Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway

Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery.

primary structure prediction of SEL1L, interspersed among other sequences in the luminal domain and form three SLR domain clusters. Therefore, the way in which these unique structural features of SEL1L are related to its critical function in ERAD remains to be elucidated.
To clearly understand the biochemical role of the SLR domains of SEL1L in ERAD, we determined the crystal structure of the central SLR domain of SEL1L. We found that the central domain of SEL1L, comprising SLR motifs 5 through 9 (SEL1L cent ), forms a tight dimer with two-fold symmetry due to domain swapping of the SLR motif 9. We also found that SLR-C, consisting of SLR motifs 10 and 11, directly interacts with the N-terminus luminal loop of HRD1. Based on these observations, we propose a model wherein the SLR domains of SEL1L contribute to the formation of stable oligomers of the ERAD translocation machinery, which is indispensable for ERAD.

Results
Structure Determination of SEL1L cent . The Mus musculus SEL1L protein contains 790 amino acids and has 17% sequence identity to its yeast homolog, Hrd3p. Mouse SEL1L contains a fibronectin type II domain at the N-terminus, followed by 11 SLR motifs and a single transmembrane domain at the C-terminus (Fig. 1A) 18 . The 11 SLR motifs are located in the ER lumen and account for more than two thirds of the mass of full-length SEL1L. The SLR motifs can be grouped into three regions due to the presence of linker sequences among the groups of SLR motifs: SLR-N (SLR motifs 1 to 4), SLR-M (SLR motifs 5 to 9), and SLR-C (SLR motifs 10 to 11) (Fig. 1A). Sequence alignment of the SLR motifs revealed that there is a short linker sequence (residues 336-345) between SLR-N and SLR-M and a long linker sequence (residues 528-635) between SLR-M and SLR-C (Fig. 1A). We first tried to prepare the full-length mouse SEL1L protein, excluding the transmembrane domain at the C-terminus (residues 735-755), by expression in bacteria. However, the full-length SEL1L protein aggregated in solution and produced no soluble protein. To identify a soluble form of SEL1L, we generated serial truncation constructs of SEL1L based on the predicted SLR motifs and highly conserved regions across several different species. Both SLR-N (residues 194-343) and SLR-C (residues 639-719) alone could be solubilized with an MBP tag at the N-terminus, but appeared to be polydisperse when analyzed by size-exclusion chromatography. However, the central region of SEL1L, comprising residues 337-554, was soluble and homogenous in size, as determined by size-exclusion chromatography. To define compact domain boundaries for the central region of SEL1L, we digested the protein with trypsin and analyzed the proteolysis products by SDS-PAGE and N-terminal sequencing. The results of this preliminary biochemical analysis suggested that SEL1L residues 348-533 (SEL1L cent ) would be amenable to structural analysis (Fig. 1A).
Crystals of SEL1L cent grew in space group P2 1 with four copies of SEL1L cent (a total of 82 kDa) in the asymmetric unit. The structure was determined by the single-wavelength anomalous diffraction (SAD) method using selenium as the anomalous scatterer (Table 1 and Methods). The assignment of residues during model building was aided by the selenium atom positions, and the structure was refined with native data to 2.6 Å resolution with R work /R free values of 20.7/27.7%. Statistics for data collection and refinement are presented in Table 1.
Overall Structure of SEL1L cent . The mouse SEL1L cent crystallized as a homodimer, and there were two homodimers in the crystal asymmetric unit (Fig. 1B,C, Supplementary Fig. 1). The two SEL1L cent molecules dimerize in a head-to-tail manner through a two-fold symmetry interface resulting in a cosmos-like shaped structure (Fig. 1B). The resulting structure resembles the yin-yang symbol with overall dimensions of 60 × 60 × 25 Å, where a SEL1L cent monomer corresponds to half the symbol. The dimer formation buries a surface area of 1670 Å 2 for each monomer, and no significant differences between the protomers were displayed (final root mean square deviation (RMSD) of 0.6 Å for all Cα atoms). Each protomer is composed of ten α -helices, which form the five SLRs, resulting in an elongated curved structure, confirming the primary structure prediction (Fig. 1D).
