Structure of the Mouse Peptide N-Glycanase-HR23 Complex Suggests Co-evolution of the Endoplasmic Reticulum-associated Degradation and DNA Repair Pathways

Abstract

Peptide N-glycanase removes N-linked oligosaccharides from misfolded glycoproteins as part of the endoplasmic reticulum-associated degradation pathway. This process involves the formation of a tight complex of peptide N-glycanase with Rad23 in yeast and the orthologous HR23 proteins in mammals. In addition to its function in endoplasmic reticulum-associated degradation, HR23 is also involved in DNA repair, where it plays an important role in damage recognition in complex with the xeroderma pigmentosum group C protein. To characterize the dual role of HR23, we have determined the high resolution crystal structure of the mouse peptide N-glycanase catalytic core in complex with the xeroderma pigmentosum group C binding domain from HR23B. Peptide N-glycanase features a large cleft between its catalytic cysteine protease core and zinc binding domain. Opposite the zinc binding domain is the HR23B-interacting region, and surprisingly, the complex interface is fundamentally different from the orthologous yeast peptide N-glycanase-Rad23 complex. Different regions on both proteins are involved in complex formation, revealing an amazing degree of divergence in the interaction between two highly homologous proteins. Furthermore, the mouse peptide N-glycanase-HR23B complex mimics the interaction between xeroderma pigmentosum group C and HR23B, thereby providing a first structural model of how the two proteins interact within the nucleotide excision repair cascade in higher eukaryotes. The different interaction interfaces of the xeroderma pigmentosum group C binding domains in yeast and mammals suggest a co-evolution of the endoplasmic reticulum-associated degradation and DNA repair pathways.Degradation of misfolded and misassembled proteins is essential for homeostasis and cell growth. In the endoplasmic reticulum (ER) 2 a quality control system sorts proteins on the basis of their conformation (1).Proteins that fail to mature in the ER are retro-translocated into the cytosol, polyubiquitinated, and targeted to the proteasome for degradation as part of the ER-associated degradation pathway (2). Defects in the ER-associated degradation system have been associated with many diseases including ␣-1 antitrypsin deficiency, cystic fibrosis, neurodegenerative disorders, tyrosinase deficiency, and cancer (3-6).Most of the misfolded glycoproteins are deglycosylated by a cytosolic peptide-N-glycanase (PNGase) activity before proteasomal degradation (7,8). PNGase hydrolyzes the ␤-aspartylglycosylamine bond of N-linked glycoproteins, which facilitates their degradation by the proteasome (9). PNGase is highly conserved from yeast to human, supporting its functional significance. In addition to the catalytic core, in higher eukaryotes PNGase contains one additional domain each at the N and C terminus, which may function as binding partners for other proteins (10).PNGase closely associates with the proteasome by either directly binding to components of the proteasome or through its interaction with HR23A/HR23B, the mammalian homologs of Ra...

Structural and biochemical studies of the C-terminal domain of mouse peptide-N-glycanase identify it as a mannose-binding module

“…For comparison, the same ITC experiments were also carried out with yeast PNGase (data not shown). The yeast enzyme neither binds to mannopentaose, which is consistent with the absence of the C-terminal domain in yeast PNGase, nor interacts with chitobiose as one would expect based on the close structural relationship between the core domain of mouse PNGase and full-length yeast PNGase (9,10).…”

“…2C). The oligomannose-binding site is apparently formed by ␤-strands 8, 9, and 16, which provide the concave part of the saddle, and the loops between ␤8 and ␤9, as well as ␤15, ␤16, and the second 3 10 helix, which form the ridges on either side of the saddle. Trp-532 resides in the loop between ␤-strands 8 and 9 and Trp-624 in the 3 10 -2 helix, and these residues are on either side of the binding site.…”

“…Both mouse and yeast PNGase have been reported to interact with HR23B͞Rad23, a protein that is also involved in DNA damage recognition (5)(6)(7). Recently the structures of yeast and mouse PNGase in complex with Rad23͞ HR23B and an inhibitor (8), carbobenzyloxy-Val-Ala-Asp-␣-fluoromethyl ketone (Z-VAD-fmk), have been solved (9,10). The yeast protein corresponds to the central region of mouse PNGase.…”

Identification and Characterization of a Novel Prokaryotic Peptide

“…Mouse PNGase has N-and C-terminal domains in addition to its central catalytic domain. The N-terminal domain is known to be involved in protein-protein interaction (29), whereas the C-terminal domain may function as a mannose-binding module (30). Allen et al reported that the N-terminal PUB domain of human PNGase consists of five ␣ helices that pack into a short threestranded antiparallel ␤ sheet.…”

Ubiquilin-1 Is a Molecular Chaperone for the Amyloid Precursor Protein

“…The central region of ubiquilin-1 contains two regions of similarity to the co-chaperone Sti-1 (also known as Hop). Sti-1 domains mediate hydrophobic protein-protein interactions and may possess intrinsic chaperone activity (26,27).…”