Structural Basis for Oligosaccharide Recognition of Misfolded Glycoproteins by OS-9 in ER-Associated Degradation

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1 Article Structural Basis for Oligosaccharide Recognition of Misfolded Glycoproteins by OS-9 in ER-Associated Degradation Tadashi Satoh, 1 Yang Chen, 2 Dan Hu, 2 Shinya Hanashima, 1 Kazuo Yamamoto, 2 and Yoshiki Yamaguchi 1, * 1 Structural Glycobiology Team, RIKEN Advanced Science Institute, Saitama , Japan 2 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba , Japan *Correspondence: yyoshiki@riken.jp DOI /j.molcel SUMMARY Misfolded glycoproteins are translocated from endoplasmic reticulum (ER) into the cytosol for proteasome-mediated degradation. A mannose-6- phosphate receptor homology (MRH) domain is commonly identified in a variety of proteins and, in the case of OS-9 and XTP3-B, is involved in glycoprotein ER-associated degradation (ERAD). Trimming of outermost a1,2-linked mannose on C-arm of highmannose-type glycan and binding of processed a1,6-linked mannosyl residues by the MRH domain are critical steps in guiding misfolded glycoproteins to enter ERAD. Here we report the crystal structure of a human OS-9 MRH domain (OS-9 MRH ) complexed with a3,a6-mannopentaose. The OS-9 MRH has a flattened b-barrel structure with a characteristic P-type lectin fold and possesses distinctive double tryptophan residues in the oligosaccharide-binding site. Our crystallographic result in conjunction with nuclear magnetic resonance (NMR) spectroscopic and biochemical results provides structural insights into the mechanism whereby OS-9 specifically recognizes Mana1,6Mana1,6Man residues on the processed C-arm through the continuous double tryptophan (WW) motif. INTRODUCTION Potentially toxic misfolded proteins are translocated from the endoplasmic reticulum (ER) into the cytosol for ubiquitin proteasome-mediated degradation, the so-called ER-associated degradation (ERAD). In the system, N-linked glycan plays an important role as a destination signal (Ellgaard et al., 1999; Helenius and Aebi, 2004). Trimming of mannose residues functions as a molecular timer for the recognition of terminally misfolded polypeptides in the ERAD system (Helenius and Aebi, 2004; Hosokawa et al., 2010a). ER-degradation-enhancing a-mannosidase-like protein (EDEM) in mammals and Htm1p in yeast were originally identified as Man 8 GlcNAc 2 isomer B (M8B)- binding lectins that enhance glycoprotein ERAD (Hosokawa et al., 2001; Jakob et al., 2001). More recent studies suggest that they have mannosidase activity (Hirao et al., 2006; Olivari et al., 2006; Quan et al., 2008; Clerc et al., 2009; Hosokawa et al., 2009, 2010b). Trimming of the outermost a1,2-linked mannose on the C-arm of high-mannose-type glycan by EDEMs/ Htm1p seems a critical process for glycoprotein ERAD. In parallel, additional players were discovered that function as lectins in the ERAD machinery. Yos9p was identified as a candidate for involvement in glycoprotein ERAD by a yeast genetic screen (Buschhorn et al., 2004). Yos9p contains a mannose-6- phosphate receptor homology (MRH) domain that is homologous to mannose-6-phosphate receptors (MPRs) (Munro, 2001). In addition to Yos9p, the MRH domain is commonly identified in a variety of proteins such as ER glucosidase II b subunit (GIIb) and Golgi GlcNAc-phosphotransferase g subunit. Cationdependent (CD-) and cation-independent (CI-) MPRs recognize terminal mannose-6-phosphate (M6P) residues presented on the N-linked glycans of lysosomal enzymes (Dahms et al., 2008; Kim et al., 2009). It has been shown that Yos9p binds to misfolded ERAD substrates in the ERAD-luminal pathway (Bhamidipati et al., 2005; Kim et al., 2005; Szathmary et al., 2005) and forms part of a large Hrd1p-Hrd3p ubiquitin ligase core complex in the ER membrane (Carvalho et al., 2006; Denic et al., 2006; Gauss et al., 2006). In the case of mammals, two MRH domain-containing lectins, OS-9 and XTP3-B, are Yos9p orthologs. These proteins are ER-resident luminal proteins involved in ERAD, and associate with a HRD1-SEL1L-ubiquitin ligase complex (Bernasconi et al., 2008; Christianson et al., 2008; Hosokawa et al., 2008; Mueller et al., 2008). Its components are conserved from yeast to human, emphasizing the biological significance of the complex. M8B and Man 5 GlcNAc 2 are predicted to function as ligands for Yos9p (Szathmary et al., 2005). Further, it has been reported that Yos9p binds not only N-glycan but also misfolded polypeptide segments (Bhamidipati et al., 2005) and that the recognition of misfolded polypeptide is mediated through the ER chaperone Kar2p, which also forms an E3 complex with Yos9p (Xie et al., 2009). Christianson et al. reported that human OS-9 MRH (OS-9 MRH ) and XTP3-B MRH domains are required for interaction with SEL1L with multiple N-glycans but not with glycoprotein ERAD substrates (Christianson et al., 2008). Studies using MRH mutants (R188A) also suggest that the lectin activity of OS-9 is not required for binding to misfolded glycoproteins (Bernasconi et al., 2008; Christianson et al., 2008). In contrast, Hosokawa et al. reported that the Molecular Cell 40, , December 22, 2010 ª2010 Elsevier Inc. 905

