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. 2015 Apr 28;4:e07367. doi: 10.7554/eLife.07367

Figure 1. PCH-2/TRIP13 is a distinct class of hexmeric AAA+ ATPase.

(A) Phylogenetic tree of selected AAA+ ATPases, colored by clade (Erzberger and Berger, 2006). (B) Conserved AAA+ sequence motifs in Pch2/TRIP13, the ‘classic remodelers’, and E. coli ClpX. Pch2/TRIP13 and ClpX lack the first of two conserved arginine residues in the Arg finger region (yellow), and possess a Sensor 2 arginine (R385, red), which the classic remodelers lack. (C) Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis of C. elegans PCH-2 in the absence of nucleotides. Hexamer molecular weight = 288.1 kDa; measured molecular weight = 252 kDa (red line). (D) SEC-MALS analysis of M. musculus TRIP13 in the absence of nucleotides. The wild-type protein (black) adopts a mixture of oligomeric states from monomer to hexamer, consistent with findings from S. cerevisiae Pch2 (Chen et al., 2014). The proportion of higher-molecular weight oligomers increases upon the addition of ATP or non-hydrolyzable analogs (not shown). The ATP hydrolysis-defective TRIP13E253Q mutant (gray, molecular weight measurements red) is predominantly hexameric both in the presence (shown) and absence of ATP. Molecular weight measurements by SEC-MALS (red) are shown for TRIP13E253Q; WT measurements (not shown) are consistent. (E) Selected negative-stain EM class averages of C. elegans PCH-2 without added nucleotides (Apo) or with added ATP. For example raw images, see Figure 1—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.07367.003

Figure 1.

Figure 1—figure supplement 1. Negative-stain electron microscopy (EM) of C. elegans PCH-2.

Figure 1—figure supplement 1.

(A) Example negative-stain EM image of C. elegans PCH-2 without added nucleotide (Apo), and selected class averages from XMIPP clustering 2D alignment (from 32 classes, 5916 total particles). (B) Example negative-stain EM image of PCH-2 in the presence of 1 mM ATP, and selected class averages (from 16 classes, 4297 total particles). PCH-2 hexamers adopt a more compact and sixfold symmetric conformation after ATP addition. Because of the strong bias toward top-down views of the hexamer, 3D reconstructions were not attempted for either state.
DOI: http://dx.doi.org/10.7554/eLife.07367.004