Highlights ► PUB domain of PNGase binds to HR23–UBL more preferentially than ubiquitin chains. ► 3D model of the PUB–UBL complex is presented based on NMR data. ► The PUB–UBL complex is mediated by electrostatic interactions. ► PUB binds the UBL surface involved in intramolecular interaction with HR23–UBA. ► PNGase-PUB could serve as a putative activator of HR23 in ERAD.