2 Figure 1. OS-9 MRH Has Flattened b-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 Man 9 GlcNAc 2 -Asn are labeled. a3,a6-man 5 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-9 MRH 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. b 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 F o F c electron density map of Man(B)-Man(4 0 ) of the a3,a6-man 5 -bound complex contoured at 2.2 s is also shown. OS-9 MRH is necessary for recognition of N-glycans of ERAD substrates rather than for SEL1L-binding activity (Hosokawa et al., 2009). Further, several research groups including ours showed oligosaccharide-binding specificity of Yos9p/OS-9/ XTP3-B MRH domains and that binding of the Mana1,6-linked C-arm of high-mannose-type glycans on ERAD substrates by the MRH domain is a critical step in guiding the entry of misfolded proteins into ERAD (Quan et al., 2008; Clerc et al., 2009; Hosokawa et al., 2009; Mikami et al., 2010; Yamaguchi et al., 2010). Although the mode of interaction between OS-9 and SEL1L still remains controversial, the OS-9 MRH has since been demonstrated to be important in glycoprotein ERAD through its recognition of specific N-glycans on ERAD substrates (reviewed by Hosokawa et al., 2010a). Many crystal structures of MPRs have been determined (Roberts et al., 1998; Olson et al., 1999, 2004a, 2004b, 2008; Brown et al., 2002) and all showed structurally similar MPR domains with a P-type lectin fold. These studies provide the structural basis for phosphomannosyl recognition by these receptors and reveal a dynamic ph-dependent load/unload mechanism for handling cargo (Dahms et al., 2008; Kim et al., 2009). Structural determination of the MRH domains is also indispensable to understanding the molecular mechanisms of how MRH domains specifically interact with a1,6-linked mannosyl residues on the C-arm and of why trimming of outermost a1,2-linked mannose on this arm is necessary for binding. Here, in order to understand the structural basis for the recognition of glycoprotein ERAD substrates by ER-luminal lectins, we report the crystal structure of an MRH domain, the OS-9 MRH (Figure 1A) complexed with Mana1,3(Mana1,6)Mana1,6(Mana1,3) Man [termed a3a6-man 5 for Man(A)(Man(B))Man(4 0 )(Man(4)) Man(3); see Figure 1B], which is a minimal mannosyl oligosaccharide structure of glycoprotein ERAD substrates processed by ER and/or Golgi a1,2-mannosidases. RESULTS The OS-9 MRH Has a Characteristic Compact P-Type Lectin Fold We expressed a recombinant OS-9 MRH (Figure 1A) protein as an inclusion body in Escherichia coli and purified it by an oxidative refolding method. The structure of OS-9 MRH complexed with 906 Molecular Cell 40, , December 22, 2010 ª2010 Elsevier Inc.

3 Table 1. Data Collection and Refinement Statistics for OS-9 MRH Native SeCys Crystallographic Data Space group P P Unit cell a/b/c (Å) 73.1/73.1/ /72.9/178.1 Data Processing Statistics Beam line NSRRC 13B1 PF-AR NE-3A Wavelength (Å) Resolution (Å) ( ) ( ) Total/unique reflections 201,235/17, ,353/7,173 Completeness (%) 99.7 (100.0) 98.1 (89.4) R merge (%) 9.0 (46.9) 12.8 (41.4) I / s (I) 28.5 (5.5) 54.8 (6.4) Redundancy Refinement Statistics Resolution (Å) R work / R free (%) 22.8/27.7 Rmsds from Ideal Bond lengths (Å) Bond angles ( ) 1.66 Ramachandran Plot (%) Favored 92.9 Allowed 7.1 a3,a6-man 5 was solved by the single-wavelength anomalous dispersion (SAD) method using a crystal of selenocysteine (SeCys)-substituted protein (see the Supplemental Experimental Procedures, available online). The final model of a3,a6-man 5 - bound OS-9 MRH refined to 2.10 Å resolution has an R work of 22.8% and R free of 27.7% (Table 1). The crystal belongs to space group P with two molecules (A and B) per asymmetric unit. The structures of molecules A and B are quite similar to each other with a root-mean-square deviation (rmsd) value of 0.58 Å for superimposed 102 Ca atoms. Molecule A, which has a lower average B value (A, 30.2 Å 2 versus B, 33.3 Å 2 ), is described hereafter. OS-9 MRH has a flattened b-barrel structure composed of two eight-stranded antiparallel b sheets, with the two b sheets in almost orthogonal arrangement (Figures 1C and 1D). Numbering of the b strands is according to the scheme of CD-MPR (Roberts et al., 1998). The first b sheet has four antiparallel b strands (b1 b4/5). Beginning with b4/5, the second b sheet is formed with five b strands (b4/5 b9). b6 and b7 are rather separated, and the N-terminal half of b6 (residues ) does not form a b sheet structure with b7. The last two b strands (b8 and b9) are in reverse order in the structure, and b9 makes a parallel b sheet with b7 and they form two disulfide bonds. All six cysteine residues (Cys110 Cys123, Cys181 Cys216, and Cys196 Cys228) in the MRH domain are involved in disulfide bridge formation. This disulfide-bonding pattern was predicted from sequence alignments (Munro, 2001). The overall structure of OS-9 MRH is essentially identical to the structures of the CD-MPR and CI-MPR domains (Roberts et al., 1998; Brown et al., 2002; Olson et al., 2004a). The rmsd values for superimposed Ca atoms between OS-9 and MPRs are Å (Table S1). Among the known MPR structures, CI-MPR domain 3 has the highest number of superimposable residues and the highest sequence identity with OS-9 MRH.A structure-based sequence alignment of OS-9 MRH with MPRs and the secondary structure assignments of OS-9 MRH and CI-MPR domain 3 are shown in Figure 2. The