Structure-guided discovery of aminopeptidase ERAP1 variants capable of processing antigens with novel PC anchor specificities.

ABSTRACT

Endoplasmic reticulum aminopeptidase 1 (ERAP1) belongs to the oxytocinase subfamily of M1 aminopeptidases (M1APs), which are a diverse family of metalloenzymes involved in a wide range of functions and have been implicated in various chronic and infectious diseases of humans. ERAP1 trims antigenic precursors into correct sizes (8-10 residues long) for Major Histocompatibility Complex (MHC) presentation, by a unique molecular ruler mechanism in which it makes concurrent bindings to substrate N- and C-termini. We have previously determined four crystal structures of ERAP1 C-terminal regulatory domain (termed ERAP1_C domain) in complex with peptide carboxyl (PC)-ends that carry various anchor residues, and identified a specificity subsite for recognizing the PC anchor side chain, denoted as the SC subsite to follow the conventional notations S1 site for P1, S2 site for P2, and so forth. In this study, we report studies on structure-guided mutational and hydrolysis kinetics, and peptide trimming assays to further examine the functional roles of this SC subsite. Most strikingly, a point mutation V737R results in a change of substrate preference from a hydrophobic to a negatively charged PC anchor residue; the latter is presumed to be a poor substrate for WT ERAP1. These studies validate the crystallographic observations that this SC subsite is directly involved in binding and recognition of the substrate PC anchor and presents a potential target to modulate MHC-restricted immunopeptidomes.