Evolutionary transition pathways for changing peptide ligand specificity and structure

We identified evolutionary pathways for the inter-conversion of three seque ntially and structurally unrelated peptides, GATPEDLNQKL, GLYEWGGARI and; F DKEWNLIEQN, binding to the same site of the hypervariable region of the ant i-p24 (HIV-1) monoclonal antibody CB4-1, Conversion of these peptides into each other could be achieved in nine or 10 single amino acid substitution s teps without loss of antibody binding. Such pathways were identified by ana lyzing all 7 620 480 pathways connecting 2560 different peptides, and testi ng them for CB4-1 binding. The binding modes of intermediate peptides of se lected optimal pathways were characterized using complete sets of substitut ion analogs, revealing that a number of sequential substitutions accumulate d without changing the pattern of key interacting residues. At a distinct s tep, however, one single amino acid exchange induces a sudden change in the binding mode, indicating a flip in specificity and conformation. Our data represent a model of how different specificities, structures and-functions might evolve in protein-protein recognition.