Abstract
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 (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat 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 p97L464P 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.
Highlights
P97, known as VCP, TER, VAT and Cdc48p, is a ubiquitous type II ATPase protein
Mutations in this region lead to the accumulation of an endoplasmic reticulum-associated degradation (ERAD) substrate, T cell receptor alpha chain fused to GFP (TCR-α-GFP) [39, 40]. p97 proteins containing only the NTD and D1 domains without the D1-D2 linker domain (ND1) exhibit only 1.4% of the ATPase activity of the fulllength wild-type p97 (p97WT) protein
We are intrigued by the interesting model and the result suggested that the D1-D2 linker region of p97 is important for motion transmission and to activate D1 activity [39,40,41]
Summary
P97, known as VCP (valosin-containing protein), TER (translational endoplasmic reticulum ATPase, in mammals), VAT (valosin-containing protein-like ATPase of Thermoplasma acidophilum, in archaea) and Cdc48p (in yeast), is a ubiquitous type II ATPase protein It is highly conserved and plays essential roles in multiple cellular processes, including protein quality and cell cycle control [1,2,3,4,5], ubiquitin-proteasome system (UPS) [6, 7], transcriptional activation [8], DNA-damage repair [9] and autophagy [10,11,12,13]. Previous work on p97 suggests that flexibility in the D1-D2 linker region is required for ATP hydrolysis-dependent motion transmission between D1 and D2. Mutations in this region lead to the accumulation of an endoplasmic reticulum-associated degradation (ERAD) substrate, T cell receptor alpha chain fused to GFP (TCR-α-GFP) [39, 40]. MSP1 disease mutations caused differential binding of adaptor proteins (p37 and p47) to the NTD of p97 in cells [44, 45]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