The α -helices subdivide the structure into five pairs (A and B) as shown in a number of TPRs 19 and SLRs 20,21 . Helices A and B are 14 and 13 residues long, respectively, and the two helices are connected by a short turn and loop (Fig. 1D). In addition, a longer loop, consisting of approximately eight amino acids, is inserted between helix B of one SLR and helix A of the next SLR. This arrangement is a unique feature for SLRs among the major classes of repeats containing an α -solenoid. Starting from its N-terminus, the α -solenoid of SEL1L extends across a semi-circle in a right-handed superhelix fashion along the rotation axis of the yin-yang circle. However, the last helix, 9B, at the C-terminus adopts a different conformation, lying parallel to the long axis of helix 9A instead of forming an antiparallel SLR. This unique conformation of helix 9B most likely contributes to formation of the dimer structure of SEL1L cent , as detailed below. With the exception of the last SLR, the four α -helix pairs possess similar conformations, with RMSD values of 0.7 Å for all Cα atoms. Although the sequence similarity for the pairwise alignments varies between 25% and 35%, all the residues present in the SLR motifs are conserved among the five pairs. The SLR domain of SLR-M ends at residue 524, and C-terminal amino acids 525-533 of the protein are not visible in the electron density map, suggesting that this region is highly flexible.
Since no information regarding dimer formation by SEL1L through its SLR motifs is available, we tested whether the SEL1L cent dimer shown in our crystal structure is a biological unit. First, we cross-linked SEL1L cent or a longer construct of SEL1L (SEL1L long , residues 337-554) using various concentrations of glutaraldehyde (GA) or dimethyl suberimidate (DMS) and analyzed the products by SDS-PAGE. We detected bands at the mass of a dimer for both SEL1L cent and SEL1L long when cross-linked with low concentrations of GA (0.005%) or DMS (0.3 mM) (Supplementary Fig. 2A,B). Next, we conducted analytical ultracentrifugation of SEL1L cent . Consistent with the cross-linking data, analytical ultracentrifugation revealed that the molecular weight of SEL1L cent corresponds to a dimer ( Supplementary Fig. 2C). Taken together, these data indicate that some kind of dimer is formed in solution.
Dimer Interface of SEL1L cent . In contrast to a previously described SLR motif containing proteins that exist as monomers in solution 20,21 , SEL1L cent forms an intimate two-fold homotypic dimer interface (Figs 1B and 2A). The concave surface of each SEL1L domain comprising helix 5A to 9A encircles its dimer counterpart in an interlocking clasp-like arrangement. However, no interactions were seen between the two-fold-related protomers through the concave inner surfaces themselves. Rather, the unique structure of SLR motif 9, consisting of two parallel helices (9A and 9B), is located in the space generated by the concave surface and provides an extensive dimerization interface between the two-fold-related molecules ( Fig. 2A). Helix 9B from one protomer inserts into the empty space surrounded by the concave region in the other monomer, forming a domain-swapped conformation. The diagram shows the domain structure of Mus musculus SEL1L, as defined by proteolytic mapping and sequence/structure analysis. The 11 SLR motifs were divided into three groups (SLR-N, SLR-M, and SLR-C) due to the presence of linker sequences that are not predicted SLR motifs. Putative N-glycosylation sites are indicated by black triangles. We determined the crystal structure of the SLR-M, residues 348-533. (B) Ribbon diagram of the biological unit of the SEL1L cent , viewed along the two-fold NCS axis. The crystal structure was determined by SAD phasing using selenium as the anomalous scatterer and refined to 2.6 Å resolution (Table 1). (C) SEL1L cent ribbon diagram rotated 90° around a horizontal axis relative to (B). (D) One protomer of the SEL1L cent dimer. This view is rotated about 90° anticlockwise from the bottom copy in (B), along the two-fold NCS axis. Starting from the N-terminus, SEL1L cent has five SLR motifs comprising ten α helices. Each SLR motif (from 5 to 9) is indicated in a different color. (E) Evolutionary conservation of surface residues in SEL1L cent , calculated using ConSurf 22 , from a structure-based alignment of 135 SEL1L sequences. The surface is colored from red (high) to white (poor) according to the degree of conservation in the SEL1L phylogenetic orthologs. The ribbon diagram of the counterpart protomer is drawn to show the orientation of the SEL1L cent dimer.
