The cellular machine Cdc48 functions in multiple biological pathways by segregating its protein substrates from a variety of stable environments such as organelles or multi-subunit complexes. Despite extensive studies, the mechanism of Cdc48 has remained obscure, and its reported structures are inconsistent with models of substrate translocation proposed for other AAA+ ATPases (adenosine triphosphatases). Here, we report a 3.7-angstrom–resolution structure of Cdc48 in complex with an adaptor protein and a native substrate. Cdc48 engages substrate by adopting a helical configuration of substrate-binding residues that extends through the central pore of both of the ATPase rings. These findings indicate a unified hand-over-hand mechanism of protein translocation by Cdc48 and other AAA+ ATPases.

The p97 AAA+ATPase is an essential and abundant regulator of protein homeostasis that plays a central role in unfolding ubiquitylated substrates. Here we report two cryo-EM structures of human p97 in complex with its p47 adaptor. One of the conformations is six-fold symmetric, corresponds to previously reported structures of p97, and lacks bound substrate. The other structure adopts a helical conformation, displays substrate running in an extended conformation through the pore of the p97 hexamer, and resembles structures reported for other AAA unfoldases. These findings support the model that p97 utilizes a “hand-over-hand” mechanism in which two residues of the substrate are translocated for hydrolysis of two ATPs, one in each of the two p97 AAA ATPase rings. Proteomics analysis supports the model that one p97 complex can bind multiple substrate adaptors or binding partners, and can process substrates with multiple types of ubiquitin modification.

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Cdc48 (also known as p97 or VCP) is an essential and abundant AAA+ ATPase. It acts to separate proteins from a variety of contexts, including organelle membranes, ribosomes, protein complexes, and chromatin 1. This "segregase" activity is best described for unfolding proteins that are presented to the proteasome for degradation. Inhibition of human Cdc48 is an emerging therapeutic strategy for cancer treatment 1 and mutations in the human homolog of Cdc48 are linked to diseases such as multisystem proteinopathy 2. Cdc48 has a general structure of two ATPase domains capped by an N-terminal domain. Six Cdc48 subunits bind together in order to form a hexamer that has a central pore with pore loops from each subunit reaching into the pore. The N-terminal domain that sits atop Cdc48 is responsible for the binding of most adaptor proteins of Cdc48 3. Until recently, all published structures show Cdc48 in a six-fold symmetric conformation with no substrate bound. This is true regardless of the nucleotide state of these structures 4-5 。 However, these symmetrical structures most likely do not represent the active state of Cdc48.