UFD1 Recognition of Initiator and Proximal Ubiquitin Drives p97-Mediated Substrate Unfolding enhanced by FAF1, FAF2, and UBXD7

Sato, Y.; Miyauchi, S.; Hirano, A.; Yasumoto, N.; Goto, Y.; Akizuki, Y.; Koyano, F.; Hino, T.; Nagano, S.; Matsuda, N.; Ohtake, F.; Hayashi, G.

doi:10.64898/2026.03.13.711538

bioRxiv · 10.64898/2026.03.13.711538

UFD1 Recognition of Initiator and Proximal Ubiquitin Drives p97-Mediated Substrate Unfolding enhanced by FAF1, FAF2, and UBXD7

Sato, Y.·Miyauchi, S.·Hirano, A.·Yasumoto, N.·Goto, Y.·Akizuki, Y.·Koyano, F.·Hino, T.·Nagano, S.·Matsuda, N.·Ohtake, F.·Hayashi, G.

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Abstract

The AAA-ATPase p97 and its primary adaptor UFD1-NPL4 (UN) unfold ubiquitinated substrates for their proteasomal degradation. Although cryo-EM has shown that substrate processing begins with unfolding of the initiator ubiquitin and its capture by NPL4, the specific contribution of UFD1 to K48-linked ubiquitin chain engagement has not been fully defined. Here we report a 1.31 [A] crystal structure of the human UFD1-UT3 bound to a K48-linked di-ubiquitin mimic, revealing that UFD1-UT3 engages the unfolded initiator ubiquitin via its Nc subdomain and the proximal ubiquitin via its Nn subdomain. AlphaFold3-guided analyses further demonstrate that accessory adaptors FAF1, FAF2, and UBXD7 enhance p97-UN activity through divergent strategies. FAF1 and FAF2 scaffold UFD1-UT3 near the NPL4-tower, whereas UBXD7 stabilizes transient interactions between UFD1 and the ubiquitin chain. Our results provide a comprehensive molecular model for the initiation of p97-mediated substrate unfolding.

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Sato, Y., Miyauchi, S., Hirano, A., Yasumoto, N., Goto, Y., Akizuki, Y., Koyano, F., Hino, T., Nagano, S., Matsuda, N., Ohtake, F., Hayashi, G.. 2026-03-16. UFD1 Recognition of Initiator and Proximal Ubiquitin Drives p97-Mediated Substrate Unfolding enhanced by FAF1, FAF2, and UBXD7. https://doi.org/10.64898/2026.03.13.711538

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