SH004 - Bag-1S directly binds p97/VCP and decouples ATPase activation from ERAD output
SH004
Bag-1S directly binds p97/VCP and decouples ATPase activation from ERAD output
E. Baştürk1,*, H. D. Boncuk1, G. Şahinbaş1, A. Takasu2, E. Destan3, I. Yapıcı3, H. Akkulak4, M. Çalıseki5, M. Öztuğ6,7, S. Özcan4, B. V. Kabasakal5,8, T. Senda2, H. DeMirci2,3,9, G. Dinler Doğanay7
1Molecular Biology-Genetics and Biotechnology, Istanbul Technical University, Istanbul, Türkiye, 2Structural Biology Research Center (SBRC), High Energy Accelerator Research Organization (KEK), Tsukuba, Japan, 3Molecular Biology and Genetics, Koç University, Istanbul, 4Chemistry, Middle East Technical University, 5Turkish Accelerator and Radiation Laboratory (TARLA), Ankara, 6National Metrology Institute (UME), TUBITAK, 7Molecular Biology and Genetics, Istanbul Technical University, Istanbul, 8Biological Sciences, Middle East Technical University, Ankara, Türkiye, 9SLAC National Laboratory, Stanford PULSE Institute, California, United States
Rationale: Proteostasis is maintained by coordinated protein folding and degradation pathways, whose dysregulation contributes to cancer and other complex diseases. The AAA+ ATPase p97/VCP is a central regulator of protein extraction and degradation, yet how its activity is modulated by co-chaperones such as Bag-1S remains poorly understood.
Methods: Recombinant p97 and Bag-1S proteins and domain variants were produced and purified using multi-step chromatography. Protein–protein interactions were analyzed using biochemical assays, ATPase activity measurements, hydrogen–deuterium exchange mass spectrometry (HDX-MS), and cross-linking mass spectrometry (XL-MS). Cryo-electron microscopy (cryo-EM) was employed in an integrative framework to support structural interpretation of the p97 complex. Structure–function relationships were further examined using site-directed mutagenesis and functional assays in mammalian cell models.
Results: Biochemical analyses demonstrated that Bag-1S modulates p97 ATPase activity. Consistent with the established role of the BAG domain in chaperone regulation, HDX-MS revealed localized conformational changes in Bag-1S upon interaction with p97, while XL-MS identified interaction interfaces involving both the BAG and ubiquitin-like domains. Cryo-EM–informed structural analysis supported the contextual interpretation of these interfaces within the p97 complex. Mutations targeting these regions altered p97 activity and impaired proteostasis-related functions in cells, confirming their functional relevance.
Conclusions: Our findings demonstrate that Bag-1S modulates p97 activity through a dynamic and context-dependent interaction involving both its BAG and UbL domains. This interaction alters p97 ATPase activity and ERAD efficiency in cancer cells, supporting a regulatory rather than purely activating role. These results provide mechanistic insight into how Bag-1S fine-tunes p97-dependent proteostasis pathways.
Disclosure of Interest: None declared