Structural Basis for Ovarian Tumor Domain-containing Protein 1 (OTU1) Binding to p97/Valosin-containing Protein (VCP)*

Background: Ovarian tumor domain-containing protein 1 (OTU1) acts as a deubiquitinating enzyme in the endoplasmic reticulum-associated degradation (ERAD) pathway by associating with valosin-containing protein (VCP). Results: One OTU1 binds to a VCP hexamer with a KD of 0.71 μm. Conclusion: The 39GYPP42 (S3/S4) loop of OTU1 is critical for interaction with VCP. Significance: This is the first structural study of VCP and OTU1 complex. Valosin-containing protein (VCP), also known as p97, is an AAA+ ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXLOTU1) complexed to the N-terminal domain of VCP (NVCP) at 1.8-Å resolution reveals that UBXLOTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of NVCP using the 39GYPP42 loop of UBXLOTU1 with the two prolines in cis- and trans-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a KD of 0.71 μm. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.

dependent on a multitude of regulatory binding partners of VCP that interact with the N-terminal domain and/or the C-terminal tail of VCP; these binding partners can be classified as either substrate-recruiting or substrate-processing proteins (10). Defects in VCP or its binding partners have been linked to several diseases (8,9).
Recently, a number of ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP (11,12). Among these enzymes is ovarian tumor domaincontaining protein 1 (OTU1). OTU1 is known as a deubiquitinating enzyme as well as a substrate-processing factor of VCP that is required for the retrotranslocation of the ERAD pathway (13). It also plays a role in controlling ubiquitinated or unubiquitinated substrates in the ERAD pathway (14). However, the exact mechanisms are not yet well understood. It harbors three domains: an N-terminal ubiquitin regulatory X-like (UBXL) domain, an OTU domain, and a C-terminal zinc finger domain of unknown function. It binds to the N-terminal domain of VCP (N VCP ) through the UBXL domain and removes ubiquitin chains using the OTU domain (15). In particular, the UBXL domain of OTU1 (UBXL OTU1 ) consists of about 80 residues similar to the ubiquitin regulatory X (UBX) domain, which is one of the best characterized adaptor domains of VCP. UBX interacts with N VCP via a conserved arginine from strand ␤1 and the three residues (Phe-Pro-Arg) in a four-residue-long loop formed between strands ␤3 and ␤4 often referred to as the S3/S4 loop (16,17). These residues, "R . . . FPR," form the signature motif of UBX. However, UBXL has low sequence identity of less than 15% to UBX and furthermore lacks the signature motif of UBX. So far, the structure of the OTU domain from yeast complexed with ubiquitin has been produced (18), and recently it has been demonstrated that YOD1, the mammalian homolog of OTU1, has high specificity for Lys 11 -linked ubiquitin chains in a report that shows the complex structure of YOD1 with Lys 11 -linked diubiquitin (19). To understand how OTU1 interacts with VCP on a molecular level and to gain insight into how this interaction regulates the ERAD pathway, we carried out a structural study on N VCP complexed with UBXL OTU1 and performed negative staining electron microscopy (EM) and biochemical studies of the full-length proteins.

EXPERIMENTAL PROCEDURES
Cloning, Mutagenesis, and Protein Preparation-All human VCP and yeast OTU1 proteins were cloned into the expression vector pET28a (Novagen) that appends an N-terminal histidine tag and thrombin site to the target protein under T7 promoter control. Substitution of Gly-Tyr-Pro-Pro to Thr-Phe-Pro-Arg at positions 39 -42 of the full-length OTU1 (residues 1-301) was introduced by a PCR method in clone OTU1 39 TFPR 42 . For expression in mammalian cells, the constructs encoding pCS2-HA-OTU1 WT and pCS2-HA-OTU1 39 TFPR 42 were prepared. A ribophorin I mutant (RPN-I N299T ) with the asparagine at position 299 replaced by threonine was cloned into the pEGFP-C1 vector (Clontech). For analytical size exclusion chromatography, the full-length UFD1 (residues 1-307) and NPL4 (residues 1-608) were cloned into the pET28a and pET22b vectors (Novagen), respectively, which are named pET28a-UFD1 and pET22b-NPL4. The glutathione S-transfer-ase (GST) protein was fused to the N terminus of the full-length OTU1, and pGST-OTU1 was derived from pGEX-4T-1 (GE Healthcare). All proteins were expressed in Escherichia coli Rosetta (DE3). Cells were grown in LB medium at 37°C, and protein expression was induced by 0.5 mM isopropyl ␤-D-1thiogalactopyranoside at 18°C overnight. Harvested cells were lysed in buffer A (20 mM Tris hydrochloride, pH 8.0, 100 mM sodium chloride, 0.5 mM tris(2-carboxyethyl)phosphine with 1 mM PMSF) and purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (GE Healthcare). Afterward, the histidine tag was proteolytically cleaved by addition of thrombin (Enzyme Research Laboratories), and protein was further purified using size exclusion chromatography on a HiLoad Superdex 75 or 200 26/60 column (GE Healthcare) in buffer A or buffer B (20 mM HEPES, pH 7.5, 150 mM sodium chloride). 2 mM ATP and 5 mM magnesium chloride were added to the full-length VCP (residues 1-806). A UFD1/NPL4 heterodimeric complex was formed by mixing pET28a-UFD1 and pET22b-NPL4. For the formation of complexes using the fulllength proteins, VCP and either OTU1 (VCP/OTU1) or UFD1/ NPL4 (VCP⅐UFD1/NPL4) were incubated at a molar ratio of 1:3 for 1 h at 4°C, and then VCP/OTU1 and VCP⅐UFD1/NPL4 complexes were analyzed by size exclusion chromatography.
