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. 2005 Jun 28;102(26):9144-9.
doi: 10.1073/pnas.0502082102. Epub 2005 Jun 17.

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

Jung-Hoon Lee  1 , Jung Min Choi, Changwook Lee, Ki Joung Yi, Yunje Cho
Affiliations

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

Jung-Hoon Lee et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Fig. 1.
Fig. 1.
Schematic representation of the yPNGase-yRad23 complex, providing two different views of the yPNGase-yRad23 complex structure. yPNGase is shown in blue and yRad23 is in yellow. yPNGase comprises three domains, an N-terminal Rad23-binding, a core, and a Zn-binding domain. Three sucrose molecules (green) are located in the deep cleft. A Zn atom (red) is coordinated by Cys-129, -132, -165, and -168 in yPNGase.
Fig. 2.
Fig. 2.
Structural alignment and conservation in yRad23XBD and yPNGase. Shown at the top is the sequence identity between Saccharomyces cerevisiae Rad23XBD (yRad23XBD) and its orthologues from human (Hs) and mouse (Mm). Blue dots indicate yRad23XBD residues that contact yPNGase. Every 10th residue is marked with an asterisk. The secondary structural elements are indicated above the alignment. At the bottom, sequence identity between S. cerevisiae PNGase (yPNGase) and its orthologues from human (Hs), mouse (Mm), and fly (Dm, bottom) is shown. Conserved residues are shaded in yellow, and the catalytic triad is highlighted in red. The four Cys residues that coordinate the Zn atom are shown in green. Orange dots indicate yPNGase residues that contact yRad23XBD. Omitted regions of HsPNGase (residues 64-174), MmPNGase (residues 64-171), and DmPNGase (residues 68-165) are indicated by arrows.
Fig. 3.
Fig. 3.
Effects of the metal chelating agent EDTA on the deglycosylation activity and stability of yPNGase. (A) Deglycosylation activities of the wild-type (WT) and mutant yPNGase proteins. Lane 1, standard molecular mass; lane 2, WT yPNGase and native RNase B; lane 3, WT yPNGase and the denatured RNase B; lane 4, WT yPNGase and the denatured RNase B in the presence of EDTA; lane 5, five residues were mutated simultaneous in yPNGasemis5; lane 6, seven residues were mutated simultaneous in yPNGasemis7. RNaseB + CHO and RNaseB - CHO represent glycosylated and deglycosylated RNase B, respectively. (B) Thermal melting curves of yPNGase without and with EDTA are determined by circular dichroism.
Fig. 4.
Fig. 4.
Active site of yPNGase and the surface representation of the yPNGase-yRad23XBD complex. (A) Interactions between yPNGase and the inhibitor (Left) or sucrose molecules (Center and Right). A sucrose molecule in site 1 of yPNGase (Center) is replaced by an inhibitor, Z-VAD-fmk, but the other two sucrose molecules in sites 2 and 3 remained in the same position upon inhibitor binding (Right). H-bonds are represented by dashed lines. O, N, and S atoms are shown in red, blue, and orange, respectively. Residues that replaced in mutational analyses are marked with red circles. (B) The molecular surfaces of yPNGase and yRad23XBD are colored in white and yellow, respectively. The surface of the yPNGase residues that is >80% conserved in four yPNGase orthologues (Fig. 2) is colored in purple. A catalytic triad is shown in yellow. Axes indicate the close-up views for the inhibitor and sugar-binding sites.
Fig. 5.
Fig. 5.
Close-up view of the yPNGase-yRad23 interface. Residues from the hydrophobic patch of yRad23XBD bind to the residues from both N- and C-terminal helices of yPNGase. The secondary structures of yPNGase (blue) and yRad23XBD (magenta) and the side chains of yPNGase (cyan) and yRad23XBD (yellow) are shown. O and N atoms are shown in red and blue, respectively. The dotted lines indicate intermolecular H-bonds and ion pairs between yPNGase and yRad23XBD.
Fig. 6.
Fig. 6.
Molecular model of the yPNGase-substrate complex. (A) A glycan moiety containing two GlcNAc residues and three mannose molecules was modeled on the active site of the cleft. The dotted lines indicate the three conserved regions where the additional carbohydrate molecules could bind. The common structure of a glycan motif is shown at the top. The close-up view on these conserved regions is shown in Fig. 11. (B) Three Cα atoms from Val-366, Arg-367, and Asn-368 and the GlcNAc residue of yeast carboxypeptidase Y (CPY; Protein Data Bank ID code IYSC) were superimposed onto those from three residues of the inhibitor, Z-VAD-fmk, and a fructose molecule in the active site of yPNGase, respectively. The glycosylated Asn residue of CPY in native form is prevented from accessing the active site by both sides of the deep cleft, which are formed by the strands S2 and S3 in the Zn-binding domain and two loops between H10 and H11 helices and between H9 and S10 of the core domain.
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