Molecular ruler of tripeptidylpeptidase II: Mechanistic principle of exopeptidase selectivity

► The E312/E343 double-Glu motif of tripeptidylpeptidase II is highly asymmetrical. ► E312 both blocks the active-site cleft at position P4 and acts as the prime docking residue. ► A salt bridge involving E312 conveys high exopeptidase selectivity. ► Substrate binding to the prime-site subsites enables weak endopeptidolysis.