The sequence-specific peptide-binding activity of the protein sulfide isomerase AGR2 directs its stable binding to the oncogenic receptor EpCAM
M. Aiman Mohtar , Lenka Hernychova , J. Robert O'Neill , Melanie L. Lawrence , Euan Murray , Borek Vojtesek , Theodore Hupp
AbstractAGR2 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.
|Journal series||Molecular & Cellular Proteomics, ISSN 1535-9476, (A 40 pkt)|
|Publication size in sheets||1.3|
|License||Other; published final; ; with publication|
|Score|| = 40.0, ArticleFromJournal|
= 45.0, ArticleFromJournal
|Publication indicators||: 2017 = 5.232 (2) - 2017=6.323 (5)|
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