Practical Fragments has previously written about the concept of hot spots – regions on a protein where fragments are particularly prone to bind. It’s always nice to have one of these when starting a program, since it’s a good indication that the protein is ligandable.
But there’s another type of hot spot too. When two proteins interact, they often do so through very large interfaces comprising dozens of amino acid residues. This is daunting from a molecular recognition perspective, but it turns out that most of these residues contribute very little energetically to the binding affinity, as assessed by alanine scanning mutagenesis. The few residues that do matter often cluster into hot spots, which are generally much smaller than the entire interface.
In a new paper in J. Chem. Inf. Model., Sandor Vajda, Adrian Whitty, Dima Kozakov, and colleagues at Boston University ask how these two types of hot spots are related.
The researchers used the program FTMap to look for fragment-binding hot spots on the protein ribonuclease A (RNase A). They found four in the vicinity of the binding site for the protein ribonuclease inhibitor (RNI), three of which had also been shown experimentally to bind small organic (solvent) molecules.
Having shown that FTMap can find fragment hot spots, they next turned to a set of 15 protein-protein complexes for which alanine scanning data were available; amino acid side chains were considered hot spots if mutation to alanine decreased binding by more than 2 kcal/mol. Applying FTMap to the receptor of each protein-protein pair showed that 92% of alanine-scanning hot spot residues map onto FTMap hot spots. Moreover, there were very few false negatives: 92% of “unimportant” residues did not map onto FTMap hot spots. In other words, the two types of hot spots seem to overlap considerably.
Although these results may make sense intuitively, the researchers discuss several reasons why this was not a foregone conclusion. First, a residue identified as a hot spot by alanine scanning might not be important for binding per se, but may instead be important for imposing long-range structure on the protein. Second, alanine scanning only identifies important side chains; backbone atoms are not considered, so a fragment may bind to a hot spot that is invisible by alanine scanning. Finally, hot spots identified by alanine scanning reflect the interactions between two proteins, whereas hot spots identified by fragments (or their virtual equivalents) look at only a single protein. This is important because if a residue protrudes from one protein into a cavity on the other, the protruding residue may be a hot spot for interactions but, because of its geometry, not be a good binding site for small molecules. As the authors put it:
A convex surface site on a protein typically will not bind small molecules strongly no matter how much binding energy the region generates in an interaction with a complementary concave site on its protein binding partner. Thus, observation of a hot spot by alanine scanning mutagenesis does not necessarily imply the existence of a small molecule fragment consensus site at that region.
And of course, as we’ve noted before, proteins can be wriggly little things, with new pockets opening up where you least expect them. So despite all these caveats, it’s reassuring to see that, for the most part, there is some constancy in the hotness of spots.