Plant UBX domain-containing proteins use distinct strategies to stably engage the unfoldase CDC48A.

The AAA+ ATPase CDC48A is a central regulator of proteostasis in plants, functioning through interactions with a diverse set of cofactors. Among these, the plant-specific ubiquitin regulatory X (UBX) domain-containing proteins (PUX) are key adaptors that direct CDC48A to specific substrates and pathways. The molecular basis of PUX-CDC48A interactions remains incompletely understood. Here, we combined structural, biophysical, and computational approaches to dissect the binding modes of representative PUX proteins from different subfamilies in Arabidopsis thaliana. Although all PUX proteins tested exhibit low micromolar affinities for CDC48A, they form unexpectedly stable complexes, suggesting additional mechanisms of interaction. We identified two distinct strategies for complex stabilization, producing different dynamic features. One relies on combining two weak associations: PUX5 employs a SHP-UBX module that engages the CDC48A N domain at two proximal sites, whereas PUX2 uses a SHP motif and a distant PUB domain to engage the N- and C-termini of CDC48A. In contrast, PUX6, PUX7, and PUX9 allosterically stabilize the association between their UBX domain and the CDC48A N domain. These multi-pronged strategies likely enable durable, yet reversible associations, facilitating fine-tuned competitive regulation of CDC48A activity across diverse cellular contexts. Our findings provide a mechanistic framework for understanding how PUX proteins achieve specificity, stability, and regulatory flexibility in directing CDC48A function.