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Updated: May 19, 2026

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.
None:
VCP/p97 binds the Npl4-Ufd1 heterodimer adaptor to extract polyubiquitinated substrates for proteasomal degradation, but how it decodes K48-linked chain length and how D1-coupled events license downstream D2 power strokes remain unclear. Here we introduce smUbiRAD, or single-molecule ubiquitin recognition and dynamics, and identify a sharp chain-length threshold: Npl4 binds transiently to short chains but switches to long-lived, multivalent engagement on tetra- and penta-ubiquitin. Ufd1 and p97 further stabilize these complexes mainly by suppressing Npl4 dissociation without affecting initial encounter. In fully assembled p97-Ufd1-Npl4-substrate complexes, D1 ATP hydrolysis-rather than D2-drives rapid Npl4 exchange. These results support a model in which D1-powered conformational changes promote cofactor Npl4, but not Ufd1, turnover and gate iterative coupling to downstream D2-driven substrate processing. Finally, we show that multisystem proteinopathy variants R155H and A232E bias p97 toward a high-affinity resting state and accelerate Npl4 exchange, implicating hyperactive cofactor cycling as a disease-linked dysregulation.
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