Pervasive degeneracy and epistasis in a protein-protein interface

Mapping protein sequence space is a difficult problem that necessitates the analysis of 20(N) combinations for sequences of length N. We systematically mapped the sequence space of four key residues in the Escherichia coli protein kinase PhoQ that drive recognition of its substrate PhoP. We generated a library containing all 160,000 variants of PhoQ at these positions and used a two-step selection coupled to next-generation sequencing to identify 1659 functional variants. Our results reveal extensive degeneracy in the PhoQ-PhoP interface and epistasis, with the effect of individual substitutions often highly dependent on context. Together, epistasis and the genetic code create a pattern of connectivity of functional variants in sequence space that likely constrains PhoQ evolution. Consequently, the diversity of PhoQ orthologs is substantially lower than that of functional PhoQ variants.