1. An AAA Motor-Driven Mechanical Switch in Rpn11 Controls Deubiquitination at the 26S Proteasome 
Evan J. Worden, Ken C. Dong, Andreas Martin 
Molecular Cell 
Volume 67, Issue 5, Pages e8 (September 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 67, 799-811.e8DOI: (10.1016/j.molcel.2017.07.023)
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3. Figure 1 Structure of Ubiquitin-Bound Rpn11-Rpn8
(A) Schematic of full-length Rpn8 and Rpn11 (top) and the Rpn11-Rpn8 crystallization construct (bottom). The MPN domains of each protein are shown as colored rectangles, and the Ins-1 loop of Rpn11 is highlighted as an orange box. The C-terminal helices of Rpn11 and Rpn8 (orange cylinders), along with the N terminus and Ins-2 region of Rpn11 (yellow box), are disordered outside the proteasome context and were removed from the final crystallization construct.
(B) Structure of the Rpn11-Rpn8 heterodimer bound to mono-ubiquitin. Rpn11, the Rpn11 Ins-1 loop, Rpn8, and ubiquitin are colored green, orange, blue, and pink, respectively. The linker that replaces Rpn11’s Ins-2 is depicted as a green dashed line. Zinc-coordinating residues in the active site and residues that help mediate the Rpn11-ubiquitin interaction are shown in stick representation.
(C) 2|Fo|-|Fc| electron density surrounding the Rpn11 active site is shown as a gray mesh contoured at 1σ with a carve of 1.8 Å surrounding the model. The structure is depicted in stick representation and colored as in (B).
(D) Close-up view of the Rpn11 active site. Side chains of Rpn11 catalytic resides and the ubiquitin C-terminal Gly are shown in stick representation. The catalytic zinc ion is shown as a gray sphere, and its coordination sites are shown as gray dashed lines.
See also Figure S1.
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4. Figure 2
A Ubiquitin-Induced Conformational Switch of the Ins-1 Loop Is Rate-Limiting for Rpn11 Deubiquitination
(A) Structure of the Rpn11 active site without ubiquitin (PDB: 4O8X; Worden et al., 2014) in front view (left) and side view (right). Rpn11 is shown in gray surface and green ribbon representation, with the Ins-1 loop exposed and colored in orange. M50 and E48 form a hydrophobic pocket and are shown as green sticks.
(B) Structure of the Rpn11-Rpn8 heterodimer bound to mono-ubiquitin oriented and colored as in (A), with ubiquitin colored pink.
(C) Michaelis-Menten analyses of Ub-GC-TAMRA substrate cleavage by the WT Rpn11-Rpn8 heterodimer (▵) and its Rpn11(G77P) (○) and Rpn11(V80A) (■, inset) mutants.
(D) Ramachandran plot based on values from the program RAMPAGE ( Lovell et al., 2003) showing the allowed dihedral angles for glycine (blue) and proline (green). Dihedral angles for Rpn11 G77 in the ubiquitin-free and ubiquitin-bound states are shown as black dots.
See also Figures S2 and S3 and Table S1.
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5. Figure 3
Rpn11’s Ins-1 Loop Conformation Depends on the Proteasome State
(A) EM reconstruction of the yeast 26S proteasome in the ATP-bound S1 state (left; EMD-6575; Luan et al., 2016). The core particle, base, and lid are colored gray, tan, and orange, respectively. The MPN domains of Rpn11 and Rpn8 are colored green and blue, respectively. Center: crystal structures of ubiquitin-free Rpn11 (PDB: 4O8X; Worden et al., 2014; light green) and ubiquitin-bound Rpn11 (dark green, ubiquitin omitted) are docked into the EM density of the S1-state proteasome contoured at a threshold (transparent gray surface). The Ins-1 loop of Rpn11 in the ubiquitin-free and ubiquitin-bound state is colored magenta and orange, respectively. The entrance to the central translocation channel is indicated with a black dashed circle. The EM-derived model of the base ATPases (PDB: 3JCP; Luan et al., 2016) is docked into the EM density of the base and depicted as tan cylinders. Right: close-up view of Rpn11 showing the lack of EM density for the Ins-1 loop in the S1 state proteasome.
(B) Michaelis-Menten analyses of Ub-GC-TAMRA cleavage by WT Rpn11 (▵) and Rpn11(G77P) (○) incorporated into the S1-state proteasome.
(C) EM reconstruction of the yeast 26S proteasome in the S3 state (left; EMD-5669; Matyskiela et al., 2013), colored as in (A). The path of substrate though the base and into the core is depicted as a black dashed line. Center: crystal structures of ubiquitin-free and ubiquitin-bound Rpn11 (colored as in A, docked into the EM density of the S3-state proteasome (EMD-6574; Luan et al., 2016) contoured at a threshold (transparent gray surface). The putative contact between the Ins-1 loop of Rpn11 in the closed state and the Rpt5 coiled coil is indicated. Right: close-up view showing the bridging density that corresponds to the closed Ins-1 loop.
(D) Michaelis-Menten analyses of Ub-GC-TAMRA cleavage by WT Rpn11(▵) and Rpn11(G77P) (○) incorporated into the S3-state proteasome.
See also Figure S3 and Table S1.
