1. Volume 24, Issue 8, Pages 1257-1270 (August 2016)
Structures of Rpn1 T1:Rad23 and hRpn13:hPLIC2 Reveal Distinct Binding Mechanisms between Substrate Receptors and Shuttle Factors of the Proteasome 
Xiang Chen, Leah Randles, Ke Shi, Sergey G. Tarasov, Hideki Aihara, Kylie J. Walters 
Structure 
Volume 24, Issue 8, Pages (August 2016)
DOI: /j.str
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2. Structure 2016 24, 1257-1270DOI: (10.1016/j.str.2016.05.018)
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3. Figure 1
hRpn13Pru Preferentially Binds to hPLIC2UBL over K48 Diubiquitin
(A) Model of the proteasome highlighting substrate receptors Rpn1, Rpn10, and Rpn13Pru (light blue) generated by using a cryoelectron microscopy-based model (PDB: 4CR2). The Rpn1 T1 and Rpn13Pru ubiquitin-binding loops are indicated in indigo, whereas cartoon representations are displayed for the hRpn10 UIMs. The ATPase ring and CP are burgundy and gray, respectively, with the remaining RP constituents colored white. The three shuttle factors are represented as green cartoon images. Green arrows indicate shuttle factor binding sites in the proteasome RP.
(B) Superimposed structure of hRpn13Pru (periwinkle blue) and mRpn13Pru (orange, PDB: 2R2Y) with secondary structural elements labeled.
(C) GST pull-down assay with GST-hRpn13Pru and M1, K6, K11, K27, K29, K33, K48, and K63 diubiquitin, as indicated (top). Immunoblotting was done with anti-ubiquitin (top panel) or anti-GST (second panel) antibodies. GST protein was used as a negative control with K48 diubiquitin, as indicated. Direct loading for 15% of the diubiquitin input for each chain type with immunoblotting by anti-ubiquitin antibody is included (third panel). The pull-down assay was repeated three times and the diubiquitin signal intensities were separately normalized to the strongest signal using ImageJ and the average value and SD (as error bars) plotted (bottom). ∗∗Statistical significance with a p value < 0.05 by a two-tailed, two-sample Student's t-test.
(D) hRpn13Pru I75 and F76 demonstrate intermediate-slow exchange upon binding to the hPLIC2UBL. A color code for the molar ratio of hRpn13Pru to hPLIC2UBL is indicated above the spectra.
(E) 1H, 15N HSQC spectra of 15N-labeled hRpn13Pru (black) and with 2-fold molar excess unlabeled hPLIC2UBL (orange). hRpn13Pru signals that shift away from their unligated state following the addition of hPLIC2UBL are labeled, with amino acids from the ubiquitin-binding region highlighted in blue.
(F) Selected regions from 1H, 15N HSQC spectra of 15N-labeled hRpn13Pru (black) with equimolar K48 diubiquitin (orange, left) or hPLIC2UBL (orange, right), and both hPLIC2UBL and K48 diubiquitin (blue).
(G) ITC analysis of the hRpn13Pru binding to K48 diubiquitin (left) or hPLIC2UBL (right). 1.12 mM K48 diubiquitin or 1.07 mM hPLIC2UBL was injected into a calorimeter cell containing  mM hRpn13Pru and the data were fit to a one-site binding mode with the indicated thermodynamic parameters.
Structure  , DOI: ( /j.str )
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4. Figure 2
Structure of hRpn13Pru:hPLIC2UBL Reveals Similarities with Ubiquitin
(A) Selected 15N planes from a 3D 15N-dispersed NOESY spectrum recorded on a sample of 0.5 mM 2H, 15N-labeled hPLIC2UBL and equimolar unlabeled hRpn13Pru. Labels inside and outside of the strips correspond to hRpn13Pru and hPLIC2UBL, respectively.
(B) Superposition of the 20 lowest-energy hRpn13Pru:hPLIC2UBL structures depicted as backbone trace diagrams with hRpn13Pru in blue and hPLIC2UBL in pink. The rmsd to the average structure for this bundle is 0.45 Å.
(C) Superposition of the hRpn13Pru (periwinkle blue):hPLIC2UBL (pink) and mRpn13Pru (indigo):ubiquitin (yellow) (PDB: 2Z59) complexes.
Structure  , DOI: ( /j.str )
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5. Figure 3
Dsk2 Proteins Mimic and Enhance Ubiquitin Interactions with hRpn13
(A) Sequence alignment of hPLIC2UBL and ubiquitin by ClustalW2 and Boxshade3.21. Identical and conserved residues are highlighted against a yellow and gray background, respectively. hPLIC2UBL residues that interact with Rpn13Pru and their aligned residues in ubiquitin are boxed with red rectangles.
