A Major Conformational Change in p97 AAA ATPase upon ATP Binding Isabelle Rouiller, Virginia M Butel, Martin Latterich, Ronald A Milligan, Elizabeth M Wilson-Kubalek  Molecular Cell  Volume 6, Issue 6, Pages 1485-1490 (December 2000) DOI: 10.1016/S1097-2765(00)00144-1

Figure 1 Purification and Imaging of p97 (A) Coomassie blue–stained SDS-PAGE gel of native p97 purified from bovine liver and murine p47 purified as a recombinant protein from E. coli. (B) Cryoelectron micrograph showing a field of p97 complexes purified in the absence of nucleotide. Scale bar, 200 Å. Molecular Cell 2000 6, 1485-1490DOI: (10.1016/S1097-2765(00)00144-1)

Figure 2 Conformations of p97 and Comparison with NSF D2 Six-fold symmetrized average projection maps of p97 complexes, in the absence of nucleotide (A), bound to AMP–PNP (B), and to ADP (C). Gray scale maps show the protein densities as white on a dark background. Contour plots of the nucleotide-free p97 (D), p97–AMP–PNP (E), and p97–ADP (F) emphasize the main features: a central hole, six regions of low density (l), surrounded by six inward-pointing peaks (p1), six outward-pointing peaks (p2), and six small peaks (p3) at the periphery. Upon binding of nucleotide, the domain encircled in (D) rotates (arrows in E). One of the six areas, which contains the small but statistical difference between the AMP–PNP and ADP state, is circled in (F). The NSF D2 domains from the PDF files of NSF D2 (Protein Data Bank ID code 1NSF; Yu et al. 1998) were imported into Spider, converted into densities, and low-pass filtered at 20 Å resolution. The en face projection was calculated, and displayed as a gray scale map (G) and contour map (I). The calculated projection map formed by six NSF D2 domains was split in two halves: (H1) showing only the N subdomains and (H2) only the C subdomains. Scale bar, 40 Å. Molecular Cell 2000 6, 1485-1490DOI: (10.1016/S1097-2765(00)00144-1)

Figure 3 Conformations of the p97–p47 Complex The 6-fold symmetrized average projection map of the p97–p47 complex in the presence of ATP was displayed as a gray scale map (A) and a contour map (B). This average has been aligned with the average of p97–AMP–PNP (Figure 2B). The areas of weak densities (l) and the peaks of density p1 and p2 are indicated. Six additional areas of density at the periphery of the complex are clearly visible. The difference map between p97–p47–ATP and p97–AMP–PNP was calculated, and a contour map representing one sixth of the extra mass is shown on projection map A. In the absence of nucleotide, the average projection map of the p97–p47 complex is less ordered, and the densities attributed to p47 are not well defined and appear to be less rigidly bound. Four independent averages of the p97–p47 (nucleotide-free) complexes are shown in (C). For comparison, four similarly obtained averages of p97–p47–ATP are shown in (D). Scale bar, 40 Å. Molecular Cell 2000 6, 1485-1490DOI: (10.1016/S1097-2765(00)00144-1)

Figure 4 Model for p97–p47 in Action Model for the p97–p47 function in disassembling an organelle (t-t-SNARE complex or a SNARE inhibitor–t-SNARE complex). In the absence of nucleotide, the p47 molecules (shadowed in gray) are weakly bound to the periphery of the p97 hexamer and are mobile. This freedom of movement allows them to bind to the organelle complex (schematically represented here as two intertwined structure elements). Upon ATP binding, the p47 molecules become rigidly attached at the periphery of the p97 hexamer. This change from a disordered to an ordered complex could serve to pull the organelle apart. Molecular Cell 2000 6, 1485-1490DOI: (10.1016/S1097-2765(00)00144-1)