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Structure of a peptide:N-glycanase-Rad23 complex: insight into the deglycosylation for denatured glycoproteins.

http://www.ncbi.nlm.nih.gov/pubmed/15964983

In eukaryotes, misfolded proteins must be distinguished from correctly folded proteins during folding and transport processes by quality control systems. Yeast peptide:N-glycanase (yPNGase) specifically deglycosylates the denatured form of N-linked glycoproteins in the cytoplasm and assists proteasome-mediated glycoprotein degradation by forming a complex with 26S proteasome through DNA repair protein, yRad23. Here, we describe the crystal structures of a yPNGase and XPC-binding domain of yRad23 (yRad23XBD, residues 238-309) complex and of a yPNGase-yRad23XBD complex bound to a caspase inhibitor, Z-VAD-fmk. yPNGase is formed with three domains, a core domain containing a Cys-His-Asp triad, a Zn-binding domain, and a Rad23-binding domain. Both N- and C-terminal helices of yPNGase interact with yRad23 through extensive hydrophobic interactions. The active site of yPNGase is located in a deep cleft that is formed with residues conserved in all PNGase members, and three sugar molecules are bound to this cleft. Complex structures in conjunction with mutational analyses revealed that the walls of the cleft block access to the active site of yPNGase by native glycoprotein, whereas the cleft is sufficiently wide to accommodate denatured glycoprotein, thus explaining the specificity of PNGase for denatured substrates.

Pubmed ID: 15964983 RIS Download

Mesh terms: Amino Acid Chloromethyl Ketones | Amino Acid Sequence | Animals | Binding Sites | Biophysical Phenomena | Biophysics | Caspase Inhibitors | Circular Dichroism | Crystallography, X-Ray | Cytoplasm | DNA Mutational Analysis | DNA-Binding Proteins | Enzyme Inhibitors | Glycoproteins | Glycosylation | Humans | Models, Molecular | Molecular Sequence Data | Mutagenesis | Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase | Proteasome Endopeptidase Complex | Protein Binding | Protein Conformation | Protein Denaturation | Protein Folding | Protein Structure, Secondary | Protein Structure, Tertiary | Recombinant Proteins | Saccharomyces cerevisiae | Saccharomyces cerevisiae Proteins | Sequence Homology, Amino Acid | Spectrophotometry, Atomic | Substrate Specificity | Zinc