Structural basis of Zn²⁺-mediated tethering that stabilizes ERp44-client complexes in protein quality control

ERp44 plays a critical role in protein quality control in the early secretory pathway by retrieving ER enzymes and immature secretory proteins from the Golgi to the ER. This function is regulated by Zn2+- and pH-dependent conformational changes in ERp44. However, the detailed mechanisms by which Zn2+ governs client binding in the Golgi and client release in the ER remain to be elucidated. Here, we report cryo-EM structures of complexes between Zn2+-bound ERp44 and two representative clients, ERAP1 and Ero1α. These structures reveal that, beyond the mixed disulfide-mediated interactions, Zn2+ is coordinated by conserved histidine residues of ERp44 together with residues in client loops, forming two distinct types of Zn2+-mediated tethers that stabilize the complexes. Consistently, removal of Zn2+ promotes complex dissociation more effectively than reduction of the mixed disulfide bonds. In the physiological ER environment, millimolar concentrations of glutathione, together with picomolar levels of labile Zn²⁺maintained by the ER-localized Zn2+ transporter ZIP7, promote complex dissociation by removing Zn2+ from the complex. The present findings demonstrate that Zn2+ plays dual roles in protein quality control by allosterically activating ERp44 and directly mediating client binding.