Scientific RepoRts | 6:20261 | DOI: 10.1038/srep20261 Three major contact interfaces are involved in the interactions, and all interfaces are symmetrically related between the dimer subunits ( Fig. 2A). Structure-based sequence alignment of 135 SEL1L phylogenetic sequences using a ConSurf server revealed that the surface residues in the dimer interfaces were highly conserved among the SEL1L orthologs ( Fig. 1E) 22 . First, helix 9B of each SEL1L cent subunit interacts with residues lining the inner groove from the SLR α -helices (5B, 6B, 7B, and 8B) from its counterpart. In this interface, Leu 516 and Tyr 519 on helix 9B are located in the center, making hydrophobic interactions with Trp 478 on helix 8B, Val 444 on helix 7B, Phe 411 on helix 6B, and Leu 380 on helix 5B from the SEL1L cent counterpart ( Fig. 2A, Interface 1 detail). In addition to hydrophobic interactions, the side chain hydroxyl group of Tyr 519 and the main-chain oxygen of Ile 515 form H-bonds to the side chain of the conserved Gln 377 and His 381 on helix 5B of the two-fold-related protomer. The side chain of Gln 523 forms an H-bond to the side chain of Asp 480 on the two-fold-related protomer ( Fig. 2A, Interface 1 detail). Second, the residues from helix 9A interact with the residues from helix 5A of its counterpart in a head-to-tail orientation. In this interface, the interacting residues on helix 9A, including Leu 503, Tyr 499, and the aliphatic side chain of Lys 500, form an extensive network of van der Waals contacts with the hydrophobic residues of the counterpart helix 5A, including Tyr 360, Leu 356, Tyr 359, and Leu 363. In addition to hydrophobic interactions, the side chains of Asn 507 and Ser 510 on helix 9A make H-bonds with highly conserved Arg 384 in the loop between helix 5B and 6A from the two-fold-related protomer ( Fig. 2A, Interface 2 detail). Third, the helix 9B from each protomer is involved in the dimer interaction by forming a two-fold antiparallel symmetry. In particular, the side chains of hydrophobic residues, including Phe 518, Leu 521, and Met 524, are directed toward each other, where they make both inter-and intramolecular contacts ( Fig. 2A, Interface 3 detail).
To further investigate the interactions observed in our crystal structure, we generated a C-terminal deletion mutant (SEL1L 348-497 ) lacking SLR motif 9 (helix 9A and 9B) from SEL1L cent for comparative analysis. The deletion mutant and the wild-type SEL1L cent showed no difference in spectra by CD spectroscopy, indicating that the deletion of the SLR motif 9 did not affect the secondary structure of SEL1L cent ( Supplementary Fig. 3). However, the mutant behaved as a monomer in size-exclusion chromatography and analytical ultracentrifugation experiments   Supplementary Fig. 2C). Additionally, to further validate the key residues involved in dimer formation, we generated a triple point mutant (Interface 1, I515A, L516A, and Y519A) of the hydrophobic residues that are involved in dimerization. The triple point mutant eluted at the monomer position upon size-exclusion chromatography, while the negative control point mutant (Q460A) eluted at the same position as the wild-type. Notably, a single-residue mutation (L521A in interface 3) abolished the dimerization of SEL1L cent (Fig. 2B). Leu 521 is located in the dimerization center of the antiparallel 9B helices in the SEL1L cent dimer. Taken together, these structural and biochemical data demonstrate that SEL1L cent exists as a dimer in solution and that SLR motif 9 in SEL1L cent plays an important role in generating a two-fold dimerization interface. The Two Glycine Residues (G512 and G513) Create a Hinge for Domain Swapping of SLR Motif 9.
SLRs of mouse SEL1L were predicted using the TPRpred server 23 . Based on the prediction, full-length SEL1L contains a total of 11 SLR motifs, and our construct corresponds to SLR motifs 5 through 9. Although amino acid sequences from helix 9A and 9B correctly aligned with the regular SLR repeats and corresponded to SLR motif 9 (Fig. 3A), the structural arrangement of the two helices deviated from the common structure for the SLR motif. According to our crystal structure, the central axis of helix 9B is almost parallel to that of helix 9A (Fig. 3B). However, this unusual conformation of SLR motif 9 seems to be essential for dimer formation, as described earlier. For this structural geometry, two adjacent residues, Gly 512 and Gly 513, in SEL1L confer flexibility at this position by adopting main-chain dihedral angles that are disallowed for non-glycine residues. The phi and psi dihedrals are 100° and 20° for Gly 512, and 110° and − 20° for Gly 513, respectively (Fig. 3C). Gly 513 is conserved among other SLR motifs in the SEL1L cent , but Gly 512 is present only in the SLR motif 9 of SEL1L cent (Fig. 3A). Thus, the Gly-Gly residues generate an unusual sharp bend at the C-terminal SLR motif 9. The involvement of a glycine residue in forming a hinge for domain swapping has been reported previously 24 . The significance of Gly 513 is further highlighted by its absolute conservation among different species, including the budding yeast homolog Hrd3p.
