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. 2016 Nov 30;5:e21516. doi: 10.7554/eLife.21516

Figure 6. Functional coupling of the two AAA rings of Hsp104.

(A) The PS1-hairpin of AAA2, which was well-defined by electron density (inset: omit density map contoured at 1.0 σ), forms specific contacts within the AAA1* active site. Bound ADP and interacting residues are shown in stick mode. (B) Characterization of the PS1 deletion (∆PS1) and the G731R mutant showing that the PS1-hairpin is essential for unfoldase and disaggregase activity, but not for ATPase activity. (C and D) ATPase and mant-ATP binding assays reveal the role of the PS1-hairpin in adjusting the activities and nucleotide binding affinities of AAA1 and AAA2 to each other. Strongest effects of the ∆PS1 mutation are highlighted (red arrow). The used AAA variants (WA/WB combined with wildtype) are indicated. Error bars represent standard deviations.

DOI: http://dx.doi.org/10.7554/eLife.21516.017

Figure 6.

Figure 6—figure supplement 1. Position of the PS1-hairpin in hexameric Hsp104.

Figure 6—figure supplement 1.

Top and middle row: Ribbon presentation of molecular models showing CtHsp104 in the planar and helical conformation. Models were constructed by overlying the AAA1 part of the crystallized CtHsp104 L/S* rigid bodies onto the respective AAA1 modules of the hexameric ClpC (top, PDB 3pxi [Wang et al., 2011]) or ScHsp104 (middle, PDB 5kne [Yokom et al., 2016]). Of note, the subunit that closes the hexamer by forming an uncanonical AAA1S-AAA2L* interface is left out from the alignment. The zoomed in windows illustrate the position of the PS1-haripin with nucleotides and functional residues shown in stick mode. Models are colored according to Figure 1B and ScHsp104 is shown in blue. Bottom row: Ribbon presentation showing the alignment of one Ctsp104 rigid body (yellow) onto the ScHsp104 hexamer (L/S* modules colored in blue and grey, respectively) with one functional unit magnified. As the MD is only partially resolved in the ScHsp104 EM structure, this domain was omitted from the presentation.
DOI: http://dx.doi.org/10.7554/eLife.21516.018
Figure 6—figure supplement 2. Effect of PS1-hairpin deletion on ScHsp104 activity.

Figure 6—figure supplement 2.

ATPase and GFP unfolding activity of wt and ΔPS1 mutants of Ct and ScHsp104 (Ct: C. thermophilium, Sc: S. cerevisiae). Error bars represent standard deviations and arrows indicate the decrease in unfolding activity.
DOI: http://dx.doi.org/10.7554/eLife.21516.019
Figure 6—figure supplement 3. SEC profiles of CtHsp104 dWA and dWB mutants.

Figure 6—figure supplement 3.

SEC profiles confirm the formation of stable hexamers for CtHsp104 (wt) and the catalytic inactive double Walker A (dWA: K230A/K641A) and double Walker B (dWB: D295A/E296A) mutants. Elution volumes of marker proteins are indicated.
DOI: http://dx.doi.org/10.7554/eLife.21516.020
Figure 6—figure supplement 4. Effect of casein on ATPase activity.

Figure 6—figure supplement 4.

ATPase activity assays confirm the importance of the PSI-hairpin in coordinating activities between AAA1 and AAA2 in the presence of casein. The used AAA variants (WA/WB combined with wildtype) are schematically indicated. The strongest effect of the ∆PSI mutation is highlighted (red arrow). Error bars represent standard deviation.
DOI: http://dx.doi.org/10.7554/eLife.21516.021