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

Abstract

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.

Structures of TorsinA and its disease-mutant complexed with an activator reveal the molecular basis for primary dystonia

“…2F o −F c electron density is contoured at 1σ. The cysteine pair adopts three alternate conformations, but remains reduced in all of them. DOI: http://dx.doi.org/10.7554/eLife.17983.003

10.7554/eLife.17983.004Figure 1—figure supplement 1.Structural comparisons.( A ) Human TorsinA-ATP (left) displayed as a cartoon, compared to the D2 domain of the double-ringed AAA+ ATPase ClpB-AMPPCP (right) from Thermus thermophilus (Zeymer et al, 2014) (PDB code 4LJ9) in the same orientation. Important structure motifs are labeled.

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10.7554/eLife.17983.004Figure 1—figure supplement 1.Structural comparisons.( A ) Human TorsinA-ATP (left) displayed as a cartoon, compared to the D2 domain of the double-ringed AAA+ ATPase ClpB-AMPPCP (right) from Thermus thermophilus (Zeymer et al, 2014) (PDB code 4LJ9) in the same orientation. Important structure motifs are labeled.

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Structural basis for the disaggregase activity and regulation of Hsp104

“…Secondly, the central β-sheet of AAA2 appears to be slightly distorted in the Hsp104 filament. Structural comparison with the isolated AAA2 domain from ClpB (Zeymer et al, 2014) suggests that in the Hsp104 oligomer, contacts to the AAA1 domain, as well as to neighboring AAA2 subunits, induce a distortion of the central β-sheet, in particular of the two capping strands (Figure 7A and Figure 7—figure supplement 1). Notably, parallel β-sheets are presumed to be less stable than the anti-parallel counterparts (Richardson, 1977) and may thus be amenable to such deformation.…”

“…The zoomed-in windows illustrate structural details of how contacts with AAA1 and neighboring subunits may destabilize the central β-sheet of AAA2. For comparison, the structure of Hsp104 (yellow) is aligned with the AAA2 domain of ClpB, which was crystallized in isolated form (red, PDB 4lj5 [Zeymer et al, 2014]). DOI: http://dx.doi.org/10.7554/eLife.21516.023

10.7554/eLife.21516.024Figure 7—figure supplement 2.PS1+ and PP mutations partially uncouple the ATPase activity of AAA1 and AAA2.ATPase activity assay of wt, PS1+ and PP mutants. WA and WB mutations were introduced as indicated.

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Disruption ofCLPBis associated with congenital microcephaly, severe encephalopathy and 3-methylglutaconic aciduria

“…Key residues in this domain have been shown to be important for ATP binding and hydrolysis as well as for oligomerisation in most AAA+ proteins 8 10 18 19. Moreover, truncating the D2-Small domain completely abolishes the oligomerisation of the prokaryotic Clpb 20.…”