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

doi:10.1073/pnas.0502082102

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

National Creative Research Center for Structural Biology and Department of Life Science, Pohang University of Science and Technology, Hyo-ja dong, San 31, Pohang, KyungBook, South Korea

Edited by William J. Lennarz, Stony Brook University, Stony Brook, NY, and approved May 12, 2005 (received for review March 14, 2005)

Abstract

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.

Footnotes

↵ * To whom correspondence should be addressed. E-mail: yunje{at}postech.ac.kr.
Author contributions: J.-H.L. and Y.C. designed research; J.-H.L., J.M.C., C.L., and K.J.Y. performed research; J.-H.L., J.M.C., and Y.C. analyzed data; and J.-H.L. and Y.C. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ER, endoplasmic reticulum; GlcNAc, N-acetylglucosamine; PNGase, peptide:N-glycanase; yPNGase, yeast PNGase; Z-VAD-fmk, carbobenzyloxy-Val-Ala-Asp-α-fluoromethylketone.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 1X3W (yPNGase-yRad23) and 1X3Z (yPNGase-yRad23 inhibitor)].