structural arrangement before b1 of OS-9 MRH is similar to that of CI-MPR domain 1 that lacks 2b and 1b strands (Brown et al., 2002). Except for the CI-MPR domain 1, all MPR domains have an additional a helix (CD-MPR) or b sheet ( 2b and 1b, CI-MPR) before b1 at the N-terminal region (Figure 2 and Figure S1). Structural conservation of disulfide bridges between OS-9 and MPRs is observed in the posterior two disulfide bonds (Cys181 Cys216 and Cys196 Cys228 in OS-9), whereas the anterior disulfide bond (Cys110 Cys123) of OS-9 is characteristic of the MRH domain. b1 3 and b7 9 of OS-9 MRH are structurally comparable with the corresponding strands of MPRs, whereas b4/5 and b6of OS-9 MRH are different. In OS-9 MRH, a continuous twisted b strand (b4/5) connects the b sheets with b3 and b6 forming a compact b-barrel structure, whereas in the MPR structures b4 and b5 present separately and b5 6 tilt outward to associate with the N-terminal a helix or b sheet. Thus OS-9 MRH has characteristic b4/5 and b6 strands as well as the N-terminal disulfide bond of the P-type lectin fold. These characteristics and the structural similarity of OS-9 MRH with MPRs lead us to conclude that OS-9 can be placed in the P-type lectin family (Drickamer and Taylor, 1993). Oligosaccharide-Binding Site of OS-9 MRH Is Composed of Conserved Canonical Residues and Characteristic Double Tryptophan Residues In the a3,a6-man 5 -binding site of OS-9 MRH, two mannose residues are clearly visible in the electron density map and are traced as Mana1,6Man (Figure 3). Because additional electron density is followed from the 3-OH and 1-OH groups of the second Man residue, Mana1,6Man is assigned as Man(B)-Man (4 0 ) in the a3,a6-man 5 structure (Figures 1B and 3). Although the glycosidic bond is clearly visible (Figure 3 and Figure S2A), the ring moiety of a1,3-linked Man(A) has poor electron density, especially in molecule A. This is due to the fact that Man(A) is turned toward the solvent and mobile. Man(3) residues in molecules A and B overlap each other, resulting in continuous electron density of the two bound a3,a6-man 5 ligands (Figure S2A). The two bound Man(3)s are disturbed by crystal contacts and their positions could not be determined. However, we hypothesized that Man(3) is indeed recognized by Trp117 in solution, and hence we performed solution NMR analyses to investigate the interaction (described in the next section). Man(4) following Man(3) is completely disordered. The oligosaccharide-binding site of OS-9 MRH is located at the C-terminal opening of the b-barrel, which is composed of b3, b8, and b9, one loop between b1 and b2, and one loop between b6 and b7 (Figures 1 and 2). The oligosaccharide-binding mode is almost identical in molecules A and B (Figure S2B). In the nonreducing Man(B)-binding site, all hydroxyl groups of the mannose are involved in binding through extensive hydrogen bonds, namely with Gln130, Asp182, Arg188, Glu212, and Tyr218 Molecular Cell 40, , December 22, 2010 ª2010 Elsevier Inc. 907

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. (Figure 3). Additionally, Leu183 has a hydrophobic interaction with the C6 atom of Man(B). Among these interactions, the 2-OH group has a significant role in recognition through a large Figure 3. OS-9 MRH Interacts with Mana1,6Man through the Unique WW Residues and Conserved Canonical Residues Omit F o F c electron density map of Mana1,6Man, which corresponds to Man (B)-Man(4 0 ) of the high-mannose-type glycan (Figure 1B) and of the a3,a6- Man 5 -bound complex contoured at 2.2 s 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-9 MRH 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. number of hydrogen bonds with three amino acid residues. As for the Man(4 0 )-binding site, a lateral Trp118 ring interacts with the a1,6-linked mannobiose structure mainly through hydrophobic interactions. The hydrophobic a1,6-linked glycosidic bond including C1 and C6 atoms participates in the binding. Furthermore, the 2-OH group of the Man(4 0 ) makes a hydrogen bond with the Trp118 N3-1 atom, while the C1 atom participates in the hydrophobic interactions with Trp117. The a1,6-linked Man(4 0 ) binding site is composed of a triad of Trp117, Trp118, and His132, which mutually stabilize each other. The Trp118 ring is stabilized by the lateral Trp117 ring through a vertical CH-P interaction, while the Trp117 ring interacts with the His132 ring through parallel P- P and/or P-cation interactions. Indeed, mutation of His132 abolishes ligand binding (Mikami et al., 2010). OS-9 MRH Recognizes Mana1,6Mana1,6Man on C-Arm of High-Mannose-Type Glycan through the Double Tryptophan Motif To demonstrate an interaction between Man(3) and Trp117 and verify that the crystallographically observed OS-9 MRH /oligosaccharide interaction occurs in solution, we performed NMR analyses of the interaction focusing on the unique double tryptophan (Trp) residues at the Man(4 0 ) and potential Man(3)-binding sites. We first performed titration experiments monitored by onedimensional 1 H-NMR spectra (Figure S3). Upon addition of the oligosaccharide ligands, a6-man 3, a3,a6-man 5, or Man 5 GlcNAc 2 -Asn to a solution of OS-9 MRH, we observed similar spectral changes in the 1 H-NMR spectra, indicating that the 908 Molecular Cell 40, , December 22, 2010 ª2010 Elsevier Inc.

