Structural basis for ovarian tumor domain-containing protein 1 (OTU1) binding to p97/valosin-containing protein (VCP)

Abstract

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 (39)GYPP(42) 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.

Keywords: ATPases; Crystal Structure; Deubiquitination; ER-associated Degradation; Electron Microscopy (EM); Isothermal Titration Calorimetry; Protein-Protein Interactions; Site-directed Mutagenesis.

FIGURE 1.

Crystal structure of the N_VCP and UBXL_OTU1 complex. A, schematic representation of domain structures of VCP and OTU1. B, ribbon presentation of UBXL_OTU1 in complex with N_VCP shown in blue and orange, respectively. Secondary structure elements are labeled: α for α-helix, β for β-strand, and η for 3₁₀-helix. Disordered regions are indicated by dotted lines. ZnF, zinc finger.

FIGURE 2.

Comparison of UBXL_OTU1 with UBX_FAF1 and UBD_NPL4. A, the surface showing electrostatic charges of UBXL_OTU1, UBX_FAF1, and UBD_NPL4 with red representing negatively charged surface and blue representing positively charged surface (top). Superposition of the three structures is also shown (bottom). The S3/S4 loop of UBXL_OTU1 and UBX_FAF1 and 3₁₀-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 − F_c) of crystal form I is shown at the 1.5σ 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 α, β, η, and TT denote α-helix, β-strand, 3₁₀-helix, and β-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).

FIGURE 3.

Analysis of interaction between N_VCP and binding partners. A, superposition of the complex structures of VCP·OTU1 (crystal form I in this study), VCP·FAF1 (Ref. ; Protein Data Bank code 3QQ8), and VCP·NPL4 (Ref. ; Protein Data Bank code 2PJH) shown in blue, pink, and yellow, respectively. Only N_VCP was used for superposition. N_VCP is shown in surface representation, whereas binding partners are shown in ribbon representation. B, binding surface of N_VCP. 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.

FIGURE 4.

ITC and substrate degradation assay for OTU1 wild type and ³⁹TFPR⁴² mutant. A, ITC raw data and fitted data for the interaction between the full-length VCP and OTU1. The raw data are shown for injection of OTU1 WT (left), ³⁹TFPR⁴² (right), and a dilution control of OTU1 in buffer (curves on the top are offset for clarity). The lower panels show the integrated heat data against the molar ratio of VCP to OTU1. The closed squares were fitted to a one-site model, and the solid lines represent the best fit results. No measurable interaction was detected between VCP and OTU1 ³⁹TFPR⁴². B, influences of OTU1 on degradation of RPN-I^N299T. Left, OTU1 WT; right, OTU1 ³⁹TFPR⁴². Substrate was totally degraded in the case of OTU1 WT, whereas the degradation process was retarded, resulting in proteolytic fragments, when the S3/S4 loop was mutated. IB, immunoblot.

FIGURE 5.

EM analysis on single particle of the VCP and OTU1 complex. A, raw images of VCP complexed with gold-labeled OTU1. Bottom panels show VCP with no label as a control (left) and particles with one (middle) and two (right) gold particles with black arrows indicating the gold particle. B, electron micrograph of the VCP and OTU1 complex. The bottom panel shows the representative two-dimensional class average for VCP alone (left) and VCP/OTU1 complex (middle and right). VCP/OTU1 complex is marked by a red box. The arrow indicates the OTU1 molecule.

FIGURE 6.

Dependence test of OTU1 binding to VCP on UFD1/NPL4. A, size exclusion chromatography profile and Coomassie-stained SDS-PAGE of fractions of VCP, GST-OTU1, and UFD1/NPL4. To hexameric VCP, GST-OTU1 and UFD1/NPL4 (top) and UFD1/NPL4 and GST-OTU1 (bottom) were added. SDS-PAGE fractions are shown below the chromatograms. V, O, U, and N stand for VCP, OTU1, UFD1, and NPL4. B, ITC binding data for UFD1/NPL4 binding to VCP/OTU1 complex (left) and for OTU1 binding to VCP·UFD1/NPL4 complex (right). mAU, milli-absorbance units.