Npl4 decodes polyubiquitin length and undergoes D1-gated exchange on p97/VCP

Human p97 binds the Npl4–Ufd1 heterodimer adapter 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 the 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 in disease. p97 maintains protein quality by processing ubiquitin-tagged proteins. This report shows how Npl4 reads ubiquitin-chain length and how p97 ATPase activity controls Npl4 exchange, which is altered by disease-linked variants