To further investigate the importance of Gly 512 and Gly 513 in the unusual SLR motif geometry, we generated a point mutation (Gly to Ala), which restricts the flexibility. Although the Gly 512 and Gly 513 residues are closely surrounded by helix 9B from the counter protomer, there is enough space for the side chain of alanine, suggesting that no steric hindrance would be caused by the mutation (Fig. 3C). This means that the effect of the mutation is mainly to generate a more restricted geometry at the hinge region. G512A or G513A alone showed no differences from wild-type in terms of the size-exclusion chromatography elution profile (Fig. 3D), suggesting that the restriction for single glycine flexibility would not be enough to break the swapped structure of helix 9B. However, the double mutant (G512A/G513A) eluted over a broad range and much earlier than the wild-type, suggesting that mutation of the residues involved in the hinge linking helix 9A and 9B significantly affected the geometry of helix 9B in generating domain swapping, and eventually altered the overall oligomeric state of SEL1L cent into a polydisperse pattern (Fig. 3D, Supplementary Fig. 6). When the residues were mutated to lysine (G512K/G513K), the mutant not only restricted the geometry of residues at the hinge but also generated steric hindrance during interaction with the counter protomer of SEL1L cent , thereby inhibiting self-association of SEL1L cent completely. The G512K/G513K double mutant eluted at the monomer position in size-exclusion chromatography (Fig. 3D). A previous study shows that induction of steric hindrance by mutation destabilizes the dimerization interface of a different protein, ClC transporter 25 .
Collectively, these data suggest that the Gly 512 and Gly 513 at the connection between helix 9A and 9B play a crucial role in forming the domain-swapped conformation that enables dimer formation.

SEL1L Forms Self-oligomers through SEL1L cent domain in vivo.
Next, we examined if SEL1L also forms self-oligomers in vivo using HEK293T cells. We generated full-length SEL1L-HA and SEL1L-FLAG fusion constructs and co-transfected the constructs into HEK293T cells. A co-immunoprecipitation assay using an anti-FLAG antibody followed by Western blot analysis using an anti-HA antibody showed that full-length SEL1L forms self-oligomers in vivo (Fig. 4A). To further examine whether the SEL1L cent domain is sufficient to physically interact with full-length SEL1L, we generated SEL1L cent and SLR motif 9 deletion (SEL1L 348-497 ) construct, which were fused to the C-terminus of SEL1L signal peptides. Co-immunoprecipitation analysis showed that the SEL1L cent was sufficient to physically interact with the full-length SEL1L, while SEL1L 348-497 failed to do so (Fig. 4A). Interestingly, however, the expression level of SEL1L 348-497 was consistently lower than that of SEL1L cent (Fig. 4A,B). Semi-quantitative RT-PCR revealed no significant difference in transcriptional levels of the two constructs (data not shown). We speculated that SEL1L 348-497 could be secreted while the SEL1L cent is retained in the ER by association with the endogenous ERAD complex. Indeed, immunoprecipitation followed by western blot analysis using the culture medium detected secreted SEL1L 348-497 fragment, but not SEL1L cent (Fig. 4B). We next examined if the reason why SEL1L 348-497 failed to bind to the full-length SEL1L may be because of the lower level of SEL1L 348-497 in the ER lumen compared to SEL1L cent fragment. In order to retain two SEL1L fragments in the ER lumen, we added KDEL ER retention sequence to the C-terminus of both fragments. Indeed, the addition of KDEL peptide increased the level of SEL1L 348-497 in the ER lumen (Fig. 4D,E) and the immunostaining analysis showed both constructs were well localized to the ER (Fig. 4C). We further analyzed whether SEL1L cent may competitively inhibit the self-oligomerization of SEL1L in vivo. To this end, we co-transfected the differentially tagged full-length SEL1L (SEL1L-HA and SEL1L-FLAG) and increasing doses of SEL1L cent -KDEL, SEL1L 348-497 -KDEL or SEL1L cent (L521A)-KDEL, respectively. Co-immunoprecipitation assay revealed that wild-type SEL1L cent -KDEL, indeed, competitively disrupted the self-association of the full-length SEL1L (Fig. 4E). In contrast, SEL1L 348-497 -KDEL and the single-residue mutation L521A in SEL1L cent did not competitively inhibit the self-association of full-length SEL1L (Fig. 4E,F). These data suggest that the SEL1L forms self-oligomers and the oligomerization is mediated by the SEL1L cent domain in vivo.