5 A W118 W118 1 W117 1 W154 W117 B W118 3 W118 2 W154 2 W117 2 W118 3 W154 3 W117 3 W117 3 W154 3 W118 2 W118 1 W154 2 W117 2 W117 1 Figure 4. Interaction of OS-9 MRH with Mana1, 6Mana1,6Man in Solution Is Achieved through the WW Motif 1 H- 15 N HSQC (A), 1 H- 13 C HSQC (B), and 1 H- 1 H NOESY (C) spectra of OS-9 MRH alone (black) and OS-9 MRH with five equivalents of a6-man 3 (red). All Trp residues of OS-9 MRH were labeled with 15 N and 13 C. NMR experiments were performed at 278 K. Interaction model between WW motif and Mana1, 6Mana1,6Man (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 transparentsphere models. W W154 1 C D H (ppm) W118 1 W H (ppm) W117 2 W117 3 W117 3 W117 2 W118 2 W118 2 W H (ppm) OMe H3/5 (Man(3)) H3 (Man(B)) H6a (Man(4 )) H2 (Man(B)) H1 (Man(4 )/(B)) Overhauser effect spectroscopy [NOESY]) on OS- 9 MRH in the presence and absence of a6-man 3 (Figure 4). Trp117 and Trp118 signals were assigned by use of selectively ( 13 C and 15 N) Trp-labeled OS-9 MRH (see Experimental Procedures and Supplemental Experimental Procedures). The characteristic higher-magnetic-field shift of Trp117 d1-proton at 4.15 parts per million (ppm) (Figure 4B) is well explained by the ring current effect of the vicinally oriented Trp118 side chain (Figure 3). Most of the 1 H- 15 N and 1 H- 13 C HSQC signals originating from Trp117 and Trp118 show significant chemical-shift perturbations in the presence of a6-man 3, whereas Trp154 signals show no significant chemical-shift change (Figures 4A and 4B). In 1 H- 1 H NOESY spectrum of OS-9 MRH in the presence of a6-man 3, intermolecular NOE signals between a6-man 3 and Trp117 and Trp118 are clearly observed (Figure 4C and Table S2). These NMR analyses reveal that Man(3) interacts with Trp117 and the OS-9 MRH /Mana1,6Mana1,6Man interaction at the WW motif. Based on the solution data, we modeled the Man(3) coordinate in the OS-9 MRH complex (Figure 4D). In the Man(3)-binding site, the Trp117 ring interacts with the hydrophobic side of the Man(3) ring through parallel CH/P interaction. This is often observed in carbohydrate/ protein interactions (Weis and Drickamer, 1996). minimal epitope recognized by OS-9 MRH is the a6-man 3 (Mana1, 6Mana1,6Man) unit. The OS-9 MRH /a6-man 3 or a3,a6-man 5 interactions are fast-exchange processes in terms of the chemical shift; the dissociation constants were estimated to be 160 ± 28 or 200 ± 35 mm, respectively, which are comparable with those obtained by frontal affinity chromatography (FAC) analyses (40 mmor87mm, Man 5 GlcNAc 2 -pyridylaminate [PA]) (Hosokawa et al., 2009; Mikami et al., 2010). In order to reveal the nature of the interaction in detail, we carried out NMR experiments ( 1 H- 15 N heteronuclear single quantum coherence [HSQC], 1 H- 13 C HSQC, and 1 H- 1 H nuclear OS-9 Binds Glycoprotein ERAD Substrate through the WW Motif on the MRH Domain To examine the contribution of each of the Trp residues of the WW motif to ligand binding, we performed site-directed mutagenesis experiments. We previously reported that mutations of conserved canonical residues (Gln130, Arg188, Glu212, and Tyr218) at the Man(B)-binding site abolish or diminish ligand binding (Mikami et al., 2010). Biotinylated OS-9 MRH was complexed with R-phycoerythrin (PE)- labeled streptavidin (SA) tetramer and incubated with Lec1 cells to examine whether W117A or W118A OS-9 MRH mutants have the ability to bind to the sugar chains displayed on the surface of the Lec1 cells. Lec1 cells are known to be deficient in N-acetylglucosaminyltransferase I (GnT-I) and rich in Man 5 GlcNAc 2 N-glycans (Chen and Stanley, 2003). The binding of Molecular Cell 40, , December 22, 2010 ª2010 Elsevier Inc. 909

6 Figure 5. OS-9 Binds Glycoprotein ERAD Substrate AT NHK through the WW Motif of the MRH Domain (A) Binding of OS-9 MRH -SA and its mutants to Lec1 cells. Binding of 10 mg/ml OS-9 MRH -SA (WT, filled histogram) or PE-SA (thin line) to Lec1 was measured by flow cytometry. Binding of mutated OS-9 MRH -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 null Hong Kong variant (AT NHK ) 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, AT NHK, and AT NHK -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 AT NHK or AT NHK -QQQ band (n = 3). W117A OS-9 MRH to Lec1 cells is significantly decreased (25 times) compared with that of wild-type, and the W118A mutant does not bind to Lec1 cells at all (Figure 5A). These results demonstrate that in addition to canonical residues, the OS-9 MRH WW motif, especially Trp118, contributes to oligosaccharide binding. We assessed interaction between full-length OS-9 mutants and the glycoprotein ERAD substrate, human a1-antitrypsin (AT) variant null Hong Kong (AT NHK ), which is known to be a wellestablished substrate for ERAD. AT and AT NHK have three N-linked sugar chains, and we previously showed that AT NHK, but not AT, appears to have smaller-molecular-weight highmannose-type glycans without a1,2-linked mannose(s) (Mikami et al., 2010). As previously reported (Mikami et al., 2010), AT NHK, but not AT, coprecipitates with FLAG-tagged wild-type OS-9 (Figure 5B). In contrast, the amount of AT NHK that precipitated with W117A, W118A, W117A/W118A, and R188A OS-9 mutants is significantly decreased compared with wild-type. These results strongly suggest that OS-9 recognizes glycoprotein ERAD substrates such as AT NHK through the WW motif and canonical residues of the MRH domain. In contrast, part of the interaction between OS-9 and AT NHK seems not to be mediated by sugar chains attached to AT NHK because a significant amount of AT NHK is still coprecipitated with sugar-bindingdeficient OS-9 mutants. In addition, we examined the effect of mutations of OS-9 WW motif on the nonglycosylated ERAD substrate, AT NHK -QQQ (Figure 5B). In contrast to AT NHK, almost the same amount of AT NHK -QQQ coprecipitated with wild-type or mutant OS-9 as previously reported (Bernasconi et al., 2008; Hosokawa et al., 2009). These results suggest that OS-9 binds to the polypeptide segment of ERAD substrates in addition to sugar moieties. Because glycan dependency in the interaction between OS-9 and SEL1L remains controversial (Christianson et al., 2008; Hosokawa et al., 2009), we investigated whether the interaction is affected by mutations of OS-9 MRH WW motif. SEL1L was coprecipitated with OS-9, and the interaction was significantly decreased when Ala substitution of OS-9 at WW motif or canonical Arg188 was introduced (Figure S4). These data indicate that the interaction between OS-9 and SEL1L is glycan dependent, as previously reported (Christianson et al., 2008). 910 Molecular Cell 40, , December 22, 2010 ª2010 Elsevier Inc.