Limited Proteolysis of the Complex of N VCP and UBXL OTU1 -Purified complexes of N VCP and UBXL OTU1 were digested with chymotrypsin and trypsin (Sigma) at a protein-to-protease ratio of 1,000:1 in buffer A for 15 and 5 min, respectively, at 37°C. The reactions were stopped by addition of 0.1 mM PMSF. Reaction products were resolved by 15% SDS-PAGE and visualized by subsequent Coomassie Brilliant Blue staining. The proteins were used at a concentration of 20 mg/ml for crystallization.
Crystallization of the VCP and OTU1 Complex-Crystals of crystal form I were obtained from a reservoir solution containing 14% PEG 4000, 100 mM sodium acetate, pH 5.0, and those of crystal form II were obtained from 20% PEG 2000, 10% tacsimate, pH 5.0 using the hanging drop vapor diffusion method by mixing with an equal volume of the protein solution. Crystals in a soaking solution containing 25% ethylene glycol as a cryoprotectant were flash cooled in liquid nitrogen.
Data Collection and Structure Determination-Diffraction data of the two crystal forms were collected at 100 K on beamline 5C of Pohang Light Source, Republic of Korea. The data were processed and scaled using the program suite HKL2000 (20). Crystal form I in the asymmetric unit was located by molecular replacement using the program Phaser (21) with the free VCP structure (Ref. 22; Protein Data Bank code 1E32) as a search model. The structure of crystal form II was subsequently determined using the refined structure of crystal form I as an input model. Iterative model building was performed by manually fitting into the unoccupied densities with Coot (23), and refinement was carried out by CNS (24), PHENIX (25), and REFMAC5 (26). Water molecules were added with Coot, and structures of the two crystal forms were validated using PROCHECK (27). Coordinates of the crystal structures have been deposited in the Protein Data Bank under codes 4KDI and 4KDL. All figures were generated with PyMOL (28). The data collection and processing statistics for the two structures are summarized in Table 1.
Chemical Cross-linking-VCP⅐UFD1/NPL4 complex was purified and concentrated to 0.05 mg/ml in buffer B. To improve its stability, the complex was chemically cross-linked with a final concentration of 0.1% EM grade glutaraldehyde (Sigma) for 10 min at 25°C. The reaction was stopped by adding Tris, pH 8.0 to a final concentration of 25 mM.
Isothermal Titration Calorimetry (ITC) Measurements-ITC experiments were performed on an iTC200 calorimeter (GE Healthcare) at 25°C. For the interaction between the fulllength VCP and OTU1, OTU1 wild type (WT) or the mutant ( 39 TFPR 42 ) was placed in the syringe at 300 or 500 M, respectively, and injected into the sample cell containing the fulllength VCP at a concentration of 13 M. The concentration of VCP/OTU1 and VCP⅐UFD1/NPL4 complexes was 0.01 M in the sample cell, and UFD1/NPL4 and OTU1 were titrated to a concentration of 20 M. Buffer B always matched between the syringe and the sample cell. The data were analyzed with the software Origin 7.0 using a one-site model.
