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

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. 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. The information here relates to the cross-linking mass spectrometry data portion of the project.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ian Kelsall

LAB HEAD: Yogesh Kulathu

PROVIDER: PXD078865 | Pride | 2026-07-28

REPOSITORIES: Pride

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			Action	DRS
	1-4_combined.zhrm	Other
	10-12_UFD1-NPL4-FAF2-p97hex-combined.zhrm	Other
	13-16_combined.zhrm	Other
	16-18_UFD1-NPL4-FAF2-K48-Ub6-p97hex-combined.zhrm	Other
	17-20_combined.zhrm	Other

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VCF1 is a p97 cofactor promoting recruitment of p97-UFD1-NPL4 to ubiquitylated substrates

Project description:The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discovered that FAM104A, a protein of unknown function that we named VCF1, acts as a novel p97 cofactor in human cells. Detailed structure-function studies revealed that VCF1 directly binds p97 via a conserved novel alpha-helical motif that recognizes the p97 N-domain with unusually high affinity exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 stimulates the affinity of the p97-UFD1-NPL4 complex for ubiquitin conjugates indirectly via its binding to p97 but does not itself interact with ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.

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