FAF1 and FAF2 enhance unfolding by p97-UFD1-NPL4 complex enabling rational design of p97 activators

Abstract

VCP/p97 is an AAA+ ATPase that, together with its cofactors UFD1-NPL4 (p97-UN), 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 cofactors and identify FAF2 to potently enhance substrate unfolding by p97-UN. 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, thereby stabilizing and supporting the unfolding of the initiator ubiquitin in a UFD1-dependent manner. We leverage the features of the FAF2 activation motif 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. Efficient unfolding by the ATPase p97/VCP is essential for protein quality control, yet human p97 is far less efficient than its yeast counterpart for reasons that have remained unclear. This work investigates the cofactor and the mechanism enabling efficient unfolding by the human enzyme. A cofactor screen identifies FAF2 as the strongest activator of substrate unfolding by the human p97-UFD1-NPL4 complex. FAF2 binds the p97-UFD1-NPL4 complex in a UFD1-dependent manner through its conserved activation motif. The FAF2 activation motif simultaneously engages the UFD1 UT3 domain, the substrate-proximal and initiator ubiquitin, thereby accelerating the rate-limiting initiator unfolding step. These mechanistic insights allow computational de-novo design of mini-proteins that potently activate human p97. A cofactor screen identifies FAF2 as the strongest activator of substrate unfolding by the human p97-UFD1-NPL4 complex. FAF2 binds the p97-UFD1-NPL4 complex in a UFD1-dependent manner through its conserved activation motif. The FAF2 activation motif simultaneously engages the UFD1 UT3 domain, the substrate-proximal and initiator ubiquitin, thereby accelerating the rate-limiting initiator unfolding step. These mechanistic insights allow computational de-novo design of mini-proteins that potently activate human p97. A cofactor screen identifies FAF2 as the strongest activator of substrate unfolding by the human p97-UFD1-NPL4 complex.