Mechanistic insight into substrate processing and allosteric inhibition of human p97

Abstract

p97, also known as valosin-containing protein (VCP), processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Mutations in human p97 are associated with multisystem proteinopathy (MSP), a dominantly inherited degenerative disorder that can affect muscle, bone and the central nervous system. It is also a drug target for cancer therapy with various inhibitors developed over the past decade. Despite significant structural insights into the fungal homologue of p97, Cdc48, little is known about how human p97 processes its substrates and how the activity is allosterically affected by inhibitors. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Angstrom that captured power stroke-like motions of both the D1 and D2 ATPase rings of p97. The structures elucidated how the unfolded substrate is engaged in the pore at atomic level. Critical conformational changes of the inter-subunit signaling (ISS) motifs were revealed, providing molecular insights into substrate translocation. Furthermore, we also determined cryo-EM structures of human p97 in complex with NMS-873, the most potent p97 inhibitor, at a resolution of 2.4 Angstrom. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change, thus blocking substrate translocation allosterically. Finally, using NMS-873 at a substoichiometric concentration, we captured a series of intermediate states, suggesting how the cofactor Npl4 coordinates with the D1 ring of p97 to initiate the translocation.

Structural basis of ubiquitin-independent PP1 complex disassembly by p97

“…Our structures show p97 loaded with the SPI complex at a defined position on the N-domain in the act of translocating a segment of I3. The density map (Figure 1a-c and Supplementary Figure S1a) confirms a conserved peptide threading mechanism previously proposed for p97 and yeast Cdc48 (Cooney et al, 2019; Pan et al, 2021; Twomey et al, 2019; Xu et al, 2022). Both D1 and D2 domains of p97 are in the staircase configuration with subunit A on top, followed by subunits B, C and D in a clockwise manner to subunit E at the bottom (Figure 1a and b).…”

“…The SPI structure is best resolved on subunit B, making that state the most informative. Previous structural studies have used cryo-EM to reveal the spiral conformation of p97 with a substrate trapped in the pore (Cooney et al, 2019; Pan et al, 2021; Twomey et al, 2019; Xu et al, 2022), but have not revealed a loading complex with the adaptor proteins delivering the substrate to p97 for protein complex disassembly. Our structure of p97 with a loaded substrate complex on the N-domain reveals unanticipated new principles of how a substrate is positioned and engaged onto p97 with the help of the adapter leading to dissociation of one subunit while sparing the other components.…”

“…Given that the distance between these densities is compatible with the 40 residue length of that sequence, it is tempting to speculate that these elements are connected, and that our structure represents I3 engaged in the channel and in the process of being pulled off PP1 (Model Figure 5a and b). The ATPase rings in our structure are in a spiral conformation primed to thread the peptide through the channel by the established hand-over-hand mechanism (Cooney et al, 2019; Pan et al, 2021; Twomey et al, 2019; Xu et al, 2022).…”

“…Biochemical reconstitution revealed that, as in other AAA+ unfoldases, substrate proteins are inserted in the D1 pore, threaded through the central channel of p97 by the sequential activity of the hexamer subunits and ejected from the D2 pore (Bodnar and Rapoport, 2017; Weith et al, 2018). Cryo-electron microscopy (cryo-EM) analysis showed that the active p97 hexamer is in a staircase configuration with the substrate peptide being transported by sequential interaction with the spiral of pore loops (Cooney et al, 2019; Pan et al, 2021; Twomey et al, 2019; Xu et al, 2022). The pore loops provide a non-sequence-specific interaction between aromatic residues and the peptide backbone that allows it to be threaded through the channel in a hand-over-hand mechanism.…”

AAA ATPases as therapeutic targets: Structure, functions, and small-molecule inhibitors

“…NMS-873 is thought to bind to the linker domain between the D1 and D2 ATPase domains of p97, which stabilizes D2-ADP bound p97 [63]. We performed docking study to investigate the binding mode of NMS-873, which was proposed in Figure 4B [64]. Bastola et al validated the binding site of NMS-873 by the mutations at nearby residues, which abrogated or significantly reduced the activity of NMS-873 [65].…”