Relationship between Hot Spot Residues and Ligand Binding Hot Spots in Protein–Protein Interfaces

In the context of protein–protein interactions,
the term
“hot spot” refers to a residue or cluster of residues
that makes a major contribution to the binding free energy, as determined
by alanine scanning mutagenesis. In contrast, in pharmaceutical research,
a hot spot is a site on a target protein that has high propensity
for ligand binding and hence is potentially important for drug discovery.
Here we examine the relationship between these two hot spot concepts
by comparing alanine scanning data for a set of 15 proteins with results
from mapping the protein surfaces for sites that can bind fragment-sized
small molecules. We find the two types of hot spots are largely complementary;
the residues protruding into hot spot regions identified by computational
mapping or experimental fragment screening are almost always themselves
hot spot residues as defined by alanine scanning experiments. Conversely,
a residue that is found by alanine scanning to contribute little to
binding rarely interacts with hot spot regions on the partner protein
identified by fragment mapping. In spite of the strong correlation
between the two hot spot concepts, they fundamentally differ, however.
In particular, while identification of a hot spot by alanine scanning
establishes the potential to generate substantial interaction energy
with a binding partner, there are additional topological requirements
to be a hot spot for small molecule binding. Hence, only a minority
of hot spots identified by alanine scanning represent sites that are
potentially useful for small inhibitor binding, and it is this subset
that is identified by experimental or computational fragment screening.