The interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein–protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1–α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1–α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein–protein interactions, with implications for drug development and rational engineering of these interfaces.
Bibliographical noteFunding Information:
This work was supported by the Israel Science Foundation (Grant 1454/13), a grant from the Canadian Institutes of Health Research (CIHR), the International Development Research Centre (IDRC), the Israel Science Foundation (ISF), and the Azrieli Foundation (Grant 3512/19) and by the DS Research Center at the University of Haifa.
© 2021, The Author(s).
- Amino Acid Sequence/genetics
- Binding Sites/genetics
- DNA-Binding Proteins/genetics
- Escherichia coli Proteins/chemistry
- Multiprotein Complexes/chemistry
- Protein Binding/genetics
- Protein Interaction Maps/genetics
- Protein Structure, Secondary
- RNA-Binding Proteins/genetics