Structural Comparison of SEL1L SLRs with TPRs or SLRs of Other Proteins. Previous studies
reveal that TPRs and SLRs have similar consensus sequences, suggesting that their three-dimensional structures are also similar 18 . The superposition of isolated TPRs from Cdc23 (S. pombe, cell division cycle 23 homolog, PDB code 3ZN3) and SLRs from HcpC (Helicobacter Cysteine-rich Protein C, PDB code 1OUV) yields RMSDs below 1 Å, confirming that the isolated repeats are indeed similar 20 . This is relevant to SLR motifs in SEL1L, as isolated SLR motifs from SEL1L cent showed good structural alignment with isolated TPRs (RMSD 1.6 Å for all Cα chains) from Cdc23 N-term and SLRs (RMSD 0.6 Å for all Cα chains) from HcpC (Fig. 5A). However, superimposing the structure of SLR motifs 5 to 9 from SEL1L cent onto the overall Cdc23 N-term or full-length HcpC structures revealed that SLR motifs 5 to 9 in SEL1L cent have a different superhelical structure than either Cdc23 or HcpC (RMSD The 11 SLR motifs were aligned based on the present crystal structure of SEL1L cent . The sequences of SEL1L cent included in the crystal structure are highlighted by the blue box. The secondary structure elements are indicated above the sequences, with helices depicted as cylinders. Residues that are conserved in at least 7 out of 11 sequences are red. The GG sequence in SLR motif 9, which creates the hinge for domain swapping (see text), is shaded yellow. Stars below the sequences indicate the specific residues that commonly appear in SLRs. (B) Structure alignment of five SLR motifs in SEL1L cent is shown to highlight the unusual geometry of SLR motif 9. Each SLR motif is shown in a different color. The arrow indicates the direction of the helical axes. In SLR motif 9, the axes for the two helices are almost parallel, while the other SLR motifs adopt an α -hairpin structure.
(C) Stereo view shows that the Gly 512 and Gly 513 residues are surrounded by neighboring residues from helix 9B from the counterpart dimer. Oxygen and nitrogen atoms are colored red and blue, respectively. The Gly 512 and Gly 513 residues are colored green and red, respectively. (D) The following point mutations were generated to check the effect of the Gly 512 and Gly 513 residues in terms of generating the hinge of SLR motif 9: G512A, G513A, G512A/G513A, and G512K/G513K. Size-exclusion chromatography was conducted as described in Fig. 2B. The standard molecular masses are shown at the top as in Fig. 2B. HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. The full-length SEL1L-FLAG was co-immunoprecipitated with the full-length SEL1L-HA. Also, SEL1L cent was co-immunoprecipitated with the full-length SEL1L while the SLR motif 9 deletion failed to do so. (B) The HEK293T cells were transfected with the indicated plasmid constructs and the cell lysate and culture media were analyzed by western blot analysis and immunoprecipitation respectively. The SEL1L 348-497 fragment was secreted to the culture media but the SEL1L cent was retained in the ER. (C) SEL1L cent -FLAG-KDEL and SEL1L 348-497 -FLAG-KDEL localized to the ER. The nuclei were stained with DAPI in blue. The ER was visualized with the anti-calnexin antibody in green. The SEL1L fragments were stained in red. (D) HEK293T cells were transfected with the indicated plasmid constructs and the lysates were immunoprecipitated with an anti-HA antibody followed by Western blot analysis using an anti-FLAG antibody. The full-length SEL1L forms self-oligomers and the SEL1L cent -FLAG-KDEL was co-immunoprecipitated with full-length SEL1L-HA. The red asterisk indicates the expected signal for SEL1L 348-497 -FLAG-KDEL. SEL1L 348-497 -FLAG-KDEL did not co-immunoprecipitate with fulllength SEL1L-HA. The white asterisks indicate non-specific bands. (E) SEL1L cent -HA-KDEL competitively inhibited self-oligomerization of full-length SEL1L. The indicated plasmid constructs were transfected and immunoprecipitation assay was performed using an anti-FLAG antibody followed by western blot analysis using an anti-HA antibody. The red rectangle indicates competitively inhibited SEL1L self-oligomer formation by the increasing doses of SEL1L cent -HA-KDEL. (F) L521A point mutant in SEL1L cent did not inhibit the selfassociation of SEL1L. values of > 2.5 Å for Cα atoms) (Fig. 5B). The differences may result from the differing numbers of residues in the loops and differences in antiparallel helix packing. Moreover, there are conserved disulfide bonds in the SLR motifs of HcpC and HcpB, but no such bonds are observed in SEL1L cent . These factors contribute to the differences in the overall conformation of the SLR motifs in SEL1L and other SLR or TPR motif-containing proteins.
