Evolutionary paths that link orthogonal pairs of binding proteins

Some protein binding pairs exhibit extreme specificities that functionally insulate them from homologs. Such pairs evolve mostly by accumulating single-point mutations, and mutants are selected if their affinity exceeds the threshold required for function . Thus, homologous and high-specificity binding pairs bring to light an evolutionary conundrum: how does a new specificity evolve while maintaining the required affinity in each intermediate ? Until now, a fully functional single-mutation path that connects two orthogonal pairs has only been described where the pairs were mutationally close enabling experimental enumeration of all intermediates . We present an atomistic and graph-theoretical framework for discovering low molecular strain single-mutation paths that connect two extant pairs and apply it to two orthogonal bacterial colicin endonuclease-immunity pairs separated by 17 interface mutations . We were not able to find a strain-free and functional path in the sequence space defined by the two extant pairs. By including mutations that bridge amino acids that cannot be exchanged through single-nucleotide mutations, we found a strain-free 19-mutation trajectory that is completely functional . Despite the long mutational trajectory, the specificity switch is remarkably abrupt, resulting from only one radical mutation on each partner. Each of the critical specificity-switch mutations increases fitness, demonstrating that functional divergence could be driven by positive Darwinian selection. These results reveal how even radical functional changes in an epistatic fitness landscape may evolve.