Kim, Su Jin Cho, Jinhong Song, Eun Joo Kim, Soo Jin Kim, Ho Min Lee, Kyung Eun Suh, Se Won Kim, Eunice EunKyeong 2024-01-20T10:00:17Z 2024-01-20T10:00:17Z 2021-09-04 2014-05-02 0021-9258 https://pubs.kist.re.kr/handle/201004/126803 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 K-D of 0.71 m. Conclusion: The (39)GYPP(42) (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 (N-VCP) at 1.8- resolution reveals that UBXLOTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of N-VCP 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 K-D of 0.71 m. 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. English AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC UBIQUITIN-PROTEASOME SYSTEM AAA ATPASE P97/VCP ENDOPLASMIC-RETICULUM CRYSTAL-STRUCTURE DEGRADATION ERAD QUALITY CONTROL P97 COMPLEX COFACTORS SUITE Structural Basis for Ovarian Tumor Domain-containing Protein 1 ( OTU1) Binding to p97/Valosin-containing Protein ( VCP) Article 10.1074/jbc.M113.523936 1 JOURNAL OF BIOLOGICAL CHEMISTRY, v.289, no.18, pp.12264 - 12274 JOURNAL OF BIOLOGICAL CHEMISTRY 289 18 12264 12274 scie scopus 000335581400007 2-s2.0-84899751309 Biochemistry & Molecular Biology Biochemistry & Molecular Biology Article UBIQUITIN-PROTEASOME SYSTEM AAA ATPASE P97/VCP ENDOPLASMIC-RETICULUM CRYSTAL-STRUCTURE DEGRADATION ERAD QUALITY CONTROL P97 COMPLEX COFACTORS SUITE ATPases Crystal Structure Deubiquitination Electron Microscopy (EM) ER-associated Degradation Isothermal Titration Calorimetry Protein-Protein Interactions Site-directed Mutagenesis