Molecular Basis for Bre5 Cofactor Recognition by the Ubp3 Deubiquitylating Enzyme

Yeast Ubp3 and its co-factor Bre5 form a deubiquitylation complex to regulate protein transport between the endoplasmic reticulum and Golgi compartments of the cell. A novel N-terminal domain of the Ubp3 catalytic subunit forms a complex with the NTF2-like domain of the Bre5 regulatory subunit. Here, we report the X-ray crystal structure of an Ubp3–Bre5 complex and show that it forms a symmetric hetero-tetrameric complex in which the Bre5 NTF2-like domain dimer interacts with two L-shaped β-strand–turn–α-helix motifs of Ubp3. The Ubp3 N-terminal domain binds within a hydrophobic cavity on the surface of the Bre5 NTF2-like domain subunit with conserved residues within both proteins interacting predominantly through antiparallel β-sheet hydrogen bonds and van der Waals contacts. Structure-based mutagenesis and functional studies confirm the significance of the observed interactions for Ubp3–Bre5 association in vitro and Ubp3 function in vivo. Comparison of the structure to other protein complexes with NTF2-like domains shows that the Ubp3–Bre5 interface is novel. Together, these studies provide new insights into Ubp3 recognition by Bre5 and into protein recognition by NTF2-like domains.