Structures of Asymmetric ClpX Hexamers Reveal Nucleotide-Dependent Motions in a AAA+ Protein-Unfolding Machine

Abstract

Summary ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.

Structural basis for intersubunit signaling in a protein disaggregating machine

“…The existence of an ISS network that regulates ATP hydrolysis in diverse AAA+ ring complexes is also consistent with a sequential ATP-hydrolysis mechanism proposed for ClpB (45,46) and Hsp104 (47), with four out of six subunits in the ClpB homohexamer occupied by nucleotides at any one time (46). This model is similar to the staircase mechanism proposed for the T7 gene 4 ring helicase (48), and is consistent with the nucleotide occupancy observed in the crystal structure of an engineered, covalently linked ClpX hexamer (11). In our model (Fig.…”

“…Like other type II AAA+ ATPases, ClpB forms a double-ring structure, with six copies of the D1 and D2 domains each making up a homohexamer ring (5,8). Although ClpB shares similar quaternary structure with ClpA (9), ClpC (10), and the single-ring ClpX (11) and HslU (12, 13) AAA+ ATPases, which function as the protein unfoldase components of energy-dependent proteases, ClpB does not associate with a chambered peptidase to degrade proteins. Instead, ClpB cooperates with the cognate Hsp70 system (DnaKJ/GrpE) in a species-specific manner (14,15) to recover functional protein from aggregates (16)(17)(18).…”

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

“…The AAA-ATPase heterohexamer is thought to undergo largescale motions during its functional cycle (61), probably similar to the ClpX hexamer (62). One function of the hexamer formed by the PCI-containing subunits seems to be that of a scaffold, which positions Rpn11 near the mouth of the AAA-ATPase.…”

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

“…Sensor 2 motifs are only present in a subset of AAA+ proteins. We compared ClpB NBD2 with ClpX (PDB entry 3hws; Glynn et al, 2009) and also found similar distances and orientations regarding the sensor 2 arginine. Even though we cannot exclude that our single NBD2 structures somewhat differ from their counterparts in the hexamer, this comparison indicates that they may very well represent snapshots of relevant states that are also adopted in the hexameric context.…”

“…Here, a conserved lysine from the neighbouring subunit works in concert with the classic arginine finger. Furthermore, extensive mechanistic and structural studies regarding the hexameric assembly have been performed for ClpX, another prominent AAA+ unfoldase (Glynn et al, 2009(Glynn et al, , 2012Martin et al, 2005;Stinson et al, 2013), and for p97, an ERADassociated AAA+ protein (Davies et al, 2008;Li et al, 2012;Nishikori et al, 2011). To evaluate our results, we compared the average distances between the nucleotide and the catalytically relevant Walker and sensor residues in our structures with those observed in the available high-resolution hexameric AAA+ structures, using the analysis presented by Wendler and coworkers as a starting point (Wendler et al, 2012).…”