Another major difference in the structure of SLR motifs between SEL1L and HcpC is the oligomeric state of proteins. The TPR motif is involved in the dimerization of proteins such as Cdc23, Cdc16, and Cdc27 26 . In particular, the N-terminal domain of Cdc23 (Cdc23 N-term ) has a TPR-motif organization similar to that of the SLR motif in SEL1L cent . The seven TPR motifs of Cdc23 N-term are assembled into a superhelical structure, generating a hollow surface and encircling its dimer counterpart in an interlocking clasp-like arrangement (Fig. 5C) 26 . The TPR motif 1 (TPR1) of each Cdc23 N-term subunit is located in the hollow surface of the counter subunit and interacts with residues lining the inner groove TPR α -helices, generating two-fold symmetry homotype interactions. However, in this structure, a conformational change in the TPR motif itself is not observed.
Self-association of HcpC has not been reported, and there is no domain-swapped structure in the SLR motifs of HcpC, in contrast to that observed in SEL1L cent . Although SEL1L contains a number of SLR motifs comparable to HcpC, the SLR motifs in SEL1L are interrupted by other sequences, making three SLR motif clusters (Fig. 1A). The interrupted SLR motifs may be required for dimerization of SEL1L cent , as five SLR motifs are more than enough to form the semicircle of the yin-yang symbol (Fig. 1B). Helix 5A from SLR motif 5 meets helix 9A from SLR motif 9 of the counterpart SEL1L. If the SLR motifs 5 to 9 were not isolated from other SLR motifs, steric hindrance could interfere with dimerization of SEL1L. This is one of the biggest differences from TPRs in Cdc23 and from the SLRs in HcpC, where the motifs exist in tandem. TPR and SLR motifs are generally involved in protein-protein interaction modules, and the sequences between the SLR motifs of SEL1L might actually facilitate the self-association of this protein.

SLR-C of SEL1L Binds HRD1 N-terminus Luminal Loop.
Based on the structural data presented herein, a possible arrangement of membrane-associated ERAD components in mammals, highlighting the molecular functions of SLR domains in SEL1L, is shown in Fig. 6C. We suggest that the middle SLR domains are involved in the dimerization of SEL1L based on the crystal structure and biochemical data. SLR-C, which contains SLR motifs 10 to 11, might be involved in the interaction with HRD1. Indirect evidence from a previous yeast study shows that the circumscribed region of C-terminal Hrd3p, specifically residues 664-695, forms contacts with the Hrd1 luminal loops 12 . The Hrd3p residues 664-695 correspond to mouse SEL1L residues 696-727, which include the entire helix 11B (residue 697-709) of SLR motif 11 and a well-conserved adjacent region ( Supplementary Fig. 4). This observation is supported by the following: (1) the meticulous range of SLR motif 10 to 11 is newly established from a structure-guided SLR motif alignment, based on the present structure study, and (2) the relatively high sequence conservation between mammalian SEL1L and yeast Hrd3p around SLR motifs 10 to 11, which contain contact regions with HRD1 (Hrd1p) (Supplementary Figs. 4 and 5). To address this hypothesis, we prepared constructs encoding mouse HRD1 luminal fragments fused to GST as shown in Fig. 6A, and tested their ability to bind certain SLR motifs in SEL1L. The fusion proteins were immobilized on glutathione-Sepharose beads and probed for binding to SLR-N, SLR-M, SLR-C, and monomer form of SLR-M (SLR-M L521A ). Figure 6B shows that the SLR-C, consisting of SLR motifs 10 and 11, exclusively interacts with N-terminal luminal loop (residues 21-42) of HRD1.
The molecular functions of SLR-N are unclear. One possibility is that SLR-N contributes to substrate recognition of proteins to be degraded because there are a couple of putative glycosylation sites within the SLR-N domain (Fig. 1A). SEL1L cent contains a putative N-glycosylation site, Asn 427, which is highly conserved among different species and structurally exposed to the surface of the SEL1L dimer according to the crystal structure (Fig. 6C).

