Abstract
The conserved ATPase p97 (Cdc48 in yeast) and adaptors mediate diverse cellular processes through unfolding polyubiquitinated proteins and extracting them from macromolecular assemblies and membranes for disaggregation and degradation. The tandem ATPase domains (D1 and D2) of the p97/Cdc48 hexamer form stacked rings. p97/Cdc48 can unfold substrates by threading them through the central pore. The pore loops critical for substrate unfolding are, however, not well-ordered in substrate-free p97/Cdc48 conformations. How p97/Cdc48 organizes its pore loops for substrate engagement is unclear. Here we show that p97/Cdc48 can form double hexamers (DH) connected through the D2 ring. Cryo-EM structures of p97 DH reveal an ATPase-competent conformation with ordered pore loops. The C-terminal extension (CTE) links neighboring D2s in each hexamer and expands the central pore of the D2 ring. Mutations of Cdc48 CTE abolish substrate unfolding. We propose that the p97/Cdc48 DH captures a potentiated state poised for substrate engagement.
Highlights
The highly conserved molecular chaperone p97, called valosin-containing protein in humans and Cdc[48] in the budding yeast, is a member of the ATPases associated with diverse cellular activities (AAA) family of ATPases[1,2]
We focused on the structure analysis of p97 double hexamer (DH), as it had not been well studied
In contrast to a previous report claiming that human p97 DH was inactive in hydrolyzing ATP27, we showed that both p97/Cdc[48] single hexamers (SH) and DH were active in ATP hydrolysis with comparable activities (Fig. 2d; Supplementary Fig. 4g, h)
Summary
The highly conserved molecular chaperone p97, called valosin-containing protein in humans and Cdc[48] in the budding yeast, is a member of the ATPases associated with diverse cellular activities (AAA) family of ATPases[1,2]. In complex with a variety of cofactors and adaptors, p97/ Cdc[48] acts as a segregase or unfoldase that extracts polyubiquitinated proteins from membranes, chromatin, macromolecular complexes, and misfolded protein aggregates for subsequent degradation or remodeling[1,3,5,6,7] As such, it regulates diverse cellular processes, including endoplasmic reticulum-associated degradation, mitochondrial-associated degradation, membrane fusion, and DNA replication and repair[1,2,3]. The D1 and D2 domains form two stacked hexameric rings with a central pore[12,14,20,21,22] Both D1 and D2 domains can hydrolyze ATP, but only the ATPase activity of D2 domains is critical for substrate unfolding[23,24,25,26].
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