Structural and mutational studies on the importance of oligosaccharide binding for the activity of yeast PNGase

Abstract

Peptide:N-glycanase (PNGase) is an important component of the endoplasmic reticulum-associated protein degradation pathway in which it de-glycosylates misfolded glycoproteins, thus facilitating their proteasomal degradation. PNGase belongs to the transglutaminase superfamily and features a Cys, His, and Asp catalytic triad, which is essential for its enzymatic activity. An elongated substrate-binding groove centered on the active site Cys191 was visualized in the crystal structure of apo-PNGase, whereas its complex with Z-VAD-fmk, a peptide-based inhibitor of PNGase, revealed that the inhibitor occupied one end of the substrate-binding groove while being covalently linked to the active site Cys. Recently, haloacetamidyl-containing carbohydrate-based inhibitors of PNGase were developed and shown to specifically label the active site Cys. In this study, we describe the crystal structure of yeast PNGase in complex with N,N′-diacetylchitobiose (chitobiose). We found that the chitobiose binds on the side opposite to the peptide binding site with the active site Cys191 being located approximately midway between the carbohydrate and peptide binding sites. Mutagenesis studies confirm the critical role of the chitobiose-interacting residues in substrate binding and suggest that efficient oligosaccharide binding is required for PNGase activity. In addition, the N-terminus of a symmetry-related PNGase was found to bind to the proposed peptide-binding site of PNGase. Together with the bound chitobiose, this enables us to propose a model for glycoprotein binding to PNGase. Finally, deleting the C-terminal residues of yeast PNGase, which are disordered in all structures of this enzyme, results in a significant reduction in enzyme activity, indicating that these residues might be involved in binding of the mannose residues of the glycan chain. © The Author 2008. Published by Oxford University Press. All rights reserved.