Protein-protein interfaces are structural and energetic mosaics. Within these interfaces, a subset of amino acid residues contributes to a higher proportion of the binding energy, while many other residues are energetically silent. These residues are termed hot spots and contribute disproportionately to the affinity of the complex.
The binding energetics of protein-protein interactions are networked, in that some residues communicate, resulting in protein-protein binding energies that are not necessarily a sum of the binding energies from the individual residues within the interface. Furthermore, energetic networks within protein-protein interfaces display a degree of fine structure with numerous sub-networks existing within a single interface.
Several groups have shown that hot-spot residues tend to cluster together into hot regions rather than being distributed evenly across the protein-protein interface. The binding energies between residues within a hot region are thought to be cooperative, while energetics between residues in distinct hot regions are proposed to be strictly additive.
By using combinatorial mutagenesis and SPR binding analysis of yet another yeast display evolved TCR molecule in complex with its SAG target, in this case affinity-matured variant of hVb2.1 binding to toxic shock syndrome toxin-1 (TSST-1), we were able to dissect energetic additivity and cooperativity in this protein-protein interaction. The general experimental design is shown in Figure 3a. We showed that residues within distinct hot regions, and at distances >20 Å apart, contribute in a significantly cooperative manner to the binding energetics of the complex (Figure 3b).
Our results suggest that a broader consideration of cooperativity within protein-protein interactions may ultimately lead to more accurate predictions of these interactions. The presence of hot regions within these relatively planar interfaces offers prospective binding sites for small molecule inhibitors of protein-protein interactions, which have previously proven difficult to develop. If, as our results suggest, some distinct hot regions are linked energetically, the potency of a small molecule inhibitor that targets a cooperative hot region may be amplified relative to a small molecule region targeting one that is strictly additive.
In our research, SPR analysis of protein-protein interactions has been critical to our basic understanding of the molecular basis of SAG-mediated disease and has enabled us to capitalize on this understanding to develop novel protein therapeutics. Coming full circle, SPR analysis has provided an avenue by which to take the end products of our drug development process to address some outstanding fundamental questions in protein molecular recognition.