Mechanism of allosteric inhibition of human p97/VCP ATPase and its disease mutant by triazole inhibitors

Abstract

Human p97 ATPase is crucial in various cellular processes, making it a target for inhibitors to treat cancers, neurological, and infectious diseases. Triazole allosteric p97 inhibitors have been demonstrated to match the efficacy of CB-5083, an ATP-competitive inhibitor, in cellular models. However, the mechanism is not well understood. This study systematically investigates the structures of new triazole inhibitors bound to both wild-type and disease mutant forms of p97 and measures their effects on function. These inhibitors bind at the interface of the D1 and D2 domains of each p97 subunit, shifting surrounding helices and altering the loop structures near the C-terminal α2 G helix to modulate domain-domain communications. A key structural moiety of the inhibitor affects the rotameric conformations of interacting side chains, indirectly modulating the N-terminal domain conformation in p97 R155H mutant. The differential effects of inhibitor binding to wild-type and mutant p97 provide insights into drug design with enhanced specificity, particularly for oncology applications.

Fig. 1. Structure of human p97 ATPase and designed triazole inhibitors used in this study.

a Primary structure of human p97 ATPase. Location of the pathogenic mutation R155H (blue) and arginine fingers of D1 (R359, R362) and D2 domains (R635, R638) are labeled. α/β and α subdomains of the D1 and D2 ATPase domains are underlined. Highlighted regions (pink; D1 α and D2 α/β subdomains) are the domains that interact with the triazole-based inhibitors. b Structure of wild-type p97 with ADP and the triazole-based inhibitor bound (PDB code: 8UV2). The N-terminal (NTD), D1, D2, and linker domains are in green, orange, orange-red, and purple, respectively. Bound nucleotides and the triazole-based inhibitor are shown in space-filling representation in light and medium blue, respectively. The helices of the D2 domain are labeled. c Chemical structures of the lead compounds for p97 ATPase: NSC799462, NSC804515, NSC819701, NMS-873, and UPCDC30766.

Fig. 2. Cryo-EM structure of p97 with NSC799462 triazole inhibitor.

Surface representation of the two p97 monomers. Green, orange, orange-red, and purple are the N-terminal (NTD), D1, D2, and linker domains in chain A. White (chain B) is one of the adjacent p97 monomers, which interfaces with the binding site of NSC799462. a The NSC799462 terminal methylpiperidine group approaches the adjacent D2 nucleotide-binding site at a distance of approximately 8 Å. Hot pink is the D2 domain of the adjacent p97 monomer (D2’). Light blue surfaces are cryo-EM densities for NSC799462 and ADP. b NSC799462 binding site. NSC799462 is shown in ball-and-stick. Gray, red, blue, yellow, and light green represent carbon, oxygen, nitrogen, sulfur, and fluorine atoms, respectively. Side chains of the interacting protein residues are shown in sticks. Cryo-EM density is shown in light blue surfaces. c Superposition of p97^WT | _NSC799462 and p97^WT (PDB code: 5FTK) (RMSD 1.098 Å). p97^WT structure is shown in semi-transparent white. The region following the C-terminus of the α2 G helix that adopts a different conformation in the presence of inhibitor is shown in the inset. d Superposition of p97^WT (light purple, without inhibitor) (PDB code: 5FTK) and p97^WT | _NSC799462 (orange red, with NSC799462 in surface representation) illustrates the expansion of the binding site to fit the inhibitor. All distances are measured in Å.

Fig. 3. Structural and functional comparisons of p97 with various triazole inhibitors.

a NMS-873 binding site (PDB code: 7LMY). Orange-red is the p97 D2 domain. b UPCDC30245 binding site (PDB code: 5FTJ). c Structural comparison of the nucleotide-binding sites between p97|_{ADP,NSC799462} and p97^E578Q | _ATP,NMS-873. Yellow, blue, and pink sticks are the intersubunit signaling (ISS) motif, arginine fingers, and E578 (p97|_{ADP,NSC799462}) or Q578 (p97^E578Q|_ATP,NMS-873). Distances are measured in Å. d Histogram comparing IC₅₀ measurements (on a logarithmic scale) of triazole inhibitors and CB-5083 on the ATPase activities of various purified p97 mutants (n = 6). Data are presented as the mean ± standard deviation (SD). CB-5083 is an ATP-competitive inhibitor. WT represents the wild type.

Fig. 4. Structural superposition of p97^WT | _NSC799462 and p97^WT.

a Superposition of p97^WT | _NSC799462 and p97^WT (PDB code: 5FTK). Green, orange, orange-red, and purple are the N-terminal (NTD), D1, D2, and linker domains, respectively. p97^WT is shown in transparency. b Enlarged view of the interaction network between the NTD and N-D1 linker domain. The interaction network is established via hydrogen bonds between protein residue side chains, stabilizing the NTD in the down conformation. All distances are measured in Å. Density fitting is shown in Supplementary Fig. 3b.

Fig. 5. Allosteric inhibition of the p97^R155H disease mutant by triazole inhibitors.

a Binding site of the triazole inhibitors. Gold, blue and white are the structures of p97^R155H | _NSC804515, p97^R155H | _NSC819701, and p97^WT | _NSC799462. Yellow, light green, blue, and red ball representations are for sulfur, fluorine, nitrogen, and oxygen atoms. K615 side chain adopts different conformations when interacting with meta- and para-fluorobenzene rings. b Cryo-EM densities of p97^R155H | _NSC804515 and p97^R155H | _NSC819701. Green, orange, orange-red, and purple are the N-terminal (NTD), D1, D2, and linker domains, respectively. The densities were filtered to 5.0 Å⁻¹ for presentation. The NTD shows in different conformations when bound to the triazole inhibitors. Two p97^R155H | _NSC819701 structures with different NTD conformations, partially up and down conformations, were determined. c Structural superpositions of the p97^R155H | _NSC819701 (blue; State B, down NTD) and p97^R155H | _NSC804515 (light orange; up NTD) (RMSD 0.846 Å). Dark green is the NTD of p97^R155H | _NSC819701. Enlarged view shows the interaction network of the NSC819701 with the surrounding residues of helices α1 J and α1 M. d Interaction between NTD and D1 domain of the p97^R155H | _NSC819701. Dark green is the NTD of p97^R155H | _NSC819701. Gray surfaces are cryo-EM densities of p97^R155H | _NSC819701 in NTD-down conformation. All distances are measured in Å.

Fig. 6. Proposed mechanistic model of the triazole allosteric inhibition on p97 ATPase.

Two adjacent p97 monomers are shown in gray cartoons. Surface representations are the area impacted by the triazole inhibitor binding, which are the D1 domain and the adjacent D2 domain. Dark green is the N-terminal domain (NTD), orange, yellow, and coral are helices of D1 domain, orange-red are helices of D2 domain, and blue is the triazole inhibitor. R155H position is highlighted in the NTD.