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Conserved L464 in p97 D1–D2 linker is critical for p97 cofactor regulated ATPase activity

Zhang, Xiaoyi and Gui, Lin and Li, Shan and Nandi, Purbasha and Columbres, Rod Carlo and Wong, Daniel E. and Moen, Derek R. and Lin, Henry J. and Chiu, Po-Lin and Chou, Tsui-Fen (2021) Conserved L464 in p97 D1–D2 linker is critical for p97 cofactor regulated ATPase activity. Biochemical Journal, 478 (17). pp. 3185-3204. ISSN 0264-6021. doi:10.1042/bcj20210288.

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p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1–D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1–D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4–Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (k_(cat)) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the k_(cat) of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97^(L464P) and revealed the conformational change of the D1–D2 linker, resulting in a movement of the helix-turn-helix motif (543–569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ∼50 Å away.

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Chou, Tsui-Fen0000-0003-2410-2186
Additional Information:© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society. Received: April 27 2021; Revision Received: August 10 2021; Accepted: August 18 2021; Accepted Manuscript online: August 18 2021. Cryo-EM data collection was by the use of the Titan Krios TEM at the Eyring Materials Center at Arizona State University, with funding for the instrumentation by NSF MRI 1531991. Special thanks to Dewight Williams for assisting cryo-EM data collection. Computation for image processing was partly supported by the NVIDIA GPU Grant Program. Molecular graphics and analyses performed with the UCSF ChimeraX was supported by NIH R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, NIAID. This project was supported in part with funds from the National Institute of Neurological Disorders and Stroke, R01NS100815 and R01NS102279. Data availability: Cryo-EM density maps were deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers of EMD-23775 and EMD-23776 (symmetrized) and the Protein Data Bank (PDB) under accession numbers of 7MDM and 7MDO (symmetrized). All data are available from the corresponding author upon request. Author Contributions: XZ, LG, SL, PN, PLC, TFC designed the research. XZ, LG, SL, PN, RCC, DEW and DRM performed the research. LG, XZ, SL, PN, DEW, DRM, HJL, PLC and TFC analyzed the data and composed the paper. The authors have no competing financial interests to disclose.
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Subject Keywords:anti-caner, cryo-electron microscopy, enzyme kinetics, p97/VCP
Issue or Number:17
Record Number:CaltechAUTHORS:20210910-171152306
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Official Citation:Xiaoyi Zhang, Lin Gui, Shan Li, Purbasha Nandi, Rod Carlo Columbres, Daniel E. Wong, Derek R. Moen, Henry J. Lin, Po-Lin Chiu, Tsui-Fen Chou; Conserved L464 in p97 D1–D2 linker is critical for p97 cofactor regulated ATPase activity. Biochem J 17 September 2021; 478 (17): 3185–3204. doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:110799
Deposited By: Tony Diaz
Deposited On:10 Sep 2021 20:09
Last Modified:28 Oct 2021 18:57

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