Nucleotide‐dependent conformational changes of the AAA+ ATPase p97 revisited

Abstract

Edited by Peter BrzezinskiThe ubiquitous AAA-ATPase p97 segregates ubiquitylated proteins from their molecular environment. Previous studies of the nucleotide-dependent conformational changes of p97 were inconclusive. Here, we determined its structure in the presence of ADP, AMP-PNP, or ATP-cS at 6.1-7.4 A resolution using single particle cryo-electron microscopy. Both AAA domains, D1 and D2, assemble into essentially six-fold symmetrical rings. The pore of the D1-ring remains essentially closed under all nucleotide conditions, whereas the D2-ring shows an iris-like opening for ADP. The largest conformational changes of p97 are 'swinging motions' of the N-terminal domains, which may enable segregation of ubiquitylated substrates from their environment.

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

“…S1 and S2). The map shows VAT in a split-ring conformation that has not been observed previously for p97/VCP (38)(39)(40)(41)(42). For the ATPγS-loaded state, 514 micrographs were collected and subjected to a similar analysis.…”

“…The sixfold symmetrical, stacked-ring conformation of ATPγS-loaded VAT resembles the crystal structure of homologous Mus musculus p97/VCP in complex with a mixture of ADP and ADPAlF 6 (42) and recent high-resolution cryo-EM structures of p97 in different nucleotide states (38,39) and in the presence or absence of an allosteric inhibitor (38). Consequently, it was surprising to observe that in the ADP-bound conformation, the protomers arrange in a helical manner so as to generate an asymmetrical, split-ring structure.…”

“…1 E-G and Fig. 2B, where D1-D2 interactions are formed rather than the "canonical" D1/D1 and D2/D2 contacts observed in crystal structures and cryo-EM maps of p97 (38)(39)(40)(41)(42) and manifest in the split-ring arrangement seen for VAT. The result is that the D2 domain of protomer 6, in particular, lacks many of the interactions that are present in other D2 domains, and its density is notably weak (Fig.…”

“…To date, there is no atomic resolution structure of VAT. However, an atomic model is available for a mammalian homolog, mouse p97 (39)(40)(41)(42), which has ∼45% sequence identity with the T. acidophilum VAT. Thus, a homology model was built for VAT using the crystal structure of mouse p97 [Protein Data Bank (PDB) ID code 3CF3] and the NMR structure of VAT NTD (PDB ID code 1CZ4).…”

Molecular Mechanism of Substrate Processing by the Cdc48 ATPase Complex

“…Other models postulate that the polypeptide chain inserts only shallowly into the D1 ring (for review, see Xia et al, 2016). Finally, it has been suggested that the large, nucleotide-dependent conformational changes of the N domains are sufficient to move a polypeptide chain (Schuller et al, 2016). In each of these cases, it is unclear how a continuous pulling force could be generated, although such a force would likely be required to extract a subunit from a tight multimeric complex or a multi-spanning protein from the ER membrane.…”