I wrote some years ago about the case of a protein that seemed to have a completely empty binding pocket – empty, as in not even any water molecules hanging around in there. There are a number of these known, and there’s a lot of arguing about them among both experimental and computational chemists. You’d think that clearing a void like that completely of solvent would be energetically costly – I mean, surely water molecules can generally find something to interact with, right? (The flip side of that argument is that they they generally can, thus the relative rarity of these empty pockets).
Here’s a new paper investigating this phenomenon, using the well-known enzyme thermolysin. It’s known to have a binding pocket one residue’s worth away from the active site that very much prefers greasy aliphatic side chains (model protein substrates show that it particularly likes valine, leucine, and isoleucine in that spot). So there’s certainly a hydrophobic cavity (as confirmed by X-ray crystal structures), but just how hydrophobic is it? According to this new work, very. The team (from Marburg) used a weak ligand that doesn’t avail itself of the pocket, but rather bridges over it, largely covering it up. Careful X-ray work quantified just what sort of electron density remained inside the cavity, and the answer is “none”. They saw no evidence for any water molecules in there. Noble gas atoms made their way in if you exposed the system to such things, but water veers off.
If proteins really can have such regions, there’s a huge amount of favorable binding waiting to be picked up by any similarly hydrophobic ligand that can access them. And so it proves in thermolysin: as the SAR of its substrates (and inhibitors) would suggest, you can pick up 40,000-fold binding affinity by dropping an isobutyl group in there. Careful isothermal calorimetry showed that this was completely due to changes in enthalpy; entropic changes were basically zero. There is no desolvation penalty to be paid by the residues in the protein pocket, because there’s nothing to desolvate. That accounting was done when the protein folded. In fact, it’s only with the larger side chains that you start to see a desolvation penalty at all, and that comes from the water molecules around the side chains themselves. Other than that, it’s pure profit, energetically speaking.
So there really are situations where it’s more energetically favorable to have a totally empty cavity in a protein’s three-dimensional structure – a tiny zone of utter vacuum – than it is to let even one water molecule in there, even though the protein itself is swimming in the stuff. The take-home lessons of this paper are not only that these cases are real, but that they represent a huge opportunity for picking up binding affinity when they are identified. Actually, there’s at least one more lesson – that medicinal chemists’ explanations of solvation and compound binding are, at least in some cases, a bit too pat. We need to realize that, as odd as it may seem to us, that there are bubbles of vacuum in some proteins, and be ready to take advantage of that fact. And we should also be wary of any computational approaches that try to model water molecules into such spots because we think that they should be there. In some cases, they just aren’t.