7 Figure 6. Conservation of Carbohydrate-Binding Site between MRH and MPR Domains (A) Comparison between OS-9 MRH (green) and CD-MPR (cyan) carbohydrate-binding sites. Residues binding ligands are shown as ball-and-stick models. Bound Mn 2+ and Mana1,2-linked M6P in CD-MPR are shown as a pink sphere and gray stick models, respectively. (B) Comparison between OS-9 MRH (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-9 MRH, 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-9 MRH and their homologous residues in Yos9, XTP3-B, and GIIb 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. DISCUSSION The Double-Tryptophan Motif on OS-9 MRH Determines a1,6-linkage Specificity of the Oligosaccharide Binding The oligosaccharide-binding site of OS-9 MRH is compared with those of CD-MPR in complex with a1,2-linked M6P (Olson et al., 1999) and CI-MPR domain 3 in complex with M6P (Olson et al., 2004b) (Figures 6A and 6B). CI-MPR has 15 repeated homologous domains, and domains 3, 5, and 9 are involved in phosphomannosyl ligand binding (Dahms et al., 2008; Kim et al., 2009). Although the oligosaccharide-binding specificities of OS-9 and MPRs are essentially different, the structure of the primary nonreducing Man(B)-binding site, involving Gln130, Arg188, Glu212, and Tyr218 of OS-9 and M6P-binding residues of MPRs (excluding phosphate-binding residues), is conserved. Our crystallographic and NMR spectroscopic analyses in conjunction with biochemical mutagenesis experiments demonstrate that OS-9 MRH specifically recognizes Mana1,6Mana1, 6Man residues through a WW motif (Trp117/Trp118). In the CD- and CI-MPRs, Met44/Tyr45 (CD-MPR), Glu323/Tyr324 (CI-MPR domain 3), Lys620/Tyr621 (domain 5), and Glu1219/ Tyr1220 (domain 9) replace Trp117/Trp118 of OS-9. It has been shown that CD- and CI-MPRs preferentially bind to a1,2- linked M6P residues but not to a1,6-linked M6P (Olson et al., 1999, 2008; Song et al., 2009; Bohnsack et al., 2009). This a1,2-linkage-specific binding is considered to be achieved Molecular Cell 40, , December 22, 2010 ª2010 Elsevier Inc. 911

8 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 (Glc 3 Man 9 GlcNAc 2 ) immediately after polypeptides enter the ER. The glycan processing to Man 8 GlcNAc 2 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 a1,6-linked trisaccharide Man(B)-Man(4 0 )-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. mainly by the second Tyr residue. Superimposition of OS-9 MRH with CD-MPR suggests that the interaction area between Mana1,2Man and OS-9 MRH is smaller than that between Mana1,6Man and OS-9 MRH. Our data rule out interaction of a1,2-linked Man with Trp117 of OS-9 MRH although it is possible with Trp118 (Figure 6A). Furthermore, the expected inner a1,6-linked Man may be separate from the OS-9 MRH oligosaccharide-binding site (see a1/ in Figure 6A and Man(4 0 ) in Figure 7). The structural data suggest that interaction between untrimmed glycans and OS-9 MRH (two Man residues with a single Trp) is weaker than the one between trimmed glycans and OS-9 MRH (three Man residues with the Trp doublet) (Figure 7). The 2-OH group of the nonreducing Man interacts extensively with OS-9 MRH, eliminating extra space in the site and making trimming of the outermost a1,2-linked Man on the C-arm necessary if Mana1,6Mana1,6Man is to bind through the WW motif (Figure 7). This result is in agreement with previous biochemical studies, which demonstrated by using a flow cytometry assay that Mana1,2Man at low concentrations does not inhibit OS-9 MRH ligand-binding (Mikami et al., 2010) and that binding of OS-9 MRH to high-mannose-type glycans with a1,2-linked Man on the C-arm is much weaker or undetectable than binding to trimmed glycans, according to FAC assays (Hosokawa et al., 2009; Mikami et al., 2010). These findings concur with reports that trimming of the outermost a1,2-linked Man on the C-arm by a1,2- mannosidases is a critical process for glycoprotein ERAD (Quan et al., 2008; Clerc et al., 2009; Hosokawa et al., 2009, 2010b). From these observations, we propose that it is the structural difference between Trp and Tyr on b2 which is a critical determinant for linkage specificity of these P-type lectins and that OS-9 preferentially binds terminal a1,6-linked mannosyl residues processed through a1,2-mannosidases on the C-arm of high-mannose-type glycans on glycoprotein ERAD substrates. In order to investigate whether a Trp to Tyr mutation of OS-9 MRH alters the linkage specificity from Mana1,6Man to Mana1,2Man, we performed binding experiments with a W117E/W118Y OS-9 mutant, based on the appearance of Glu-Tyr in domain 3 of CI-MPR and GIIb MRH (Figure 2). Unexpectedly, this double mutation bound to neither kifunesine (ER a-mannosidase inhibitor)-treated Lec1 cells nor untreated cells (data not shown), which are rich in Man 7-9 GlcNAc 2 glycans (Kawasaki et al., 2007) and Man 5 GlcNAc 2 (Chen and Stanley, 2003), respectively. We performed least-squares alignment of OS-9 MRH