27 Jun 2019

Abstract

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. Here, we report a 3.7 Å 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.

[Image]

Fig. 1 Cryo-EM structure of the asymmetric Cdc48-Shp1-substrate complex.

(A) Cdc48 and Shp1 domain organization. Gray boxes indicate domains that are seen in the cryo-EM reconstruction. WA, Walker A; WB, Walker B; L1, pore loop 1, L2, pore loop 2; Arg, finger arginines.

(B, C) High (B) and low (C) threshold views of the cryo-EM reconstruction. Densities are segmented for the six Cdc48 subunit ATPase cassettes, N domains (tan), translocating substrate (magenta), and the Shp1 UBX domain (orchid). Side-view densities are transparent in order to visualize substrate density in the pore.

(D) Top and side views of the Cdc48-Shp1-substrate model in ribbon representation. Subunit C (yellow) is removed in the side view to visualize the substrate.

(E) Model of Cdc48 (subunit B shown) with the associated Shp1 UBX domain, substrate, and nucleotides (pink spheres).