1. The Mammalian Proteasome Activator PA28 Forms an Asymmetric α4β3 Complex 
Eva M. Huber, Michael Groll 
Structure 
Volume 25, Issue 10, Pages e3 (October 2017)
DOI: /j.str
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2. Structure 2017 25, 1473-1480.e3DOI: (10.1016/j.str.2017.07.013)
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3. Figure 1 Architecture and Key Sequences of WT and Mutant PA28 Subunits
(A) Schematic illustration of WT and mutant mouse PA28 α (gray; 249 amino acids) and β (green; 239 residues) subunits used in this study according to the sequence alignment with the human α subunit (Knowlton et al., 1997) (Figure S1A). Key sequence motifs are depicted by black double arrows. The homolog-specific insert of the α subunit is 13 amino acids longer than that of the β subunit.
(B) Mouse PA28 sequences (one-letter code) responsible for proteasome binding (activation loop and C termini) are aligned. For comparison, the sequence of the homologous PA26 subunit from Trypanosoma brucei is included. The conspicuous difference in amino acid 147 is highlighted in blue. See also Figures S1 and S2.
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4. Figure 2 Biochemical Characterization of PA28 Complexes
(A) Characteristic thermal unfolding curves and apparent melting temperatures for PA28 assemblies as determined by differential scanning fluorimetry (Figure S3C).
(B) Native PAGE of PA28 samples alone or bound to mouse iCPs. Note that PA28β7 does not bind to the proteasome. Identical results were obtained with mouse cCPs (Figure S4A).
(C) Native PAGE of chimeric PA28 samples alone or bound to mouse iCPs. Similar data for mouse cCPs are shown in Figure S4B.
(D) Homoheptameric PA28α and PA28β were mixed in defined ratios and incubated for 2 hr at room temperature prior to native PAGE. Additional bands compared with the control lanes (α, β, or αβ alone) indicate the formation of heterooligomeric PA28αβ species with varying subunit compositions.
(E) Anti-PA28β western blot of samples separated by native PAGE. iCPs were pre-incubated with PA28α in a molar ratio of 1:3 and incubated for half an hour at room temperature before addition of excess of β (1:2 molar ratio of α:β). As a control, half the amount of PA28β was loaded. For additional controls see Figure S4K.
(F) PA28αβ preparations were spiked with either PA28α7 or PA28β7 in defined ratios and the mixtures were incubated for 2 hr at room temperature prior to native PAGE analysis. Compared with the controls (α, β, or αβ alone) no additional bands were observed, indicating that PA28αβ complexes are stable with respect to their subunit composition.
The asterisks mark monomeric PA28α subunits. The molar ratio of PA28:CP was always at least 3:1, thus resulting only in double-capped species.
See also Figures S3 and S4.
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5. Figure 3 Crystal Structures of PA28 Complexes
(A) Quaternary structures of PA28 complexes as found by X-ray crystallography (see Figures S5A and S5B). The α and β subunits are shown as balls and colored in gray and green, respectively.
(B) Side and bottom views of the PA28α4β3 crystal structure shown as ribbons; coloring is according to (A).
(C) Illustration of the α and β subunits from the PA28α4β3 crystal structure as coils and their superposition. N and C termini are labeled. See also Figure S5.
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6. Figure 4 Subunit Interfaces
2FO – FC electron density maps (blue mesh contoured to 1σ) for sections of the α-β (left), the α-α (middle), and the β-β (right) subunit interfaces of PA28 crystal structures. Below: while Pro103 (α) and Met195 (β)/Gly195 (α) allow subunit association (left and middle), modeling of a β-β interface (gray) based on the PA28α4β3 crystal structure depicts steric hindrance of Tyr103 (β) and Met195 (β) (right). Superposition of the experimentally determined β-β assembly (green) onto the model (gray) visualizes reorientation of Tyr103 and Met195 upon association of the β chains. See also Figure S5 and Table S1.
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7. Figure 5
Schematic Representation of the Proposed PA28α4β3 Assembly Pathway
Isolated PA28 α and β chains exist in monomer-heptamer equilibria. In the presence of both entities, the most stable α-β interfaces are formed until the maximum number per heptamer, represented by the α4β3 state (framed), is reached. Note: in addition to the intermediates α6β1 and α5β2, also α1β6, α2β5, and α3β4 complexes transiently form when mixing α7 and β7 (see Figure 2D).
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