Marginal specificity in protein interactions constrains evolution

The evolution of novel functions in biology relies heavily on gene duplication and divergence, creating large paralogous protein families. Selective pressure to avoid detrimental cross-talk often results in paralogs that exhibit exquisite specificity for their interaction partners. But how robust or sensitive is this specificity to mutation? Here, using deep mutational scanning, we demonstrate that a paralogous family of bacterial signaling proteins exhibits marginal specificity, such that many individual substitutions give rise to substantial cross-talk between normally insulated pathways. Our results indicate that sequence space is locally crowded despite overall sparseness, and we provide evidence that this crowding has constrained the evolution of bacterial signaling proteins. These findings underscore how evolution selects for good enough rather than optimized phenotypes, leading to restrictions on the subsequent evolvability of paralogs. Significance StatementLarge paralogous protein families are found throughout biology, the product of extensive gene duplication. To execute different functions inside cells, paralogs typically acquire different specificities, interacting with only desired, cognate partners and avoiding cross-talk with non-cognate proteins. But how robust is this interaction specificity to mutation? Can individual mutations lead to cross-talk or do paralogs diverge enough such that multiple mutations would be required, providing a mutational buffer against cross-talk? To address these questions, we built mutant libraries that produce all possible single substitutions of a bacterial kinase and then screened for cross-talk to non-cognate proteins. Strikingly, we find that many single substitutions can produce cross-talk, meaning that these pathways typically exhibit only marginal specificity, and demonstrate that this restricts their evolvability.