1. Volume 34, Issue 5, Pages 580-590 (June 2009)
Structure and Activity of the N-Terminal Substrate Recognition Domains in Proteasomal ATPases 
Sergej Djuranovic, Marcus D. Hartmann, Michael Habeck, Astrid Ursinus, Peter Zwickl, Jörg Martin, Andrei N. Lupas, Kornelius Zeth 
Molecular Cell 
Volume 34, Issue 5, Pages (June 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Alignment of Proteasomal ATPases from the Three Domains of Life
(Top) For the coiled coil, the positions of the hydrophobic core residues are marked (a, d) and the regions predicted to assume coiled-coil structure in the individual sequences are underlined; the sequence of the GCN4 leucine zipper is included for reference, and the location of the trigger sequence essential for folding is indicated. (Middle) For the OB fold, the two domains of Actinobacteria are aligned beneath each other; the consensus secondary structure is shown as S = β strand, b = β bulge. Two positions are marked by arrows and labeled: the residue at the nexus of the pore at the center of the hexameric OB rings and the GD box of the connecting loop, which mediates the orthogonal transition to the C-terminal β-hairpin and forms a backbone hydrogen bond to the N-terminal β strand, providing closure (Coles et al., 2005). (Bottom) The AAA domain is shown only up to the P loop motif; its predicted secondary structure, as deduced from sequence comparison to AAA domains of known structure, is shown as h = α helix, s = β strand. Residues conserved in at least half of the aligned sequences are highlighted in black; at these positions, similar residues are highlighted in gray. The sequences are as follows: from bacteria: ReARC (Rhodococcus erythropolis gi ), MtARC (Mycobacterium tuberculosis gi ), KrARC (Kineococcus radiotolerans gi ); from archaea: AfPAN (Archaeoglobus fulgidus gi ), MjPAN (Methaocaldococcus jannaschii gi ), ApPAN (Aeropyrum pernix gi ); from eukaryotes: HsRPT1-6 (Homo sapiens RPT1, gi ; RPT2, gi ; RPT3, gi ; RPT4, gi ; RPT5, gi ; RPT6, gi ).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Structure of the ARC N Domain
(A) Side and top views of the double OB ring of ARC-N. The chains are shown as ribbon diagrams in rainbow coloring, from N (blue) to C terminus (red). The termini (residues 78 and 220–227) are not visible due to flexibility; in addition, residues 177–192 are not visible in one monomer of the asymmetric unit and residues 182–198 in the other two.
(B) Cutaway side view of a space-filling representation of the ARC-NΔcc hexamer. Positively charged residues are colored blue, and negatively charged residues are colored red. Several of the charged residues in the cavity are well conserved, e.g., Arg101, Glu163, and Arg165. The surface charge representation of an ARC-N model with its native coiled coil is shown in Figure S3.
(C) Electron density map of the interface between the OB1 and OB2 domains of one ARC-NΔcc monomer (chain A). The interface is extensive, quite hydrophobic, and includes five aromatic residues, of which four are well conserved in ARC-N domains (Phe97, Tyr136, Tyr210, and Phe212).
(D) A single ARC-NΔcc subunit (chain A) in rainbow colors. The two OB domains have the same orientation in their respective rings and are displaced by 2 nm along the six-fold axis.
(E) Stereo view of the superimposed OB1 (blue) and OB2 (orange) domains of one ARC-NΔcc subunit (chain A). The corresponding sequence alignment is shown underneath.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Structure of the PAN N Domain
(A) Side and top views of the GCN4-PAN-NΔcc hexamer. The chains are shown as ribbon diagrams in rainbow coloring. The central threefold axis is marked in the top view.
(B) Space-filling views of the GCN4-PAN-NΔcc hexamer. In the side view on the left, the space-filling shell is transparent, showing the underlying chains in ribbon representation. The two parts of the chimera are colored orange (GCN4 cc) and blue (PAN OB). In the top view on the right, positively charged residues are colored blue, and negatively charged residues are colored red. The surface charge representation of a PAN-N model with its native coiled coil is shown in Figure S3.
(C) Chains A and B of the GCN4-PAN-NΔcc structure, showing the asymmetric location of the coiled coil above the β4-β5 hairpin of chain B. The blowup shows the electron density map in the linker region.
(D) Superposition of chains A and B, showing the asymmetry of the linker. The equivalent view for the Pro61Ala mutant, including the electron density map of the linker, is shown in Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Model of the PAN ATPase Showing the Conformational Range of the Coiled Coil
Side view (A) and top view (B). The experimentally determined structure is shown in solid lines, and samplings of conformations accessible to the linker are added in transparent representation (rainbow colors). A model of the ATPase ring (gray) was added to illustrate the relative domain sizes and overall shape of the complex. Given the lack of structural knowledge on the long connector between the OB and ATPase rings, we did not attempt to model this part of the structure.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 ARC-PAN Comparisons
(A) Stereo view of the superimposed ARC OB1 and PAN OB. The side chains of key residues are shown in stick representation. The corresponding sequence alignment is underneath, with structurally equivalent residues colored magenta and identical residues connected by bars. PAN OB is only slightly more similar to ARC OB1 (1.2 Å rmsd) than to ARC OB2 (1.3 Å rmsd), despite a substantially higher sequence similarity (35% versus 19% identity).
(B) Superposition of the ARC-NΔcc and GCN4-PAN-NΔcc hexamers in side and top view. The PAN OB ring is somewhat closer to the OB2 ring of ARC (rmsd of 1.6 Å) than to the OB1 ring (rmsd of 2.1 Å), due to the more similar ring diameter, and is more similar to either than these are to each other (rmsd of 2.8 Å).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Chaperone Activity of N Domains
Each point was measured at least three times; the values are shown ±the standard error.
(A) Inhibition of thermal aggregation of citrate synthase and luciferase by ARC-N and PAN-N as a function of the molar ratio between substrate and chaperone.
(B) Inhibition of aggregation by wild-type and chimeric proteins at a molar ratio to substrate of 6:1.
(C) Inhibition of aggregation by linker mutants at a molar ratio to substrate of 6:1.
Molecular Cell  , DOI: ( /j.molcel )
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