and CD-MPR or CI-MPR domain 3 with corresponding carbohydrate-binding residues. There is a slight structural difference between OS-9 MRH WW motif and MPRs XY motif (Figures 6A and 6B). The distances between corresponding Ca carbons of Trp118 OS-9 MRH and Tyr45 CD-MPR or Tyr324 CI-MPR are 1.3 and 2.3 Å, respectively. These results suggest that a1,2- linked Man binding requires not only the Xxx-Tyr sequence but also the three-dimensional (3D) scaffold of the MRH domain. Structure-Based Alignment Provides Insights into the Oligosaccharide-Binding Mode of ER-Luminal Lectins with MRH Domain The OS-9 MRH domain shares sequence similarities with those of yeast Yos9p, human XTP3-B, and GIIb (19.0% 35.5%, Figure 2). The MRH domains of these proteins have been shown to be essential for their respective oligosaccharide binding (Quan et al., 2008; Yamaguchi et al., 2010; Hu et al., 2009). Like OS-9, Yos9p and XTP3-B recognize a1,6-linked mannobiose or mannotriose structures, particularly on the C-arm of highmannose-type glycans (Quan et al., 2008; Yamaguchi et al., 2010). In contrast, GIIb binds to an a1,2-linked mannobiose structure on the C-arm (Hu et al., 2009). In order to obtain insights into the mechanism of oligosaccharide recognition by Yos9p, XTP3-B, and GIIb, we generated structure-based sequence alignment by using structures of OS-9 MRH and MPR 912 Molecular Cell 40, , December 22, 2010 ª2010 Elsevier Inc.

9 domains as templates (Figure 2). Nonpolar residues involved in the association of the two b sheets in OS-9 MRH are highly conserved in these proteins, suggesting that these MRH domains form a similar P-type lectin fold. The primary Man(B)- binding site of OS-9 MRH including Gln130, Arg188, Glu212, and Tyr218 is similarly conserved in Yos9p, XTP3-B, and GIIb, suggesting that the Man-binding mode at the primary binding site is essentially the same as that of OS-9 MRH (Figures 2 and 6C). XTP3-B has two MRH domains, N-terminal domain 1 and C-terminal domain 2. In addition to conservation of the primary binding site, the triad sequence of OS-9 MRH that is involved in Man a1,6-linkage-specific binding (Trp117/Trp118/His132) is also conserved in XTP3-B domain 2 (Trp351/Trp352/His366). We recently showed that only domain 2 is involved in oligosaccharide binding (Yamaguchi et al., 2010). However, the alignment analysis did not give a definitive answer as to why domain 1 does not bind oligosaccharide ligands, even though the residues involved in sugar binding and polypeptide folding are highly conserved. As for the Yos9p MRH domain, the triad sequence can be aligned as Phe124/Trp125/His139, suggesting that the Trp125 binds with the Man(4 0 ) moiety through polar and nonpolar interactions and the Phe124 binds Man(3) through CH-P hydrophobic stacking. This remarkable amino acid sequence conservation with OS-9 suggests that the oligosaccharide-binding modes of XTP3-B domain 2 and Yos9p are essentially identical with that of OS-9, which specifically recognizes Mana1, 6Mana1,6Man of high-mannose-type glycans. In the GIIb MRH domain, the triad sequence of OS-9 is replaced with Glu422/Tyr423/Pro437, suggesting that GIIb does not form a hydrophobic triad as observed in the OS-9 MRH structure. Instead, the Tyr423 can be aligned with Tyr45 of CD-MPR, which is involved in a1,2-linked Man binding (Olson et al., 1999, 2008). The significant amino acid conservation between GIIb and MPRs suggests that GIIb binds to a1,2-linked mannobiose of high-mannose-type glycoproteins in the same manner as in the MPRs. Insights into Misfolded Glycoprotein Recognition by OS-9 We constructed a complex model of the larger substrate Man 5 GlcNAc 2 -Asn and OS-9 MRH (see Supplemental Experimental Procedures and Figure S5). In this model, there are no significant steric clashes between Man 5 GlcNAc 2 -Asn and OS-9 MRH. In this model, Man(A) and Man(4) seem not to participate in oligosaccharide binding, and the chitobiose residue (GlcNAc(2) and GlcNAc(1), Figure 1B) also seems to turn away from the protein surface of OS-9 MRH. Upon titration with a6-man 3, a3,a6-man 5, and Man 5 GlcNAc 2 -Asn ligands, comparable spectral changes were observed in the 1 H-NMR experiments (Figure S3). From these results, we conclude that the minimal epitope of OS-9 MRH is an a1,6-linked Man(B)-Man(4 0 )-Man(3) on the C-arm and that the interactions identified in the crystal structure and in solution are applicable to recognition of high-mannose-type glycans by OS-9 in cells. It has been suggested that OS-9 recognizes both glycan and polypeptide segments (Bernasconi et al., 2008; Christianson et al., 2008; Hosokawa et al., 2009). In our experiments, binding of sugar-binding-deficient OS-9 mutants to AT NHK was also still detected (Figure 5B), although the mutants showed a significantly decreased binding to AT NHK as compared to that of wild-type. Further, binding of OS-9 to nonglycosylated AT NHK - QQQ was observed as previously reported (Bernasconi et al., 2008; Hosokawa