In Vitro Assays of Substrate Degradation-HEK293T cells were seeded onto 12-well poly-L-lysine-coated plates at a density of 2.5 ϫ 10 5 cells/well, and then transfection with expression plasmids was performed for 2 days using Effectene reagent (Qiagen). The total amount of DNA used in each well for transfection was 600 ng. Cells were treated with 10 M MG132 for 2 h to prevent protein degradation and washed twice with complete medium. After addition of cycloheximide at a final concentration of 100 g/ml, cells were harvested with cold Dulbecco's phosphate-buffered saline (PBS) and lysed, and cell extracts were analyzed by Western blotting with antibodies against pCS2-HA, pCS2-HA-OTU1 WT, pCS2-HA-OTU1 39 TFPR 42 , and pEGFP-C1-RPN-I N299T .
Ni-NTA Nanogold Labeling of VCP and OTU1 Complex-The complex was prepared by co-purifying the full-length untagged VCP and OTU1 tagged with a hexahistidine tag at the C terminus. Freshly purified protein complex and 5-nm-diameter Ni-NTA Nanogold (Nanoprobes Inc.) were incubated together at a molar ratio of 1:10 for 10 min at 4°C. The mixture was purified with a further chromatographic step using a Superdex 200 10/300 GL column (GE Healthcare) in buffer A. Excess Ni-NTA Nanogold was removed by centrifugation at 3,000 ϫ g for 15 min using a 10-kDa-cutoff filter (Sartorius).
EM-The complex of the full-length VCP and OTU1 (VCP/ OTU1) labeled with gold was diluted to 20 g/ml in buffer A. The samples were loaded onto a glow-discharged copper grid coated with a carbon film and stained with 2% uranyl acetate solution. Images were recorded on the film using a Tecnai F20 microscope operated at 200 kV equipped with a 2,048 ϫ 2,048pixel Gatan charge-coupled device camera. The gold-labeled proteins were selected from individual digital micrographs using a semiautomatic selection procedure, and the stoichiometry of OTU1 incorporation into VCP was determined by counting the number of gold particles. For unlabeled VCP/ OTU1 complex, VCP/OTU1 complex was formed by incubating purified VCP with a 10-fold molar excess of OTU1 for 15 min at 4°C. The complex was then further purified using size exclusion chromatography (GE Healthcare) in buffer contain-ing 20 mM Tris hydrochloride, pH 8.0, 200 mM sodium chloride. 3 l of VCP/OTU1 complex fraction at 0.1 M was applied to a glow-discharged carbon-coated copper grid, and negatively stained with 2% methylamine vanadate, pH 8.0 (Nanoprobes Inc.). The specimen was examined under a Tecnai T120 microscope at 120 kV using a nominal magnification of 67,000ϫ (1.64 Å/pixel; FEI Eindhoven, The Netherlands). Images were recorded with an FEI Eagle 4,000 ϫ 4,000-pixel charge-coupled device camera using a defocus of 1.2 m and an electron dose of about 30 e Ϫ /A 2 .
EM Image Processing-Because VCP has a preferred orientation on the EM grid, we could only obtain the hexagonal view of VCP or VCP/OTU1 complex. Hexagonal particles were automatically selected with EMAN2 boxer, and bad particles were manually excluded (29). A total of 2,839 particles were selected from 50 micrographs. Subsequent processing was performed using IMAGIC (30). To separate VCP/OTU1 complex from the heterogeneous data set containing either VCP or VCP/OTU1 complex, we executed comparative projection matching with two reference sets. One reference set was the forward projection of the crystal structure of VCP hexamer (Ref. 31; Protein Data Bank code 1R7R), and the other reference set was the forward projection of the model structure of VCP hexamer with one OTU1 molecule (Protein Data Bank codes 4KDI and 4KDL in the present work and 1R7R). These two separate data sets were respectively subjected to 10 rounds of reference-free alignment and multivariate statistical analysis followed by multivariate statistical analysis classification and averaging.
Analytical Size Exclusion Chromatography-pET28a-VCP, pGST-OTU1, pET28a-UFD1, and pET22b-NPL4 were expressed and purified separately. Analytical size exclusion chromatography was carried out at 4°C on a Superdex 200 10/300 GL column (GE Healthcare) in buffer A at a flow rate of 0.5 ml/min. Elution fractions were analyzed for complexes by SDS-PAGE and Coomassie Brilliant Blue staining.

