AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.

¶Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute 656 53 Brno Czech Republic

¶Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute 656 53 Brno Czech Republic ‖University of Gdansk International Centre for Cancer Vaccine Science ul Wita Stwosza 63 80 308 Gdansk Poland

From the ‡University of Edinburgh Institute of Genetics and Molecular Medicine Edinburgh Scotland United Kingdom EH4 2XR

From the ‡University of Edinburgh Institute of Genetics and Molecular Medicine Edinburgh Scotland United Kingdom EH4 2XR §National University of Malaysia UKM Medical Molecular Biology Institute 56000 Kuala Lumpur Malaysia

From the ‡University of Edinburgh Institute of Genetics and Molecular Medicine Edinburgh Scotland United Kingdom EH4 2XR ¶Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute 656 53 Brno Czech Republic

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