Structural basis for redox dependent retention of ERAP1 by ERp44 in endoplasmic reticulum

Abstract

ERp44, a PDI family chaperone with RDEL signal sequence, sequesters proteins from cis-golgi to ER. Deletion of ERp44 in mice releases intracellular ERAP1 to plasma, and increases angiotensin II processing and blood pressure. Previous experiments in transfected cells had implicated ERp44 C29 in the thioredoxin-like a-domain and ERAP1 C498 in the domain-II “exon-10” loop as important in the retention mechanism. However, other key domain-domain interactions between the two proteins and mechanism of release of ERAP1 from ERp44 is yet to be studied. Using purified recombinant ERp44 expressed in E. coli and ERAP1 expressed in insect cells, we confirmed that the C29-C498 intermolecular disulfide formed spontaneously upon mixing ERp44 and ERAP1 resulting in a monodisperse 1:1 complex. Previously ERp44 interaction with other targets has been shown to be pH dependent, but we found that ERp44 interaction with ERAP1 was not highly pH sensitive in the range 5.7 – 7.7, suggesting a possible novel mode of interaction. The interaction was specific for ERp44, as PDI and ERp57, other oxidoreductases, did not form a complex with ERAP1. We are using cryo-electron microscopy to get an insight into the structural basis for interaction of ERAP1 and ERp44. Preliminary data collected on Talos (200kV) yielded a low-resolution 3D model map of ERAP1-ERp44 at 8.5Ǻ that resembles ERAP1 in the open conformation, with substantial density extending from the vicinity of the exon 10 loop. A model for the complex fits well into this density, positioned with ERp44 C29 adjacent to ERAP1 C498. We will examine the ERAP1-ERp4 structure to see if we can identify the structural basis for the preferential reactivity of ERp44 C29 over C63, and for ERAP1 C498 over C486, and to understand the mechanism of release of ERAP1 from the complex through a disulfide exchange reaction.