RESULTS AND DISCUSSION
Overall Structure of N VCP and UBXL OTU1 Complex-To obtain atomic details on the interaction between VCP and OTU1, a number of complexes of VCP and OTU1 using various fragments containing the full -length were subjected to crystallization trials (see Fig. 1A). However, only the complex of N VCP and UBXL OTU1 yielded diffraction quality crystals in two conditions: one with residues 21-196 and the other with residues 1-187 of N VCP , referred to hereafter as crystal form I and crystal form II, respectively. Both crystal forms I and II crystallized in space group P2 1 2 1 2 1 but with Z ϭ 1 and Z ϭ 2, respectively, and were refined at 1.81-and 1.86-Å resolution, respectively. First, the molecular replacement method using various structures such as UBX of Fas-associated factor 1 (FAF1) (Protein Data Bank code 3QX1; denoted as UBX FAF1 hereafter) and the NMR structure of UBXL OTU1 (Protein Data Bank code 2KZR) failed to give a solution. However, repeating the search using uncomplexed N VCP (residues 21-187) (Ref. 22; Protein Data Bank code 1E32) as a search model gave a solution, and the subsequent F o Ϫ F c difference electron density map revealed unambiguous density for bound OTU1. The structure of crystal form II was determined by the molecular replacement method using the structure of crystal form I. Residues that were not visible in the electron density map were left out in the final model; they include residues 1-20 of N VCP in all three molecules, 121-128 of N VCP in one molecule from crystal form II, 48 -52 of UBXL OTU1 in crystal form I, and 49 -55 in one molecule of UBXL OTU1 from crystal form II. The statistics on data collection and the refinements are given in Table 1.
The overall structure of the N VCP and UBXL OTU1 complex is shown in Fig. 1B. As seen in the figure, N VCP is composed of two subdomains (N1 and N2) connected by a seven-residue-long linker, and UBXL OTU1 binds at the interface between the N1 and N2 domains of VCP. The N1 domain (residues 23-104) consists of a double-⌿ ␤-barrel, and the N2 domain (residues 112-196) consists of a six-stranded ␤-clam with two helices on the two ends as described earlier (7). The structure of UBXL OTU1 (residues 1-73) reveals a ␤-grasp fold with a ␤-␤-␣-␤-␤-␣-␣-␤ secondary structural organization that resembles ubiquitin and ubiquitin-like proteins as well as UBX and the ubiquitin D domain (UBD) (18,32). However, it differs significantly, especially in the conformation of the loops and the electrostatic surface, and these differences will be discussed later.
The three crystallographically independent molecules, one from crystal form I and two from crystal form II, are almost identical. When the molecule of crystal form I is taken as a reference, the two molecules in crystal form II show root mean square deviation (r.m.s.d.) values of 0.63 and 0.64 Å for N VCP and 0. 45    C␣ atoms. Although these proteins, namely UBXL OTU1 , UBX FAF1 , and UBD NPL4 , share structural homology at the C␣ level, the electrostatic surfaces are quite different (Fig. 2A). The electrostatic surface of UBXL OTU1 is particularly distinct: one side shows a positively charged patch, whereas the opposite side shows a broad negatively charged patch. The positively charged patch is formed by Lys 2 , Lys 4 , Arg 37 , Arg 44 , and Lys 69 from the various strands of the ␤-sheet, whereas the negatively charged patch is formed by residues such as Asp 24 , Glu 27 , Asp 60 , and Glu 61 from ␣1 and 1 of UBXL OTU1 (data not shown).
Despite the fact that the overall structures are similar, a detailed comparison reveals differences in four particular regions, and these are highlighted in Fig. 2, A and C. The four regions are well defined in the electron density maps and are almost the same in all three structures of crystal forms I and II. First, the ␤1-␤2 loop, highlighted the region 1 of Fig. 2A, is tilted more toward the body of the molecule by about 20°in UBXL OTU1 . Second, the ␣1 helix of UBXL OTU1 is shorter than The S3/S4 loop of UBXL OTU1 and UBX FAF1 and 3 10 -helix of UBD NPL4 are circled, and the binding region for N VCP is marked by a green line. Four regions (1-4) with significant differences are indicated by circles. B, the electron density map (2F o Ϫ F c ) of crystal form I is shown at the 1.5 level. C, structure-based sequence alignment of UBXL OTU1 , UBX FAF1 , and UBD NPL4 with the secondary structural elements of UBXL OTU1 shown above the sequence, and ␣, ␤, , and TT denote ␣-helix, ␤-strand, 3 10 -helix, and ␤-turn, respectively. Well conserved residues are boxed, and the predominant residues shown in red. Residues of the signature motif of UBX are marked by green inverted triangles. The four regions in A are highlighted by boxes. Residues involved in N VCP binding are indicated by a colored circle below the sequence. The color code is the same as in A. The alignment was generated by STRAP (6) and ESPript (49). that of UBX FAF1 and UBD NPL4 by nearly one turn. This is due to deletion of four residues in UBXL OTU1 . In addition, UBXL OTU1 has an additional ␣-helix formed by residues 53-56 with an insertion of five residues after ␤4 (region 4), and this region extends outward from the body of the molecule. In fact, it is about 11 Å away from the corresponding loop position of UBX FAF1 .
