Oxytocin exhibited a distinct binding pose due to its cyclic core structure, interacting with residues distal to the active site (see Figure and Figure ). Both peptides, however, demonstrated analogous N-terminal interactions, including robust hydrogen bonding between the NH3 + group and the glutamate residues Glu295, Glu431, and Glu487. Additionally, the scissile bond oxygen interacted with Zn 2+ and Tyr549. The GAMEN loop facilitated multiple hydrogen bonds, notably between the scissile NH group and the Ala429 backbone carbonyl, enabling the peptides to adopt an antiparallel β-strand conformation (Figure ). This interaction is crucial for both inhibitor complex formation and substrate catalytic activity. It has been shown that the GAMEN loop, part of the D2 domain, undergoes substantial conformational changes upon inhibitor binding, inducing the catalitically active closed conformation of the IRAP active site. This conformational plasticity is essential for inhibitor and substrate binding. Site-directed mutagenesis studies on M1 aminopeptidases have demonstrated that mutations of Glu431 67 and Tyr549 significantly reduce the catalytic activity of IRAP and ERAP1, respectively.