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Updated: May 19, 2026
07:58
In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination
Published on: January 2, 2026
Npl4 decodes polyubiquitin length and gates D1-D2 coupling in human VCP/p97
Laxmikanta Khamari1, Jingxuan Tang1, Stephanie L Moon2,3
1Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Biorxiv : the Preprint Server for Biology
|May 18, 2026
Summary
The VCP/p97 enzyme recognizes polyubiquitin chain length using a novel single-molecule method. This reveals how p97 (valosin-containing protein) selects substrates for degradation and how disease mutations impact its function.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The VCP/p97 AAA+ ATPase is crucial for protein homeostasis, extracting polyubiquitinated proteins for proteasomal degradation.
- Understanding how p97 recognizes polyubiquitin chain length and how its ATPase domains (D1 and D2) coordinate substrate processing is essential.
Purpose of the Study:
- To elucidate the mechanism by which VCP/p97 decodes polyubiquitin chain length.
- To investigate the roles of D1 and D2 ATPase cycles in cofactor dynamics and substrate processing.
- To explore the impact of disease-associated mutations on p97 function.
Main Methods:
- Development and application of a single-molecule ubiquitin recognition and dynamics (smUbiRAD) assay.
- Biochemical reconstitution of p97-Ufd1-Npl4-ubiquitin complexes.
- Characterization of wild-type and mutant p97 variants.
Main Results:
- A sharp chain-length threshold was identified: Npl4 exhibits transient binding to short ubiquitin chains but stable, multivalent engagement with tetra- and penta-ubiquitin.
- Ufd1 and p97 stabilize these complexes by inhibiting Npl4 dissociation.
- D1 ATPase hydrolysis, not D2, drives rapid Npl4 exchange in assembled complexes, suggesting a model where D1-powered conformational changes gate cofactor turnover.
- Mutations R155H and A232E associated with multisystem proteinopathy shift p97 towards a high-affinity state and accelerate Npl4 exchange.
Conclusions:
- smUbiRAD reveals a critical chain-length sensing mechanism for p97 substrate selection.
- D1 ATPase activity licenses substrate processing by regulating cofactor Npl4 turnover.
- Hyperactive cofactor cycling due to specific p97 mutations may underlie multisystem proteinopathy pathogenesis.
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