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bioRxiv · 10.64898/2026.02.24.707758

FAF1 and FAF2 enhance human p97-UFD1-NPL4 complex unfoldase activity enabling rational design of p97 activators

Dasgupta, P.·Kelsall, I. R.·Anand, G.·Perez-Rafols, A.·Knebel, A.·Gourlay, R.·Masson, G. R.·Kulathu, Y.
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Abstract

VCP/p97 is an AAA+ ATPase that, together with its cofactors UFD1-NPL4 (p97-UN), binds and unfolds ubiquitylated substrates to maintain cellular homeostasis. The human p97-UN complex associates with additional cofactors, but how these cofactors modulate p97-UN activity is not fully understood. Here, we screen for cofactors that enhance p97-UN activity and identify FAF2 as the strongest activator. Using biochemical and structural approaches, we show how FAF2 engages p97-UN and polyubiquitin to promote unfolding. We define a conserved activation motif in FAF2 that contacts both UFD1 and the ubiquitin proximal to the initiator, stabilizing and initiating unfolding in a UFD1-dependent manner. We leverage the features of FAF2 AM to engineer de novo proteins that potently enhance unfolding, providing a rational strategy to boost p97 activity. Our findings reveal how cofactors can provide additional adaptive control, fine-tuning human p97 activity to unfold challenging substrates and those modified with short ubiquitin chains.

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Dasgupta, P., Kelsall, I. R., Anand, G., Perez-Rafols, A., Knebel, A., Gourlay, R., Masson, G. R., Kulathu, Y.. 2026-02-25. FAF1 and FAF2 enhance human p97-UFD1-NPL4 complex unfoldase activity enabling rational design of p97 activators. https://doi.org/10.64898/2026.02.24.707758

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