The most intriguing difference is the ␤3-␤4 loop conformation of UBXL OTU1 (region 3 in Fig. 2A). This loop corresponds to the S3/S4 loop, which is part of the signature motif of UBX. This loop is composed of Gly 39 -Tyr 40 -Pro 41 -Pro 42 , and all the residues are well defined in the electron density map as shown in Fig. 2B. The two prolines in this loop are found in the cis-and trans-configurations, respectively. The loop has a type VIb ␤-turn and is stabilized by intramolecular hydrogen bonds formed between Phe 38 and Gln 43 (N-O, 3.07 Å; O-N, 2.83 Å) as well asstacking interactions between proline and the preceding tyrosine. This is quite similar to that of UBX FAF1 ; i.e. the corresponding Thr-Phe-Pro-Arg forms a VIb ␤-turn, and the proline adopts a cis-configuration. However, the proline of UBX FAF1 does not form similarstacking interactions with the preceding phenylalanine, and the distances between the first and the fourth C␣ atoms in the loop are 4.96 and 3.80 Å in UBXL OTU1 and UBX FAF1 , respectively, resulting in different conformations (Fig. 2B). This may be due to the additional proline in UBXL OTU1 . Conversely, the proline in the S3/S4 loop of UBX p47 was reported to be in trans-configuration (36). The corresponding region in UBD NPL4 is longer by six residues and forms a 3 10 -helix instead of the loop as shown in Fig. 2B. Taken together, the structure of UBXL OTU1 exhibits distinctive features from UBX FAF1 or UBD NPL4 , and these regions, especially regions 1 and 3, appear to serve as a platform for different interactions.
Comparison of Interaction between N VCP and UBXL OTU1 / UBX FAF1 /UBD NPL4 -To understand how UBXL OTU1 differs from UBX FAF1 and UBD NPL4 in N VCP binding, the complex structure of N VCP and UBXL OTU1 (hereafter referred to as VCP⅐OTU1) was compared with those of N VCP and UBX FAF1 (Ref. 35; Protein Data Bank code 3QQ8; VCP⅐FAF1) and N VCP and UBD NPL4 (Ref. 34; Protein Data Bank code 2PJH; VCP⅐NPL4). The change in accessible surface area (⌬ASA) upon complex formation is 590 Å 2 for VCP⅐OTU1; this is much smaller than the corresponding values for VCP⅐FAF1 and VCP⅐NPL4, namely 810 and 1,050 Å 2 , respectively. The residues involved in the interaction with N VCP are positioned in three regions, namely ␤1-␤2, ␤3-␤4, and ␤5, as indicated in Fig.  2C. The three complex structures are superposed using only the structures of N VCP . Surprisingly, the three VCP-binding partners are at significantly different positions with respect to each other; i.e. they do not overlap at all (Fig. 3A). The most pronounced difference is in the position of the ␣1 helix. The relative positioning of the ␣1 helix from VCP-binding partners is somewhat different as mentioned above ( Fig. 2A) but is significantly different when the complexes are superimposed; e.g. in The region that all three binding partners bind is colored in brown, whereas the region unique to UBXL OTU1 binding is shown in blue, and the region that is irrelevant to UBXL OTU1 binding but important in UBX FAF1 and UBD NPL4 is shown in green. C, details of the interaction between N VCP and binding partners. The interactions formed around the S3/S4 loop region (in boldface) of binding partners are shown with the key residues in stick presentation. Hydrogen bonds are indicated by dashed lines.

Structural Basis for OTU1 Binding to p97/VCP
the complex structures, the ␣1 helix of UBD NPL4 is more than 5 Å shifted from that of UBX FAF1 , whereas that of UBXL OTU1 is somewhat twisted from the other two (Fig. 3A).
