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bioRxiv · 10.64898/2026.05.01.722286

Npl4 decodes polyubiquitin length and gates D1-D2 coupling in human VCP/p97

Khamari, L.·Tang, J.·Moon, S.·Walter, N. G.
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Abstract

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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Khamari, L., Tang, J., Moon, S., Walter, N. G.. 2026-05-05. Npl4 decodes polyubiquitin length and gates D1-D2 coupling in human VCP/p97. https://doi.org/10.64898/2026.05.01.722286

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