine. When they solved the crystal structure, they found that the mutation had created a surface pocket that was filled with a molecule of 1,2-ethanediol (EDO). EDO is an ingredient in antifreeze because of its ability to prevent ice formation, and this property also makes it a common cryoprotectant in crystallography. The EDO molecule was making both van der Waals contacts as well as a hydrogen bond with the protein. The researchers found similar results when they used propylene glycol. (See here for a related discussion of MiniFrags, the smallest of which are the size of propylene glycol.)
To see whether water could make these same interactions, the researchers determined another crystal structure in the absence of EDO. Surprisingly, a phenylalanine side chain rotated and closed the pocket. Had this been the only structure solved, the possibility of pocket formation would not have been suspected.
Next, the researchers conducted molecular dynamics simulations. Starting from the closed state, the pocket remained occluded by the phenylalanine, giving no hint of its potential presence. Starting from the open state and removing EDO, the pocket also rapidly closed. In other words, in the absence of a ligand, the pocket appears to collapse in upon itself.
Importantly, these observations are not limited to a mutant bacterial protein. The researchers looked at published crystal structures of four unrelated proteins with known cryptic pockets and found that EDO could bind in all of them. They also ran molecular dynamic simulations on two proteins in which EDO was included as a virtual cosolvent. For both NPC-2 and IL-2, addition of EDO was able to open up cryptic pockets that had been previously found using other molecules; we’ve discussed earlier computational work on IL-2 here.
This is a nice example of following up on an unexpected observation, and is well-suited for further study. For example, it would be interesting to do a systematic study of EDO and
propylene glycol binding sites throughout the
entire protein data bank. For those of you doing molecular dynamics or crystallography,
it may be worth adding EDO – virtually or experimentally – to see if it reveals
any surprises in your favorite proteins.