et al., 2009), suggesting that part of the interaction occurs through the polypeptide segments. It has not been shown whether OS-9 directly distinguishes between folding intermediates of ERAD substrates or whether OS-9 simply functions as a lectin together with ER chaperones BiP and GRP94 (Hosokawa et al., 2010a). In yeast, Yos9p also has bipartite functions (Bhamidipati et al., 2005) and the recognition of misfolded polypeptide is mediated through the Kar2p, BiP ortholog (Xie et al., 2009). In addition to an MRH domain (residues ), human OS-9 protein has N- and C-terminal domains of unknown function (N: , C: , variant2: D ) (Kimura et al., 1998). Mapping of hydrophobic residues on OS-9 MRH shows no large hydrophobic areas on the molecular surface (Figure S5), suggesting that a putative folding sensor domain exists in the N and/or C domain(s) but not in the MRH domain. Based on these results, we suggest that the MRH domain simply functions as a lectin domain that binds high-mannose-type glycans, on which the outermost a1,2-linked Man on the C-arm is trimmed by ER and/or Golgi a1,2-mannosidases. Here we showed that OS-9 binds to SEL1L as well as AT NHK through the WW motif in a glycan-dependent manner (Figure S4). Although the carbohydrate structures of SEL1L remain unknown, the glycans on SEL1L are possibly trimmed by EDEM1 and are capable of binding OS-9 MRH. Recently, it was reported that EDEM1 interacts with SEL1L (Cormier et al., 2009) and has a1,2-mannosidase activity (Hosokawa et al., 2010b). If OS-9 is homotrimeric like its yeast ortholog Yos9p (Quan et al., 2008), the multiple MRH domains in the oligomer may enable simultaneous binding of SEL1L and ERAD substrate glycans. These data lead us to suggest that OS-9 serves as a molecular hinge between misfolded proteins and the SEL1L-containing ER membrane dislocation complex. In summary, we determined the 3D structure of the MRH domain of OS-9 complexed with a a3,a6-man 5 ligand, which is a minimal mannosyl oligosaccharide structure of the glycoprotein ERAD substrates. Our results provide structural insights into the mechanism of a specific oligosaccharide recognition of high-mannose-type glycans of glycoprotein ERAD substrates by OS-9 MRH in an a1,6-linked C-arm-specific manner through a double-trp motif. Further biochemical and biophysical studies on OS-9, together with its cognate partners including ER chaperones and components such as GRP94, BiP, and SEL1L, are underway in our laboratories. Such studies should further the understanding of the mechanism of translocation of misfolded glycoproteins by the ER-luminal surveillance lectins in the ERAD system. EXPERIMENTAL PROCEDURES Expression and Purification of Native and Selenocysteine-Substituted OS-9 Proteins His 6 -MBP-tagged OS-9 MRH (Gly97 Pro229 and Ala108 Pro229) were purified from E. coli Rosetta2(DE3)pLysS inclusion bodies and refolded by an oxidative Molecular Cell 40, , December 22, 2010 ª2010 Elsevier Inc. 913

10 refolding method. Further details are in the Supplemental Experimental Procedures. Expression of SeCys-substituted OS-9 MRH (Ala108 Pro229) was performed according to a method previously described (Müller et al., 1994; Sanchez et al., 2002). The auxotrophic E. coli host cell BL21(DE3) selb::kan Cys51E (Müller et al., 1994), referred to as BL21(DE3)cys, was used for the SeCys labeling experiments. The BL21(DE3)cys cells harboring an OS-9 MRH plasmid were cultured in minimal medium (Sanchez et al., 2002) containing 100 mg/l L-SeCys. Subsequent expression and purification were performed as for the native proteins. Crystallization, X-Ray Data Collection, and Structure Determination The crystal of native a3,a6-man 5 -bound OS-9 MRH (Ala108 Pro229) was obtained in a buffer containing 7 mg/ml protein, 4 mm a3,a6-man 5, 1.9 M ammonium sulfate, 0.1 M Tris-HCl (ph 7.5), and 2% PEG400 on incubation at 303 K for 5 days. All crystals were cryoprotected with crystallization mother liquor supplemented with 20% glycerol. The diffraction limit was improved by approximately 0.5 Å by using a flash annealing technique (Yeh and Hol, 1998); the nitrogen stream was blocked for 2 s once. The crystal of native OS-9 MRH belonged to space group P and diffracted to 2.10 Å resolution. The crystal of SeCys-substituted a3,a6-man 5 -bound OS-9 MRH was obtained in a buffer containing 6.5 mg/ml protein, 4 mm a3,a6-man 5, 1.9 M ammonium sulfate, 0.1 M Bis-Tris (ph 6.5), and 2% PEG400 on incubation at 303K for 5 days. The crystal of SeCys-OS-9 MRH belonged to space group P and diffracted to 2.85 Å resolution. All diffraction data were processed with HKL2000 (Otwinowski and Minor, 1997). The native and SeCys-substituted crystal parameters of OS-9 MRH are shown in Table 1. The crystal structure of OS-9 MRH was solved by using the SAD method with a crystal of the SeCys-substituted protein, and the final model was refined to 2.10 Å resolution. Further details are in the Supplemental Experimental Procedures. The final refinement