Discussion
Many reports demonstrate that membrane-bound ERAD machinery proteins in yeast, such as Hrd1p, Der1p, and Usa1p, are involved in oligomerization of ERAD components [27][28][29] . The Hrd1p complex forms dimers upon sucrose gradient sedimentation 5,30 and size-exclusion chromatography 30 . Previous data show that HA-epitope-tagged Hrd3p or Hrd1p efficiently co-precipitate with unmodified Hrd3p and Hrd1p, respectively, suggesting that both Hrd1p and Hrd3p homodimers are involved in self-association of the Hrd complex. Considering that the functional and structural composition of ERAD components are conserved in both yeast and mammals, we propose that the mammalian ERAD components also form self-associating oligomers. This hypothesis is supported by cross-linking data suggesting that human HRD1 forms a homodimer 31 . Consistent with the previous data, our crystal structure and biochemical data demonstrate that mouse SEL1L cent exists as a homodimer in the ER lumen via domain swapping of SLR motif 9. We need to further test whether there are contacts involved in dimer formation in SEL1L in addition to those in the SLR-M region.
In yeast, Usa1p acts as a scaffold for Hrd1p and Der1p, in which the N-terminus of Usa1p interacts with the C-terminal 34 amino acids of Hrd1p in the cytosol to induce oligomerization of Hrd1p, which is essential for its activity 30,32 . However, metazoans lack a clear Usa1p homolog. Although mammalian HERP has sequences and domains that are conserved in Usa1p, the molecular function of HERP is not clearly related to that of Usa1p 4,31 . Rather, recent research shows that a transiently expressed HRD1-SEL1L complex alone associates with the ERAD lectins OS9 or XTP-B and is sufficient to facilitate the retrotranslocation and degradation of the model ERAD substrate α -antitrypsin null Hong-Kong (NHK) and its variant, NHK-QQQ, which lacks the N-glycosylation sites 33 . Assuming that the correct oligomerization of ERAD components may be critical for their function, we hypothesize that homodimer formation of SEL1L in the ER lumen may stabilize oligomerization of the HRD complex, given that SEL1L forms a stoichiometric complex with HRD1 10,13,15,30 . This is further supported by our data showing that the SLR-C of SEL1L directly interacts with the luminal fragment of HRD1 in the ER lumen.
Although the organization of membrane-bound HRD complex components may be very similar between metazoans and yeast, the molecular details of interactions between the components may not necessarily be conserved. In yeast, it is unclear whether self-association of Hrd3p is due to SLR motifs because the sequence of Hrd3p does not align precisely with the SLR motifs in SEL1L 18 . Furthermore, we are uncertain whether self-association of Hrd3p contributes to formation of the active form of the Hrd1p complex. Recently, a truncated version of Yos9 was shown to form a dimer in the ER lumen and to contribute to the dimeric state of the Hrd1p complex 34 . This interaction seems to be weak because direct Yos9-Yos9 interactions were not detected in immunoprecipitation experiments from yeast cell extracts containing different epitope-tagged variants of Yos9.
However, the dimerization of Yos9 could provide a higher stability for the Hrd1p complex oligomer. Likewise, the dimerization of SEL1L might provide stability for the mammalian HRD oligomer complex. Further cell biological studies are required to clarify whether SEL1L (Hrd3p) dimerization could be cooperative with the oligomerization of the HRD complex. in the ER membrane. The 11 SLR motifs of SEL1L were expressed with red cylinders and grouped into three parts (SLR-N, SLR-M, and SLR-C) based on the sequence alignment across the motifs and the crystal structure presented herein. We hypothesized that the interrupted SLR motifs of SEL1L have distinct functions such that the SLR-M is important for dimer formation of the protein, and SLR-C is involved in the interaction with HRD1 in the ER lumen. The surface representation of SEL1L cent is placed in the same orientation as that shown in the schematic model to show that the putative N-glycosylation site, residue N427 (indicated in yellow), is exposed on the surface of the protein. The yellow arrow indicates self-association among the respective components.
Considering that it is very important for the function of the HRD complex that the components assemble as oligomers, we believe that the self-association of SEL1L strongly contributes to generating active forms of the HRD complex, even in the absence of Usa1p, in metazoans. These findings should provide a foundation for molecular-level studies to understand the membrane-associated HRD complex assembly in ERAD.

