Understanding the Roles of Secondary Shell Hotspots in Protein–Protein Complexes

Hotspots are interfacial residues in protein-protein complexes that contribute significantly to complex stability. Methods for identifying interfacial residues in protein-protein complexes are based on two approaches, namely, (a) distance-based methods, which identify residues that form direct interactions with the partner protein and (b) Accessibility Surface Area (ASA)-based methods, which identify those residues that are solvent-exposed in the isolated form of the protein and become buried upon complex formation. In this study, we introduce the concept of secondary shell hotspots, which are hotspots uniquely identified by the distance-based approach, staying buried in both the bound and isolated forms of the protein and yet forming direct interactions with the partner protein. From the analysis of the dataset curated from Docking Benchmark 5.5, comprising 94 protein-protein complexes, we find that secondary shell hotspots are more evolutionarily conserved and have distinct Chou-Fasman propensities and interaction patterns compared to other hotspots. Finally, we present detailed case studies to show that the interaction network formed by the secondary shell hotspots is crucial for complex stability and activity. Further, they act as potentially allosteric propagators and bridge interfacial and non-interfacial sites in the protein. Their in silico mutations to any other amino acid types cause significant destabilization. Overall, this study sheds light on the uniqueness and importance of secondary shell hotspots in protein-protein complexes.