The molecular surface of N VCP is mapped by interaction of different binding partners (Fig. 3B). The binding region common to all three VCP-binding partners is colored in brown, the unique surface to UBXL OTU1 binding is colored in blue, and the region irrelevant to UBXL OTU1 , i.e. relevant to only UBX and UBD, is colored in green. As seen in Fig. 3B, interaction in the three complexes is in the same general region, but there are clear differences. In particular, Ser 37 and Arg 144 of N VCP are only specific to UBXL OTU1 binding. They make non-bonded contacts with Tyr 40 of UBXL OTU1 , which makes a hydrogen bond to the side chain of Asp 35 of N VCP (Fig. 3C). Interestingly, Tyr 40 is part of the S3/S4 loop. On the other hand, the green region involves residues from the ␤6-␤7 and ␤11-␤12 loops of N VCP that comprise mainly hydrophobic residues. They interact with the residues from the ␤1-␤2 loop of UBX FAF1 and UBD NPL4 , i.e. region 1 (Fig. 2, A and C). However, the ␤1-␤2 loop of UBXL OTU1 clearly adopts a different conformation from the other two ( Fig. 2A) and does not make any interactions with N VCP , whereas there are numerous interactions involving both hydrophobic and polar residues between N VCP and UBX FAF1 (or UBD NPL4 ) (Fig. 3B).
The primary interaction is via the S3/S4 loop of UBXL OTU1 as in UBX (Fig. 3C). However, binding of UBXL OTU1 is somewhat different as the aromatic ring of Tyr 40 of UBXL OTU1 is packed against the following cis-Pro 41 on one side and by the side chains of Val 38 and Leu 72 of N VCP on the other side. Also, Tyr 40 is positioned further between the N1 and N2 domains of N VCP , the hydroxyl oxygen of which makes hydrogen bonds with the carboxyl oxygens of Asp 35 (2.76 and 3.29 Å) of N VCP (Fig. 3C). In addition, arginine in the signature motif of UBX is replaced by threonine in UBXL OTU1 and does not seem to be conserved. In the case of VCP⅐FAF1, the corresponding residue Phe 619 of UBX FAF1 is packed by Val 38 and Ile 70 of N VCP on one side, and its carbonyl oxygen makes a hydrogen bond with Arg 53 of N VCP (3.23 Å). The guanidinium group of Arg 621 of UBX FAF1 makes hydrogen bonds to the carbonyl oxygens of Pro 137 (3.31 Å) and Leu 141 (2.52 Å) of N VCP . Alternatively, in UBD NPL4 , a 3 10 -helix is formed instead of the S3/S4 loop found in UBX FAF1 and UBXL OTU1 , and this facilitates N VCP binding (Figs. 2C and 3C). Another major interaction region involves the C-terminal region of the binding partners (Fig. 2C). They interact with Phe 52 , Arg 53 , Gly 54 , and Asp 55 of N VCP , which is colored in brown (Fig. 3B). As seen in the figure, this region is equally important. However, the degree of interactions varies per the three complex structures. For example, Arg 53 of N VCP makes six hydrogen bonds in VCP⅐OTU1, whereas the same residue of N VCP forms only three and two hydrogen bonds in VCP⅐FAF1 and VCP⅐NPL4, respectively. Taken together, our data show that UBXL OTU1 binds in a similar position of N VCP as UBX FAF1 and UBD NPL4 , but the interaction mode is specific unlike the others. This implies that OTU1 is a particular binding partner of VCP.
The S3/S4 Loop of OTU1 Critical for VCP Binding and the ERAD Pathway-To confirm the importance of the S3/S4 loop of OTU1 in VCP binding based on the complex structure, the mutant was made by using the full-length OTU1. The S3/S4 loop of OTU1 was replaced with that of FAF1, which showed the closest resemblance as shown above; i.e. the mutant (OTU1 39 TFPR 42 ) has the S3/S4 loop sequence corresponding to Thr 618 -Phe-Pro-Arg 621 of FAF1. The interactions between the full-length VCP and OTU1 were measured using ITC, and the results are shown in Fig. 4A. The binding affinity (K D ) between VCP and OTU1 WT is 0.71 M, whereas the corresponding value for OTU1 39 TFPR 42 could not be measured possibly due to weak binding. This could be understood based on the modeling study, which showed physical clashes with N VCP when the Gly-Tyr-Pro-Pro loop of OTU1 was replaced by Thr-Phe-Pro- Arg (data not shown). In addition, the two prolines in the S3/S4 loop of OTU1 may impose rigidity in the backbone conformation and prevent conformational changes from propagating to the rest of the protein. The substitution of proline with other residues such as glycine or arginine relieves the rigidity of the backbone and increases the flexibility of the loop. Therefore, forming a negative loop of UBXL OTU1 leads to low binding affinity between VCP and OTU1.