statistics of the OS-9/a3,a6-Man 5 complex are summarized in Table 1. Figures were prepared with PyMOL (DeLano, 2002). NMR Spectroscopy Synthesis of Mana1,6Mana1,6Mana-OMe (a6-man 3 ) was performed as described in the Supplemental Experimental Procedures. L-[ 13 C 11 / 15 N 2 ] Trp was purchased from Taiyo Nippon Sanso. Trp-selective labeling was performed as described previously (Nishida et al., 2006). OS-9 MRH (Gly97 Pro229) was expressed in E. coli Rosetta2(DE3)pLysS by using M9 minimal medium supplemented with L-[ 13 C 11 / 15 N 2 ] Trp and other unlabeled amino acids. Refolding and purification of Trp-labeled OS-9 MRH was performed by the same procedure as for the unlabeled protein. For NMR measurements, purified proteins were first concentrated to mm in a final volume of 0.5 ml with 10 mm sodium phosphate buffer (ph 6.5) containing 50 mm NaCl and 10% 2 H 2 O. All NMR experiments were carried out on a DRX-600 spectrometer (BrukerBiospin) equipped with a triple-resonance probe with the temperature set to 278 K or 293 K. 1 H chemical-shift values were given in ppm calibrated with external reference DSS (4,4- dimethyl-4-silapentane-1-sulfonic acid) at 0 ppm, while indirect referencing was used for 15 N and 13 C according to the absolute frequency values. Data processing and analysis were performed by XWIN-NMR ver. 3.5 (BrukerBiospin). Dissociation constants between OS-9 MRH and a6-man 3 or a3,a6-man 5 were calculated by 1 H chemical-shift changes of OS-9 MRH. Assignment of NMR signals was carried out as described in the Supplemental Experimental Procedures. Binding Assays with Flow Cytometry and Immunoprecipitation Binding assays using flow cytometry were carried out as previously described (Mikami et al., 2010). Preparation of biotinylated recombinant OS-9 MRH protein and its mutants were performed as described in Supplemental Experimental Procedures. To prepare an R-PE-labeled OS-9 MRH -SA tetramer, we mixed biotinylated OS-9 MRH with SA conjugated to PE (SA-PE; BD Biosciences Phar- Mingen, San Jose, CA) at a molar ratio of 4:1 for 1 hr on ice. Ten microliters of Lec1 cells were incubated with the indicated concentration of PE-labeled OS-9 MRH -SA in 20 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH (ph 7.4) and 150 mm NaCl (HEPES-buffered saline [HBS]) containing 1 mm CaCl 2, 0.1% NaN 3, and 0.1% BSA at 25 C for 30 min. After washing with HBS, cells were suspended in 200 ml of HBS containing 1 mg/ml propidium iodide. The fluorescence intensity of stained cells was measured by using a FACS Caliber and CellQuest software (BD Biosystems). The fluorescence at 575 nm associated with PE on the surface of the cells was recorded and converted to a mean fluorescence intensity. Immunoprecipitation of human AT, AT variant null Hong Kong (AT NHK ), AT NHK -QQQ, and SEL1L with FLAG-tagged OS-9 expressed in 293T cells was carried out as described in the Supplemental Experimental Procedures. ACCESSION NUMBERS The coordinate and structure factor have been deposited in the Protein Data Bank under accession number 3AIH. SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures, two tables, and Supplemental Experimental Procedures and can be found with this article online at doi: /j.molcel ACKNOWLEDGMENTS We thank Drs. Mayumi Kanagawa (RIKEN, Wako, Japan), Hideyuki Miyatake (RIKEN), Masato Kawasaki (KEK, Tsukuba, Japan), Masahiko Hiraki (KEK), and Mr. Yuki Nakamura (PharmAxess, Inc., Osaka, Japan) for help with crystallization and X-ray data collection; Mr. Masaki Kato (RIKEN) for performing the bioinformatics analyses; Ms. Kana Matsumoto (RIKEN) for performing high-performance liquid chromatography (HPLC) and mass spectrum (MS) analyses; Dr. Yoichi Takeda (RIKEN/ERATO) for help with analyses of NMR titration data; Dr. David Waugh (National Cancer Institute, Frederick, MD) for providing the prk793 plasmid; and Dr. Marie-Paule Strub (National Heart, Lung, and Blood Institute, Bethesda, MD) for providing the BL21(DE3)cys strain. We thank beamline staff of PF/KEK (Japan), SPring-8 (Japan), and NSRRC (Taiwan) for providing data collection facilities and support. This work was supported by a Global COE program (Frontier Biomedical Science Underlying Organelle Network Biology, Osaka University) for young scientists (T.S. and S.H.) and by a Grant-in-Aid for Science Research in a Priority Area (Protein Community, T.S. and Y.Y.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Received: March 18, 2010 Revised: July 24, 2010 Accepted: September 24, 2010 Published: December 21, 2010 REFERENCES Bernasconi, R., Pertel, T., Luban, J., and Molinari, M. (2008). A dual task for the Xbp1-responsive OS-9 variants in the mammalian endoplasmic reticulum: inhibiting secretion of misfolded protein conformers and enhancing their disposal. J. Biol. Chem. 283, Bhamidipati, A., Denic, V., Quan, E.M., and Weissman, J.S. (2005). Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen. Mol. 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