Cryptic pockets stay open in a crowd

Most structural models of proteins are limited in the same way a photograph is. They show one pose.

In reality, a protein is full of moving parts, and as they move, gaps open up on its surface that are nowhere to be seen in the static picture.

We call these cryptic pockets. They matter because a great many proteins have no obvious pockets for a drug to bind in, and have therefore been written off as undruggable. If a pocket opens on such a protein even a small fraction of the time, a drug can slip in and alter the protein’s function.

Our lab found one of these pockets a few years ago on a protein from Ebola virus, first in molecular simulations and then confirmed experimentally.

One concern is whether proteins behave the same in cells as they do in test tubes. In a cell, between 8 and 40 percent of the space is taken up by other molecules — proteins, RNA, sugars, machinery of every description. It is less a solution than a traffic jam (see figure below). And the general expectation in biophysics is that crowding pushes proteins toward their most compact shapes, because compact shapes take up less room. Opening a cryptic pocket means expanding a little. If a crowded cell simply squeezes those pockets shut, then a decade of work on cryptic pockets as drug targets applies only to the dilute conditions inside a test tube.

The figure shows a model of a cell that highlights how crowded the interior is. Green is the membrane that encapsulates the cell and light blue are proteins inside the cell (called the cytosol). The density of proteins and other molecules means the proteins are constantly bumping into things. Other cellular environments are even more crowded. Image from Stevens et al. Frontiers in Chemistry 2023.

We’ve now taken the first step towards establishing whether the crowded environments within cells suppress cryptic pocket opening. We used a synthetic crowder called Ficoll 70, a branched sugar polymer that takes up space without much chemical personality of its own, and we crowded our Ebola protein with it up to 40 percent by weight — the top of the range seen in real cells. Then we watched the pocket with two different experimental techniques.

Importantly, the pockets appear to behave the same in a crowded environment as they do in the dilute conditions we typically use for studying them. You can read more in our latest preprint if you want more details.

All this work was inspired by simulations originally conducted on Folding@home. One of our goals has always been to provide insight into proteins that inspires new experiments, so it’s a big success.