A substrate degradation assay was carried out to check whether the inability of OTU1 binding to VCP has any influence on the ERAD pathway using ribophorin I (RPN-I N299T ) as an ERAD substrate. Degradation of the substrate was monitored in the presence of cycloheximide (37). OTU1 39 TFPR 42 showed relatively reduced degradation and accumulation of RPN-I N299T in comparison with OTU1 WT (Fig. 4B). This result shows that disruption of the association between VCP and OTU1 leads to the negative regulation of the ERAD pathway; thus, the S3/S4 loop of OTU1 is highly specific and is involved in a unique association with VCP.
It has been reported previously that abrogation of the deubiquitinating activity of OTU1 resulted in impairment of the degradation of misfolded RI332 as an ERAD substrate, suggesting that OTU1 positively regulates the ERAD pathway (13). Here, although UBXL OTU1 has no relation to the enzymatic activity of OTU1, disruption of the interaction between VCP and OTU1 led to defects in the degradation of ERAD substrates. Therefore, it seems that UBXL OTU1 is required for the ERAD pathway. Likewise, recent reports have suggested the importance of the association of VCP and UBX-containing proteins. For instance, the S3/S4 loop mutant of UBX FAF1 lost the ability to interact with VCP as well as with ubiquitinated substrate via its ubiquitin-associated domain, resulting in the accumulation of substrate (38). Another example is UBXD7, which separately binds both VCP and substrate HIF1␣ and recruits the substrate to VCP for degradation. However, its UBX-lacking mutant could not interact with both VCP and substrate, thus leading to a defect in the degradation of HIF1␣ (39). Therefore, the association of UBX-containing proteins with VCP is involved in their function; i.e. binding of OTU1 to VCP is needed to activate OTU1 in the ERAD pathway, demonstrating the importance of UBXL OTU1 . Furthermore, the S3/S4 loop of OTU1 is conserved as residues Gly-Tyr/Phe-Pro-Pro are found in other OTU1 homologs and VCP complex-interacting protein of 135 kDa (VCIP135) which is another known UBXL-containing protein (Refs. 40 and 41 and data not shown). Therefore, not only VCIP135 but also OTU1 homologs are expected to bind VCP in the same manner shown in this study.
Interaction between the Full-length Proteins-Because VCP exists as a homohexamer, it can possibly bind six different binding partners. Therefore, it is important to characterize the stoichiometry and interaction between the full-length VCP and its binding partners. To determine the stoichiometry of VCP and OTU1, an EM study was carried out. First, gold labeling was carried out using a hexahistidine tag. The tag was added to the C terminus of OTU1 because its N-terminal domain binds to VCP. Negative staining EM images of VCP alone and in complex with the gold-labeled OTU1 were taken. About 5,800 single particles were selected from individual micrographs, and the stoichiometry between VCP and OTU1 was analyzed statistically by counting the number of gold particles. Approximately 80% of the selected particles had one gold particle, and about 15% had two (Fig. 5A). The remaining 5% were particles with more than three gold particles (data not shown). We then took images of the complex of the full-length proteins (VCP/ OTU1 complex) without gold labeling to avoid the restrictive space constraints due to the gold particle, which has a diameter of 5 nm. Three types of negative staining solutions were tried, but methylamine vanadate, pH 8.0 gave the best result as shown in Fig. 5B. A total of 2,452 particles of VCP/OTU1 complex were separated successfully from 2,839 particles of a mixture containing VCP alone and VCP/OTU1 complex with the class average. When uranyl formate was used, the ratio between the complex and VCP alone was almost 1,134 to 4,231 possibly due to the acidic nature of the solution, namely pH 5.5 (data not shown). Representative two-dimensional class averages for VCP alone and VCP/OTU1 complex are shown in Fig. 5B, which clearly shows that one OTU1 molecule binds to one vertex of homohexameric VCP. ITC measurement using the fulllength VCP and OTU1 showed a binding stoichiometry of n ϭ 1.07 Ϯ 0.02 and a K D of 0.71 M, suggesting that the stoichiom-etry of OTU1 to VCP is near 1:6. This is in good agreement with the EM data above. In addition, when UBXL OTU1 was removed (data not shown) or mutated (Fig. 4A), it did not show measurable binding to VCP. This confirmed again that UBXL OTU1 is necessary for binding to VCP.