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6. Figure 4
Coupling of Substrate Deubiquitination and Degradation at the Proteasome
(A) Top: diagram of the full-length synthetic G3P proteasome substrate. Bottom: cleavage with thrombin protease removes potential N-terminal ubiquitination, leaving a substrate that is ubiquitinated with only one chain at a specific position.
(B) Michaelis-Menten analyses of G3P substrate degradation by WT (▵), Rpn11(G77P) (○), and Rpn11(V80A) (■) proteasomes.
(C) Fluorescence-imaged SDS-PAGE gel showing the degradation of TAMRA-labeled G3P substrate by WT and Rpn11 (G77P) proteasomes. Only the Rpn11 (G77P) proteasome releases some of the fully deubiquitinated substrate (Ub0) because of premature deubiquitination prior to substrate engagement with the AAA motor. Control reactions with isolated WT and Rpn11 (G77P) lid subcomplexes exhibited no substrate deubiquitination, ruling out that the formation of Ub0 substrate in the Rpn11 (G77P) proteasome sample was caused by dissociated lid subcomplexes.
(D) SDS-PAGE gels for degradation time courses of the G3P substrate with catalytically dead Rpn11(AXA), WT Rpn11, and Rpn11(V80A) proteasome variants. Products of Rpn11-mediated deubiquitination accumulated because the proteolytic activity of the core particle was inhibited with epoxomicin. Gel bands were imaged using in-gel fluorescence of the TAMRA-modified G3P substrate.
See also Figure S3 and Table S2.
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7. Figure 5
Rpn11 Deubiquitination Is Strongly Accelerated by Mechanical Substrate Translocation
(A) Schematic depicting the experimental procedure used to stall and restart proteasomal degradation through reversible Rpn11 inhibition. The proteasome is shown as a gray silhouette, with the core particle emphasized in dark gray. Rpn11 (green) and the core particle are inhibited (red X). Substrate is engaged and translocated by the base ATPases (light blue) until the ubiquitin chain runs into the narrow constriction of the N ring, leading to a stalled state. Deubiquitination is initiated by reactivating Rpn11 with Zn2+, which allows the continuation of substrate translocation into the core particle.
(B) SDS-PAGE gels showing the time courses for G3P substrate deubiquitination following Rpn11 reactivation (Zn2+) and for a mock reaction where Rpn11 was not reactivated (mock). Substrate bands were visualized and quantified by detecting the fluorescence of the attached TAMRA dye, as shown in Figure 4.
(C) Quantification of band intensity for the mock (▴) and Zn2+ addition (○) experiments shown in (B). Error bars correspond to the SD of the data for three separate experiments.
(D) Comparison of G3P substrate processing rates in single- and multiple-turnover experiments. Single-turnover kcat values were calculated from the exponential decrease of high molecular weight Ubn bands in control experiments where substrate was never stalled. Multiple turnover kcat values were calculated from the Michaelis-Menten analyses shown in Figure 4.
(E) Rate constants of ubiquitin removal calculated from the exponential increase of Ub0, Ub1, and Ub2 substrate bands after Rpn11 reactivation. Shown are the deubiquitination rate constants for WT, Rpn11(G77P), and Rpn11(V80A) proteasomes in the presence of ATP (light gray) or an excess of the slowly hydrolysable analog ATPγS (dark gray). Rate constants for Ub-GC-TAMRA cleavage in the context of the S3 state proteasome (WT and G77P) or the isolated dimer (V80A) are also shown.
Error bars in (D) and (E) correspond to the SE of the fitted rate constants. See also Figures S3–S7 and Table S3.
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8. Figure 6
Model for Mechanochemical Coupling of Substrate Translocation and Rpn11 Deubiquitination during Proteasomal Degradation
The cut-away overview depicts the proteasome during the individual stages of degradation, with the core shown in dark gray, the regulatory particle in light gray, the AAA+ motor and the N-ring in light blue, and the Rpn11-Rpn8 dimer in green and blue.
(A) The substrate-free proteasome adopts the S1 state, in which the Rpn11 Ins-1 loop is unstructured and the active site partially occluded by the N-terminal coiled coil of the ATPase subunits Rpt4 and Rpt5.
(B) Upon substrate engagement with the ATPase motor, the regulatory particle switches to the S3 conformation, with the Rpn11 active site positioned directly above the entrance to the central pore and the Ins-1 loop stabilized in the closed state.
(C) Substrate translocation delivers the first ubiquitin of a poly-ubiquitin chain to the Rpn11 active site.
(C′) Failure of Rpn11 to catch a ubiquitin modification leads to a robust translocation stall that can be rapidly resolved by minimal backsliding of the substrate out of the AAA+ motor, allowing another attempt of ubiquitin binding and cleavage by Rpn11.
(D) Mechanical pulling on the substrate by the AAA+ motor facilitates the conformational switching of the Ins-1 loop from the inactive closed state to the active β hairpin state, which positions the scissile isopeptide bond (red dot) in the Rpn11 active site for cleavage.
(E) After cleavage, ubiquitin dissociates from Rpn11 and the proteasomal ubiquitin receptor, the Ins-1 loop switches back to a closed conformation, and the substrate is further translocated into the core for proteolytic cleavage.
Molecular Cell  , e8DOI: ( /j.molcel )
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