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Related Experiment Video

Updated: May 19, 2026

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

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 Moon^2,3

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.

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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.