The stoichiometries between VCP and various binding partners have been reported. In the case of UFD1/NPL4, EM studies clearly show that one UFD1/NPL4 binds one VCP hexamer (5,42). On the contrary, for both p47 and UBXD1, three molecules of each bind to one VCP hexamer (43,44). Interestingly, in the case of both FAF1 and UBXD7, one molecule binds one VCP hexamer only when UFD1/NPL4 is prebound to VCP (35, 38).   (35), and 0.5 M for p47 (43). These values are comparable with what we have seen with OTU1. It is worthwhile mentioning that in some cases bipartite binding has been reported. For example, VCP binding of UFD1/NPL4 utilizes the BS1 segment, which is a short hydrophobic stretch in the C-terminal domain of UFD1, in addition to the UBD of NPL4 as described above (45). Also, p47 uses a shp1-eyc-p47 domain, which is in the middle of p47, in addition to the C-terminal UBX domain. The shp1-eyc-p47 domain is reported to form a trimer and binds at the interface between the N and D1 domains of VCP. Besides the hierarchical binding of VCP-binding partners, some binding partners such as UFD1/NPL4, p47, and UBXD1 are reported to show mutually exclusive binding (46,47), whereas others such as UFD1/NPL4 and UBX4 show simultaneous binding (48). This is understandable considering the existence of numerous binding partners and the diverse functions of VCP. Although the crystal structure of truncated proteins showed 1:1 binding between UBXL OTU1 and N VCP , the EM study using the fulllength proteins clearly showed that one OTU1 binds to the hexameric VCP, suggesting only one OTU1 molecule is sufficient to perform its function; furthermore, it may function with other binding partners.
Dependence of OTU1 Binding to VCP on VCP Adaptor Proteins UFD1/NPL4-UFD1/NPL4 plays a crucial role in the ERAD pathway together with VCP, and some VCP-binding partners show hierarchical binding as well as mutually exclusive binding to VCP. We tested the binding pattern for VCP between OTU1 and UFD1/NPL4 using size exclusion chromatography and ITC, and the results are shown in Fig. 6. A GSTtagged OTU1 (GST-OTU1) was used to avoid possible confusion caused by molecular weights on size exclusion chromatography and SDS-PAGE. Two sets of experiments were carried out. First, GST-OTU1 was added to VCP alone, and then UFD1/NPL4 was added. Second, UFD1/NPL4 was added to VCP alone, and then OTU1 was added. As expected, both OTU1 and UFD1/NPL4 form a complex with VCP alone, but in addition, both form a quaternary complex, i.e. VCP⅐GST-OTU1⅐UFD1/NPL4 (Fig. 6A). We further determined the binding affinities and the stoichiometries between these using ITC, and the results are shown in Fig. 6B. First, UFD1/NPL4 and premade VCP/OTU1 binary complex showed an apparent K D of 3.50 M and n ϭ 1.05 Ϯ 0.17, whereas OTU1 and premade VCP⅐UFD1/NPL4 ternary complex gave a K D of 0.41 M and n ϭ 1.09 Ϯ 0.02. Both suggest that there is 1:1 binding. Interestingly, the K D values are similar to those obtained for VCP alone; e.g. the K D for UFD1/NPL4 binding to VCP was reported as 1.7 M (35), and the K D for OTU1 binding to VCP was 0.71 M (see Fig. 4A). This clearly shows that VCP can bind OTU1 in both the presence and absence of UFD1/NPL4 and vice versa.
In summary, we have dissected the interaction between VCP and OTU1. One OTU1 molecule binds to one VCP hexamer with similar binding affinity both in the presence and absence of UFD1/NPL4. The UBXL OTU1 binds at the interface between the two subdomains (N1 and N2) of VCP. In particular, the conformation of the Gly 39 -Tyr 40 -cis-Pro 41 -Pro 42 loop of UBXL OTU1 is critical for N VCP binding. Although some of these properties of UBXL mimic UBX, the interaction is quite specific and has direct bearing on the proteasomal degradation of ERAD substrates. Based on these results, it is plausible that OTU1 can participate in the regulation of ubiquitinated substrates in both UFD1/NPL4dependent and -independent manners. Our data provide insights into the interaction between the full-length VCP and OTU1 as well as the interaction between N VCP and UBX-L OTU1 that could be applicable to other UBXL-containing proteins.