@article {Gurusinghe2022-kl,
	title = {Cold spots are universal in protein-protein interactions},
	journal = {Protein Sci.},
	volume = {31},
	number = {10},
	year = {2022},
	month = {oct},
	pages = {e4435},
	publisher = {Wiley},
	abstract = {Proteins interact with each other through binding interfaces that differ greatly in size and physico-chemical properties. Within the binding interface, a few residues called hot spots contribute the majority of the binding free energy and are hence irreplaceable. In contrast, cold spots are occupied by suboptimal amino acids, providing possibility for affinity enhancement through mutations. In this study, we identify cold spots due to cavities and unfavorable charge interactions in multiple protein-protein interactions (PPIs). For our cold spot analysis, we first use a small affinity database of PPIs with known structures and affinities and then expand our search to nearly 4000 homo- and heterodimers in the Protein Data Bank (PDB). We observe that cold spots due to cavities are present in nearly all PPIs unrelated to their binding affinity, while unfavorable charge interactions are relatively rare. We also find that most cold spots are located in the periphery of the binding interface, with high-affinity complexes showing fewer centrally located colds spots than low-affinity complexes. A larger number of cold spots is also found in non-cognate interactions compared to their cognate counterparts. Furthermore, our analysis reveals that cold spots are more frequent in homo-dimeric complexes compared to hetero-complexes, likely due to symmetry constraints imposed on sequences of homodimers. Finally, we find that glycines, glutamates, and arginines are the most frequent amino acids appearing at cold spot positions. Our analysis emphasizes the importance of cold spot positions to protein evolution and facilitates protein engineering studies directed at enhancing binding affinity and specificity in a wide range of applications.},
	keywords = {binding affinity, binding interfaces, cold spots of binding, hot spots of binding, protein-protein interactions},
	author = {Gurusinghe, Sagara N S and Oppenheimer, Ben and Shifman, Julia M}
}