Methods
Protein Production. The expression and purification of SEL1L was performed as described previously 35 . Crystallization and SAD Structure Determination of SEL1L cent . Crystals were grown using the hanging-drop vapor diffusion method at 4 °C. For crystallization of the M. musculus SEL1L cent , 1 μ l of protein solution (in 25 mM Tris-HCl, 150 mM NaCl, and 5 mM DTT, pH 7.5) was equilibrated with 1 μ l of well solution (30% isopropanol, 100 mM NaCl, 100 mM Tris, 5 mM DTT, and 20 mM phenol, pH 8.5). The crystals, which appeared after 4 days, contain two SEL1L cent dimers in the asymmetric unit (space group P2 1 , a = 29.13, b = 110.52, c = 109.81 Å, α = 90.00, β = 90.61, γ = 90.00, 44% solvent). For X-ray diffraction experiments, crystals were transferred to well solution plus paraffin-oil, then flash frozen in liquid nitrogen.
SAD data were collected with a Se-Met crystal at beamline 7A of the Pohang Accelerator Laboratory (PAL) and processed using HKL2000 software 36 . Native data (2.6 Å resolution) were collected from a single frozen crystal at the same beamline of PAL and were integrated and scaled as described above. The SAD data analysis was performed using Phenix software 37 using data between 50 and 2.9 Å resolution. Phenix identified 31 of the 32 selenium sites and refined these to give a mean f.o.m. = 0.472. Electron density modification, including non-crystallographic symmetry (NCS) averaging, using the RESOLVE software 38 yielded an initial electron density map of excellent quality. Model building and refinement were carried out with the Coot 39 and Phenix programs, respectively. The final model was refined to an R factor of 20.7% (R free = 27.7%) for native data between 30 and 2.6 Å resolution ( Table 1). The final model consisted of 5402 protein atoms and 47 water molecules. There were no outliers in a Ramachandran plot of the final model. The model contained four copies of SEL1L cent (residues 348-533) in the asymmetric unit. Of these, the following residues were not modeled due to weak electron densities: SEL1L cent residues 348-351, 420, 421, and 525-533 in the first copy; residues 348-351 and 525-533 in the second and third copies; and residues 348-352 and 525-533 in the fourth copy. The X-ray data and refinement statistics are summarized in Table 1.
Cell Culture and Plasmids Construction. HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS. The mouse Sel1L gene was cloned into pCS108 and the 3 × HA or 3 × FLAG tag was fused to the C-terminus of SEL1L. The signal peptide from Xenopus Sel1L was cloned into pCS108 and the mouse SEL1L cent domain, SEL1L (348-497) fragments, and SEL1L cent (L521A) were fused to the C-terminus of the signal peptide. Then, a 3 × HA or a 3 × FLAG tag was fused to the C-terminus of the constructs. For the ER retention signal, the KDEL sequences were added to the C-terminus of the fragments. The plasmids were transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer's manual.
Western Blot Analysis and Immunostaining. For western blot analysis, HEK293T cells were transfected with the indicated construct and harvested after washing in PBS. The cells were homogenized in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 5% glycerol), supplemented with protease and phosphatase inhibitor cocktails. Homogenates were cleared by centrifugation at 13,200 rpm for 15 minutes at 4 °C. The lysates were subsequently used for either co-immunoprecipitation experiment or western blot analysis. For the western blot analysis, the samples were run onto 6-12% polyacrylamide gel. Blots were blocked in 5% TBS + 0.05% Tween 20 and incubated with anti-DDDD-K (Abcam) or anti-HA (Roche) antibodies. Proteins were visualized using HRP-conjugated secondary antibodies (1:4000) and SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo) and exposed to ChemiDoc MP (Bio-Rad).
For immunostaining, the cells were fixed in 4% formaldehyde and incubated with the indicated antibodies. The coverslips were incubated in blocking solution (10% FBS + 2% DMSO in TBS + 0.1% Triton X-100) at room temperature for 30 minutes to block non-specific binding. Fluorescent labeling was performed using Alexa Fluor 555 or 488-conjugated secondary antibodies and nuclei were stained with DAPI. The samples were mounted and confocal images were obtained using a Zeiss LSM700.
GST Pull-down Assay. For pull-down experiments, 400 μ g of HRD1 luminal fragment GST-fusion proteins were incubated with 5 μ l of a 50% (v/v) slurry of glutathione sepharose 4B beads (GE Healthcare) for 50 min at 4 °C. Beads were washed twice with buffer A (150 mM NaCl, 25 mM sodium phosphate pH 7.5, 5 mM DTT), and then mixed with 100 μ g of MBP-SEL1L protein (SLR-N, SLR-M, SLR-C, and SLR-M L521A ) in buffer A, in a total assay volume of 500 μ l. The assay mix was incubated at 4 °C for 15 minutes, and beads were washed twice with 500 μ l buffer A. Proteins were eluted with SDS sample buffer, and analyzed by SDS-PAGE.