1. Structure of the AAA ATPase p97
Xiaodong Zhang, Anthony Shaw, Paul A. Bates, Richard H. Newman, Brent Gowen, Elena Orlova, Michael A. Gorman, Hisao Kondo, Pawel Dokurno, John Lally, Gordon Leonard, Hemmo Meyer, Marin van Heel, Paul S. Freemont 
Molecular Cell 
Volume 6, Issue 6, Pages (December 2000)
DOI: /S (00)00143-X

2. Figure 1 Crystal Structure of p97 N D1 Domain
(A) Ribbon representation of N D1 protomer structure. The domains are colored individually, with the N-terminal double ψ barrel domain (yellow), β barrel domain (gold), the D1 α/β domain (cyan), and the C-terminal α-helical domain (blue). Also highlighted are the Walker A (P loop) motif (black), Walker B (DExx box) motif (mauve), sensor loop (red), and the proposed hinge region between the N and D1 domains (magenta). Bound ADP nucleotide is shown as balls and sticks. For clarity, only the secondary structure elements discussed in the text are labeled.
(B) A close-up view of the N D1 interface (boxed region in [A]). A small rotation was applied to optimize the view of the interface.
(C) The N D1 hexamer forms a wheel-like structure of ∼160 Å in diameter and a central hole ∼15 Å in diameter. One protomer is colored as in (A). The α/β domain fold (cyan) forms the spokes of the wheel, while the helical domain (blue) forms the outer rim. N-terminal domains (yellow) are oriented counterclockwise off the main body of the wheel. Bound ADP moieties (brown) drawn as Van der Waal spheres are located between protomers. The arrow indicates the ADP binding pocket.
(D) The N D1 hexamer is rotated 90° (relative to Figure 1C) so that the amino-terminal side is on top. The thickness of N D1 is ∼20–40 Å. The N domain lies in the same plane as D1 and spans a similar thickness. Note the ADP moieties (brown spheres) bound in deep pockets.
(E) A close-up view of the hexamer interface (boxed in [C]). For clarity, only secondary structure elements discussed in the text are labeled. The dashed line indicates the protomer–protomer interface. The sensor residues (Asn-348, yellow; Arg-359, blue) are shown as sticks near the interface.
Molecular Cell 2000 6, DOI: ( /S (00)00143-X)

3. Figure 2 Structural Comparison with Other AAA Domains
(A) Superposition of N domain structures of NSF, Sec18, VAT, and p97. The overall topology and domain arrangements are conserved despite their low sequence identity. The arrow indicates a binding cleft for possible adaptor molecules.
(B) Comparison of p97 D1 ADP bound form (blue) with NSF D2 AMP–PNP bound form (green). P loops are superposed as are the α- and β-phosphates of ADP and AMP–PNP, respectively. However, the C-terminal α-helical domain has rotated toward the α/β domain in p97 D1 (blue). Also note the different locations of the corresponding linker region (p97 D1, magenta; NSF D2, orange).
(C) Sequence alignments of the highlighted regions in Figure 1A. Red represents conserved charged residues, while cyan represents conserved hydrophobic residues. Other conserved residues are colored in yellow. The sensor region is within the AAA minimum consensus region between Asn-348 and Arg-362. The indicated residue numbering is for p97. The linker region between the N and D1 domains contains conserved glycines at each end that could act as pivot points to allow the N domain and the linker loop to rotate as rigid bodies.
(D) Comparison of nucleotide binding pockets for p97 D1 and NSF D2. The nucleotides, as well as the residues involved in ADP or AMP–PNP binding, are drawn as balls and sticks and labeled. The surface surrounding the nucleotide is drawn (8 Å radius) to show the general shape of the binding pocket. An asterisk labels a residue from an adjacent protomer. The nucleotide has different conformations, probably due to the different sequences involved in nucleotide binding. Arg-362* interacts with Glu-305 (DExx box), while Arg-359* is available for γ-phosphate binding. Both residues could mediate any conformational changes upon ATP hydrolysis or ADP release.
Molecular Cell 2000 6, DOI: ( /S (00)00143-X)

4. Figure 5 Speculative Model of p97 Acting as a Molecular Ratchet
(A) Superposition of p97 D1 in an ADP state (blue) with NSF D2 in an ATP state (green) on their respective P loops. A small clockwise rotation around the hexamer axis is observed between the green and blue monomers. ADP moieties are drawn as brown spheres. Also shown are the corresponding linker regions between p97 N D1 (magenta) and NSF D1/D2 (orange).
(B) Possible ratchet mechanism for p97 with D1 and D2 rings acting as interdependent and counter-balanced parts in the ATP hydrolysis cycle. Top left, the D1 and D2 rings are shown as ATP- and ADP-bound states, respectively. ATP hydrolysis of D1 would cause a clockwise rotation of D1. Top right, the rotation of D1 causes a conformational change in D2 to allow ADP release, resulting in an ATP-ready or empty state, while D1 returns to an ADP-bound state. Bottom right, ATP hydrolysis in D2 would cause a counterclockwise motion. Bottom left, the rotation caused by D2 would allow the release of ADP in D1, resulting in an ATP-ready state, while D2 returns to an ADP-bound state. The binding of ATP by D1 would initiate another hydrolysis cycle (top left).
Molecular Cell 2000 6, DOI: ( /S (00)00143-X)

5. Figure 3 Cyro-EM Reconstruction of Rat Liver Cytosol p97 at 18 Å
(A) Side view of p97, perpendicular to the 6-fold molecular axis, shows a truncated barrel-like structure with side holes. The main body of the molecule shows pseudo 2-fold symmetry along an axis perpendicular to the 6-fold, although there are protruding densities at one end (top). The overall molecular dimensions are indicated. The 145 Å diameter of the EM map is less than the 160 Å observed in the N D1 crystal structure, which we attribute to the ill-defined N domain in the EM reconstruction.
(B) Cut-open view of (A) reveals a cage-like structure formed from the two-ring layers corresponding to the N D1 (blue) and D2 hexamers (green).
(C) Top view, down the 6-fold axis, corresponding to the N D1 hexamer (blue), reveals a large annulus, surface pockets, and disconnected densities, and is different in appearance to the D2 layer. The black arrow indicates the pocket that coincides with the nucleotide binding pocket indicated by the arrow in Figure 1C.
(D) Bottom view, corresponding to the D2 hexamer (green), shows a closed annulus and several distinctive surface features.
Molecular Cell 2000 6, DOI: ( /S (00)00143-X)

6. Figure 4
Comparison and Fitting of the D1 Crystal Structure and the D2 Homology Model with the p97 EM Structure
The D1/D2 model is in yellow, fitted into the EM reconstruction (blue).
(A) Top view, showing the overall dimension of the D1 model agrees with the main body of the EM density. The partially disconnected EM density on the outer rim accounts for the flexible N domain that is not shown for the sake of clarity.
(B) Bottom view, showing good agreement between the D2 model and EM reconstruction.
(C) Side view of the p97 D1/D2 hexamer model fitted into EM density. The width and height are in agreement, though there are extra densities in the EM map between the D1 and D2 rings that could correspond to the C-terminal 73 residues. The locations of the α-helical domains are indicated by long arrows.
(D) Tilted side view highlighting one p97 D1/D2 monomer model fitted into the EM density. The C-terminal α-helical domains from both the D1 and D2 protomers are located on the surface of the molecule near the 2-fold axis and could form part of an interhexamer interface. A possible linker region between D1 and D2 is indicated as a dashed yellow line. The short arrow indicates the location of the C termini of p97 D2.
Molecular Cell 2000 6, DOI: ( /S (00)00143-X)