1. Volume 40, Issue 6, Pages 905-916 (December 2010)
Structural Basis for Oligosaccharide Recognition of Misfolded Glycoproteins by OS-9 in ER-Associated Degradation 
Tadashi Satoh, Yang Chen, Dan Hu, Shinya Hanashima, Kazuo Yamamoto, Yoshiki Yamaguchi 
Molecular Cell 
Volume 40, Issue 6, Pages (December 2010)
DOI: /j.molcel
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2. Molecular Cell 2010 40, 905-916DOI: (10.1016/j.molcel.2010.11.017)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1
OS-9MRH Has Flattened β-Barrel Structure with Multiple Disulfide Bridges
(A) Domain structure of human OS-9 variant 1 (Hosokawa et al., 2010a). The MRH domain (residues Ala108–Pro229) was crystallized in this study.
(B) Individual carbohydrate residues of Man9GlcNAc2-Asn are labeled. α3,α6-Man5 used in the crystallization is shown in green. Yellow circles indicate the mannotriose unit used in NMR spectroscopy. The deep yellow circles show residues whose positions were determined by the crystal structure, and a light yellow circle shows a residue whose interaction was demonstrated by NMR spectroscopy. Ribbon models of OS-9MRH are shown in both (C) and in (D), which are rotated by almost 90° around a horizontal axis. The positions of the N and C termini are indicated by red letters. β strands and loops are shown in green and gray, respectively. Disulfide bonds are shown as ball-and-stick models together with residue numbers. Bound oligosaccharides are shown as yellow stick models. Omit Fo–Fc electron density map of Man(B)-Man(4′) of the α3,α6-Man5-bound complex contoured at 2.2 σ is also shown.
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4. Figure 2 Structure-Based Alignment of MRH and MPR Domains
The cysteines are highlighted in yellow and the four residues essential for oligosaccharide binding to MPRs and MRH domains are highlighted in pink. Residues that are within hydrogen-bonding distance of M6P in MPRs but were not found to be essential for binding are boxed in red. Residues within hydrogen-bonding distance and involved in hydrophobic interaction with the 6-OH group of Man(B) on OS-9 are highlighted in green. Residues that may determine the linkage specificity of oligosaccharide binding on MRH and MPR domains are highlighted in cyan.
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5. Figure 3
OS-9MRH Interacts with Manα1,6Man through the Unique WW Residues and Conserved Canonical Residues
Omit Fo–Fc electron density map of Manα1,6Man, which corresponds to Man(B)-Man(4′) of the high-mannose-type glycan (Figure 1B) and of the α3,α6-Man5-bound complex contoured at 2.2 σ in molecule B. Man(A) and Man(3) show poor electron density and Man(4) is completely disordered in the structure. Bound oligosaccharide residues are shown as yellow stick models. Residues of OS-9MRH involved in binding ligand are shown as ball-and-stick models. Dashed lines indicate potential hydrogen bonds (black) and hydrophobic interactions (orange). For clarity, subsequent carbohydrate structures are shown beside each hydroxyl group.
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6. Figure 4
Interaction of OS-9MRH with Manα1,6Manα1,6Man in Solution Is Achieved through the WW Motif
1H-15N HSQC (A), 1H-13C HSQC (B), and 1H-1H NOESY (C) spectra of OS-9MRH alone (black) and OS-9MRH with five equivalents of α6-Man3 (red). All Trp residues of OS-9MRH were labeled with 15N and 13C. NMR experiments were performed at 278 K. Interaction model between WW motif and Manα1,6Manα1,6Man (D). The model of the Man(3) position is based on the NOESY spectra. The hydrogen atoms showing intermolecular NOE signals are presented as stick and transparent-sphere models.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
OS-9 Binds Glycoprotein ERAD Substrate ATNHK through the WW Motif of the MRH Domain
(A) Binding of OS-9MRH-SA and its mutants to Lec1 cells. Binding of 10 μg/ml OS-9MRH-SA (WT, filled histogram) or PE-SA (thin line) to Lec1 was measured by flow cytometry. Binding of mutated OS-9MRH-SA with W117A or W118A substitutions was analyzed in the same manner. The data shown are representative of three independent experiments with similar results. The numbers in each panel indicate the mean fluorescence intensity.
(B) Coprecipitation of AT and its nullHong Kong variant (ATNHK) with FLAG-tagged OS-9 or its mutants in 293T cells. Proteins precipitated with FLAG-tagged OS-9 or its mutants were identified by western blotting followed by staining with anti-FLAG antibody (upper). AT, ATNHK, and ATNHK-QQQ precipitated with OS-9 or its mutants were also identified by western blotting followed by staining with anti-AT antibody (lower).
(C) Quantification of intensity of each precipitated ATNHK or ATNHK-QQQ band (n = 3).
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8. Figure 6
Conservation of Carbohydrate-Binding Site between MRH and MPR Domains
(A) Comparison between OS-9MRH (green) and CD-MPR (cyan) carbohydrate-binding sites. Residues binding ligands are shown as ball-and-stick models. Bound Mn2+ and Manα1,2-linked M6P in CD-MPR are shown as a pink sphere and gray stick models, respectively.
(B) Comparison between OS-9MRH (green) and CI-MPR domain 3 (pink) carbohydrate-binding sites. The bound M6P in CI-MPR domain 3 is shown as gray stick models. Residues of OS-9MRH, CD-MPR, and CI-MPR domain 3 are labeled in black, cyan, and pink, respectively.
(C) A close-up view showing interactions between bound oligosaccharide and residues in OS-9MRH and their homologous residues in Yos9, XTP3-B, and GIIβ with residues represented as single amino acid letters. Residues are colored as in Figure 2. The coordinates of the model of Man(3) are based on NMR analyses and the moiety is shown as transparent sticks.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Model for OS-9 Recognition of Oligosaccharides on ERAD Substrates
N-linked glycan chain is initially introduced as a high-mannose-type tetradecasaccharide (Glc3Man9GlcNAc2) immediately after polypeptides enter the ER. The glycan processing to Man8GlcNAc2 was performed by glucosidase I, glucosidase II, and ER ManI, respectively. In glycoprotein ERAD processing, EDEM1/3 further trims the Man(D3) residue. OS-9 binds α1,6-linked trisaccharide Man(B)-Man(4′)-Man(3) residues through the WW motif and canonical residues (lower). To achieve this interaction, trimming of Man(D3) residue is indispensable because Man(D3) sterically clashes with the protein. Untrimmed glycans may also interact with OS-9 (upper), but through fewer contact points—only disaccharide Man(D3)-Man(B) residues could potentially interact through the canonical residues and through only a single Trp residue.
Molecular Cell  , DOI: ( /j.molcel )
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