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Engineering a stable complex of ERp44 with a designed peptide ligand for analyzing the mode of interaction of ERp44 with its clients

ERp44, a chaperone of the protein disulfide isomerase (PDI) family cycles between the endoplasmic reticulum (ER) and cis‐Golgi compartments to act on a cohort of disparate proteins either to ensure their proper cellular localization or for the quality control of the correct assembly. This process involves intermolecular disulfide bond formation between the client protein and the conserved Cys29 in ERp44. We had identified a 9‐amino acid peptide derived from an ERp44 client, adiponectin, as the motif interacting with ERp44 via a highly conserved cysteine (Cys39 of adiponectin). However, in order to reveal detailed insight into the mode of interaction, the generation of sizeable amounts of corresponding thiol‐liganded ERp44 was unsuccessful under ambient conditions. Here we describe the production of a bromopeptide variant of this peptide ligand that led to large amounts of a pure, complex of peptide sufficient for structural studies in which Cys29 of Erp44 is covalently linked via a stable thioether bond. This method can be potentially used to rapidly probe putative, short, interacting peptide regions in other ERp44 clients to reveal their, hitherto unknown, mechanism of interaction with ERp44.