Nucleotide Binding and Conformational Switching in the Hexameric Ring of a AAA+ Machine

Stinson, Benjamin M.; Nager, Andrew R.; Glynn, Steven E.; Schmitz, Karl R.; Baker, Tania A.; Sauer, Robert T.

doi:10.1016/j.cell.2013.03.029

Cited by 98 publications

(174 citation statements)

References 33 publications

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“…ATP binding would be initiated on one side of the ring at the nucleotide-free sites where the subunits are most widely spaced, a model reminiscent of the rotary mechanism proposed for the F1 ATPase (49). Interestingly, in the case of ClpX, there may also be one or two nucleotide-free sites in the ring (10).…”

Section: Discussionmentioning

confidence: 99%

“…For the F1 ATPase, it has been established that ATPase domains hydrolyze ATP continuously in a strictly consecutive manner around the ring (2). In contrast, in another single-ring ATPase, ClpX, several ATPase domains hydrolyze ATP in sporadic bursts, either simultaneously or in short succession (3,(8)(9)(10). For double-ring ATPases, the coordination between ATPase subunits is even more complex, as there may be communication both within a given ring and between the two rings.…”

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confidence: 99%

“…Unfortunately, only a few full-length structures are available at a resolution that allows fitting of the polypeptide chain (10,(19)(20)(21). Crystallography has proven difficult, particularly because crystallization is favored by symmetric structures in which all subunits are in the same nucleotide state.…”

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confidence: 99%

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Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Blok

Tan

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.Pex1 | Pex6 | AAA ATPase | cryo-electron microscopy | peroxisome T he AAA (ATPases associated with diverse cellular activities) family of ATPases contains a large number of proteins that perform important functions in cells (1). Many members of this family form hexamers. These hexamers consist either of a single ring of ATPase domains or of stacked rings formed by tandem ATPase domains in a single polypeptide chain (type I and II ATPases, respectively). Both classes of AAA ATPases often use the energy of ATP hydrolysis to move macromolecules. Examples of single-ring ATPases include the F1 ATPase (2), which rotates a polypeptide inside its central pore, ClpX (3), which moves polypeptides into the proteolytic chamber of ClpP, and the helicase gp4 of T7 phage (4), which pulls a DNA strand through its center. Double-ring ATPases generally act on polypeptides. Prominent members of this family include N-ethylmaleimide sensitive factor (NSF), p97/VCP (valosin-containing protein), and Hsp104 in eukaryotes and ClpB in bacteria (5-7). NSF dissociates SNARE complexes generated during membrane fusion, p97/VCP plays a role in many processes, including ERassociated protein degradation (ERAD), and Hsp104 and ClpB disassemble protein aggregates.The molecular mechanism of many hexameric AAA ATPases is only poorly understood, particularly for those that move polypeptide chains. In addition, the mechanism appears to differ among the known examples. For the F1 ATPase, it has been established that ATPase domains hydrolyze ATP continuously in a strictly consecutive manner around the ring (2). In contrast, in another single-ring ATPase, ClpX, several ATPase domains hydrolyze ATP in sporadic bursts, either simultaneously or in short succession...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Blok

Tan

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Such a configuration is consistent with a proposed rotary hydrolysis mechanism [8,9] in which ATP hydrolysis takes place in coordinated waves counterclockwise around the ring. However, it is at odds with nucleotide binding configurations known for other AAA+ family ATPases that unfold proteins, such as the bacterial ClpX [10]. This suggests that the catalytic cycle of the proteasome may diverge from that of related ATPases.…”

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confidence: 47%

Proteasomes, caught in the act

Tomko

2017

Cell Res

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Although energy-dependent protein destruction by the proteasome has been known for over 30 years, how this intricate molecular machine uses ATP to power protein degradation has remained very poorly understood. In a recently published paper, Ding et al. present a snapshot of the proteasome mid-catalysis, yielding new and unexpected insights into the catalytic mechanism of this ATPpowered multisubunit machine.Cells rely on large multisubunit molecular machines to conduct many complex biological processes, such as protein synthesis, folding, and degradation. In eukaryotes, most regulated protein degradation is conducted by the proteasome, a massive 66-subunit ATP-dependent protease complex. Although recent advances in cryo-electron microscopy (cryo-EM) have yielded an onslaught of information about the proteasome, none have yet revealed how ATP drives the proteasome's catalytic cycle. By pharmacologically trapping the proteasome in a transition state, Ding et al. [1] provide a snapshot of the proteasome's molecular motor in action. This snapshot yields answers as well as poses new questions regarding the catalytic mechanism of this fascinating multisubunit machine.The proteasome consists of three subcomplexes ( Figure 1A), the barrelshaped core particle (CP), which houses the peptidase active sites; the ringshaped regulatory particle (RP) base, which abuts the ends of the CP; and the RP lid, which embraces the base and CP and hovers over their central pores. These subcomplexes function together much like an assembly line to bind the

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“…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).…”

Section: Comparison With Other Aaa+ Protein Structures Regarding the mentioning

confidence: 99%

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

Zeymer

Barends

Werbeck

et al. 2014

Acta Cryst D Biol Crystallogr

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ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotidebinding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg 2+ ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.

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Nucleotide Binding and Conformational Switching in the Hexameric Ring of a AAA+ Machine

Cited by 98 publications

References 33 publications

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Proteasomes, caught in the act

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

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