p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitinmodified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly.ATPase | electron microscopy | mechanism | structure | asymmetric complex T he protein p97 (also known as VCP and Cdc48 in yeast) is a ubiquitous type II AAA ATPase essential for cell viability (1). In complex with a large number of alternative adaptors the ATPase participates in numerous cellular activities including cell cycle regulation, membrane traffic, protein quality control, DNA metabolism, signaling, and apoptosis (reviewed in ref.2). One of the best-studied adaptors is the heterodimer Ufd1-Npl4, the only essential cofactor in yeast (3-6), which directs p97 into cellular processes regulated by ubiquitin-proteasome degradation such as endoplasmic reticulum-associated degradation (ERAD) (7-10), mitotic progression (11, 12), replication (13), and transcription factor activation (14, 15). In ERAD (16), mitosis (17), and nucleus reformation (12), the p97-Ufd1-Npl4 complex has been shown to recognize ubiquitylated substrates associated with different cellular structures and, with the energy obtained from the binding and/or hydrolysis of ATP, to extract them into the cytosol to be recycled after deubiquitylation or degraded by the proteasome (10,16,18,19). However, it remains unclear how the ubiquitin-binding domains in p97-Ufd1-Npl4 are positioned to recognize the substrate and how the conformational changes in p97 are translated into mechanic force acting on substrate molecules.The monomer of p97 (∼87 kDa) consists of an N-terminal domain, two AAA domains, termed D1 and D2, and a highly flexible C-terminal region (20)(21)(22). The N domain interacts with adaptor proteins (23). The D1 domain is proposed to be responsible for oligomerization and the D2 domain for ATP hydrolysis, although both D1 and D2 are competent for binding and hydrolysis of . Several studies have shown that p97 hexamerizes into a double ring barrel with a central pore and undergoes conformational changes during the hydrolysis cycle (21,22,(27)(28)(29)(30). The role of the pore in the recruitment and extraction of substrates is still contentious. It has been shown that substrates interact with residues in the D2 pore on the distal side of the ring (31, 32), and it has been suggested that they are not threaded down the central pore as proposed for other members of the AAA family (33-36).Ufd1 (∼34 kDa) and Npl4 (∼67 kDa) form a heterodimer with a 1∶1 ...