Structure of the Cdc48 segregase in the act of unfolding an authentic substrate

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 (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.

AAA+ ATPases in Protein Degradation: Structures, Functions and Mechanisms

“…Nevertheless, ATP hydrolysis in D1 does affect the ATPase activities of D2, and also plays an important role in substrate binding and release from the complex [154,[158][159][160]. These observations are compatible with the structure of the substrate-bound Cdc48 complex [146,152]. The D1 and D2 domains are connected by the D1-D2 linker region, serving as a flexible hinge.…”

“…Because the majority of substrate-bound AAA+ ATPase structures were solved in only one conformation at high resolution as per their biochemical condition, the sequential "hand-over-hand" model of coordinated ATP hydrolysis around the ATPase ring is largely speculative and hypothetical [131][132][133]138,146]. In few studies where coexisting conformations were obtained, there were no intrinsic features that revealed the time sequence of the events along the pathway of chemical reactions [118,145,147].…”

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

“…These structures show how asymmetric hexameric rings of ClpX dock with symmetric heptameric rings of ClpP and reveal how the pore-1, pore-2 and RKH loops of ClpX function in substrate binding. ClpX adopts a spiral conformation, with neighboring pore-1 loops interacting with every two residues of substrate in the axial channel, as observed in other AAA+ unfolding and remodeling machines (Puchades et al, 2017;de la Peña et al, 2018;Dong et al, 2019;Majumder et al, 2019;Ripstein et al, 2017;Gates et al, 2017;Zehr et al, 2017;Han et al, 2017;Su et al, 2017;Sun et al, 2017;Yu et al, 2018;White et al, 2018;Cooney et al 2019;Rizo et al, 2019;Shin et al, 2019;Twomey et al, 2019). Based on these structural features, strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been proposed.…”

“…Step). Based on similar hexamer architectures and/or substrate interactions, a SC/2R translocation model has also been proposed for Lon, for PAN/20S, and for the AAA+ proteinremodeling machines ClpB/Hsp104, CDC48/p97, NSF, and Vps4 (Gates et al, 2017;Han et al, 2017;Ripstein et al, 2017;Su et al, 2017;Sun et al, 2017;Zehr et al, 2017;Yu et al, 2018;White et al, 2018;Cooney et al 2019;Majumder et al, 2019;Rizo et al, 2019;Twomey et al, 2019;Shin et al, 2019).…”

Preparing scientists for a visual future

“…In order to better make sense of the vast and . Snapshot from animation visualizing the hand-over-hand mechanism of substrate processing by the Cdc48-Shp1 complex [11].…”