PROJECT SUMMARY/ABSTRACT ERAP1, ERAP2, and IRAP are M1-family zinc aminopeptidases with important roles in trimming antigenic peptide precursors for loading onto MHC-I proteins. Common polymorphisms in the erap1 gene are associated with increased susceptibility to autoimmune diseases including ankylosing spondylitis, psoriasis, Behçet's disease, and birdshot retinopathy, increased susceptibility to certain kinds of cancer, and essential hypertension. Polymorphic residues are located distal to the enzyme active site, and the mechanism underlying their effects on enzymatic activity is unknown. ERAP2 polymorphisms are less common and more weakly associated with autoimmune diseases than for ERAP1. Key questions about ER aminopeptidases include their mechanism of action, in particular the mechanistic basis for the unique length-dependent cleavage activity, the nature of the linkage of polymorphic variants with autoimmune disease, and to what degree mechanistic insights about ERAP1 can be extended to the other members of the oxytocinase subfamily ERAP2 and IRAP. An overarching hypothesis of this proposal is that large-scale conformational alterations provide a mechanistic underpinning for the effects of ER aminopeptidase polymorphisms on enzymatic activity and disease association, and that the conformational equilibria are modulated by interactions with other proteins in the endoplasmic reticulum. One specific aim of the proposed research is to understand how interactions between ER aminopeptidase domains regulate enzyme activity. A detailed mechanistic model for ERAP1 catalytic mechanism will be developed and tested. The model couples ERAP1 binding interactions near the N- and C-termini of peptide substrates with large-scale domain closure motions that stabilize the catalytically active configuration of key active site residues. Using salt-bridge mutations known to alter ERAP1 conformational dynamics, and small-molecule inhibitors that alter ERAP1 conformational equilibria, we will test whether disease-associated polymorphisms act through differential stabilization of open and closed conformers, and we will determine whether ERAP2 and IRAP similarly utilize large-scale domain closure motions to regulate enzymatic activity. A second specific aim is to determine the structural basis and functional consequences of ERp44-mediated endoplasmic reticulum retention of ERAP1 and ERAP2. We aim to determine structures of complexes of ERp44 with ERAP1 and ERAP2, to characterize the effect of ERp44 interaction on ERAP1 and ERAP2 processing, and to evaluate the role of ERAP1-ERp44 interactions in generating new epitopes under ER stress. A third specific aim is to evaluate the influence of the ER chaperones tapsin and TAPBPR on ERAP1 trimming of epitope precursors while they are bound to MHC-I. 1