(B–E) Expanded views of the hRpn13Pru:hPLIC2UBL (B and D) and mRpn13Pru:ubiquitin (C and E) complexes to illustrate interactions at the contact surface with key amino acids displayed and labeled. Hydrogen bonds are indicated with red dashed lines and the hRpn13Pru surface displayed in light blue.
Structure  , DOI: ( /j.str )
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6. Figure 4
Structure of scRad23UBL and Its Complex with the scRpn1 T1 Site
(A) Backbone trace diagrams for the 20 lowest-energy structures of scRad23UBL with the secondary structural elements superimposed. The N- and C-terminal residues and the individual secondary structural elements are labeled.
(B and C) Selected regions from 1H, 13C half-filtered NOESY experiments acquired with either 13C-labeled scRad23UBL and equimolar unlabeled scRpn1 T1 (B) or 13C-labeled scRpn1 T1 and equimolar unlabeled scRad23UBL (C) displaying intermolecular NOE interactions, as labeled. Breakthrough diagonal peaks are labeled (blue “D”).
(D) Superposition of the ten lowest-energy scRpn1 T1:scRad23UBL structures depicted as backbone trace diagrams with scRpn1 T1 in blue and scRad23UBL in green. The rmsd to the average structure for this bundle is 0.7 Å.
Structure  , DOI: ( /j.str )
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7. Figure 5
scRad23 Occupies the Helix28/Helix30 High-Affinity Ubiquitin-Binding Site of Rpn1 T1
(A) Sequence alignment of ubiquitin and scRad23UBL by ClustalW2 and Boxshade3.21. Identical and conserved residues are highlighted against a yellow and gray background, respectively. scRad23UBL residues that interact with scRpn1 T1 and their aligned residues in ubiquitin are boxed with red rectangles.
(B and C) Ribbon diagrams of the lowest-energy scRpn1 T1:scRad23UBL (B) and scRpn1 T1:K48 diubiquitin (C) structures. scRpn1 (blue), scRad23UBL (green), and K48 diubiquitin (yellow) side chains at the contact surfaces are displayed and labeled with oxygen and nitrogen atoms in red and blue, respectively. Displayed side chains of basic and hydrophobic residues that are conserved in scRad23UBL and ubiquitin are labeled in blue and orange, respectively, whereas scRpn1 side chains are labeled in gray.
(D and E) Expanded regions to display the contact surfaces for scRpn1 T1:scRad23UBL (D) and scRpn1 T1:K48 diubiquitin (E). Side chains of scRad23UBL K10, K11, V50, and V69 are displayed and labeled (D), as are the corresponding amino acids T9, G10, Q49, and H68 from K48 diubiquitin (E). The acidic residues in scRpn1 T1 that form hydrogen bonds with scRad23UBL or K48 diubiquitin are displayed and labeled. Oxygen and nitrogen atoms are colored red and blue, respectively. Hydrogen bonds are indicated by red dashed lines.
Structure  , DOI: ( /j.str )
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8. Figure 6
Shuttle Factor Targeting to Preferred Substrate Receptors of the Proteasome
(A) Model of substrate recruitment to the proteasome. Shuttle factors (pink, hPLIC2 and green, Rad23) use ubiquitin folds (UBL domains) to interact with ubiquitin-binding surfaces (indigo and Rpn10 UIM) while carrying ubiquitinated (gray) substrates (black and brown). High affinity for specific UBL domains leads to enhanced interaction between substrates and the proteasome as UBA domains contribute additional binding surfaces for ubiquitin. This model used PDB: 4CR2 with Rpn1, Rpn10, and Rpn13 displayed in light blue, the ATPase ring in burgundy, and CP and RP in dark gray and white, respectively. Brown and black arrows indicate available ubiquitin-binding surfaces in the proteasome for an Rpn1- and Rpn13-bound substrate, respectively.
(B) The β strand face of the ubiquitin fold for ubiquitin (top) and shuttle factor UBL domains (bottom) highlighting three regions with circles (i, ii, iii) that define affinity for proteasome substrate receptors Rpn1 (right) and Rpn13 (left). Orange and red are used to highlight regions and amino acids that bind to the indicated shuttle factor equivalently and better respectively compared to ubiquitin, with a correspondingly weaker binding residue in ubiquitin in gray (Q49). Region i is indicated in gray on the left for its minimal contacts with Rpn13, but plays a role in the low affinity of hPLIC2 for Rpn1.
Structure  , DOI: ( /j.str )
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