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. 2010 Nov 12;285(46):36070-80.
doi: 10.1074/jbc.M110.145219. Epub 2010 Sep 8.

Structural basis of E2-25K/UBB+1 interaction leading to proteasome inhibition and neurotoxicity

Sunggeon Ko  1 , Gil Bu Kang, Sung Min Song, Jung-Gyu Lee, Dong Yeon Shin, Ji-Hye Yun, Yi Sheng, Chaejoon Cheong, Young Ho Jeon, Yong-Keun Jung, Cheryl H Arrowsmith, George V Avvakumov, Sirano Dhe-Paganon, Yung Joon Yoo, Soo Hyun Eom, Weontae Lee
Affiliations

Structural basis of E2-25K/UBB+1 interaction leading to proteasome inhibition and neurotoxicity

Sunggeon Ko et al. J Biol Chem. .

Abstract

E2-25K/Hip2 is an unusual ubiquitin-conjugating enzyme that interacts with the frameshift mutant of ubiquitin B (UBB(+1)) and has been identified as a crucial factor regulating amyloid-β neurotoxicity. To study the structural basis of the neurotoxicity mediated by the E2-25K-UBB(+1) interaction, we determined the three-dimensional structures of UBB(+1), E2-25K and the E2-25K/ubiquitin, and E2-25K/UBB(+1) complex. The structures revealed that ubiquitin or UBB(+1) is bound to E2-25K via the enzyme MGF motif and residues in α9 of the enzyme. Polyubiquitylation assays together with analyses of various E2-25K mutants showed that disrupting UBB(+1) binding markedly diminishes synthesis of neurotoxic UBB(+1)-anchored polyubiquitin. These results suggest that the interaction between E2-25K and UBB(+1) is critical for the synthesis and accumulation of UBB(+1)-anchored polyubiquitin, which results in proteasomal inhibition and neuronal cell death.

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Figures

FIGURE 1.
FIGURE 1.
Crystal structure of E2–25K. A, the crystal structure of E2–25K is presented as a ribbon diagram in which the E2 and UBA domains are colored forest and pale green. Cys-92, Asn-83, and Lys-14 (sumoylation site) are presented as a ball-and-stick model. Ser-86 and Asp-127, which form a hydrogen bond that modulates the active site, are also represented. The secondary structures are labeled. B, hydrophobic interactions between the E2 and UBA domains are presented. The hydrophobic residues involved in the interaction were elucidated and displayed using the PyMol program. The E2 and UBA domains are colored forest and pale green, respectively. C, the β4-α3 loop and the α4-α5 loop are represented. The Ub conjugating residue (Cys-92) and the regulatory residues (Asp-127, Ser-86, and Asn-83) are highlighted. The hydrogen bond between Ser-86 and Asp-127 is depicted as a red dashed line.
FIGURE 2.
FIGURE 2.
Structure of the E2–25K/Ub complex. A, the crystal structure of the E2–25K/Ub complex is presented with the E2 domain, UBA domain, and Ub colored forest, pale green, and brown, respectively. Lys-14, Lys-48, and Cys-92 are presented as space-filling models. B, the interaction interface between E2–25K and Ub is presented and enlarged. The side chains of the interacting residues in E2–25K and Ub are in white and orange, respectively. The red dash lines indicate hydrogen bonds. C, 15N-labeled E2–25K was titrated with Ub. Various molar ratios of E2–25K and Ub (1:0, 1:1, 1:2.5, 1:5, 1:7.5, and 1:10) are displayed as black (a), blue (b), cyan (c), green (d), gold (e), and red (f) peaks, respectively. D, GST pulldown assay was carried out using GST-Ub and E2–25K. Proteins were resolved by 15% SDS-PAGE and visualized by Western blotting with anti-E2–25K antibody.
FIGURE 3.
FIGURE 3.
NMR structure of UBB+1. A, the 20 lowest energy structures were superimposed using the backbone atoms in the Ub region (residues 1–74). Blue (residues 1–74) and yellow-red (residues 75–95) lines indicate the Ub and C-terminal tail regions, respectively. The yellow (residues 75–88) and red (residues 89–95) lines indicate the residual structured and unstructured regions, respectively. B, the result of a 1H,15N heteronuclear NOE experiment is shown. Average heteronuclear NOE values for both Ub and the C-terminal region are indicated by blue and red lines, respectively. Secondary structures are also indicated.
FIGURE 4.
FIGURE 4.
Structure of E2–25K/UBB+1 complex. A, the crystal structure of the E2–25K/UBB+1 complex is displayed as a ribbon diagram. The E2 and UBA domains of E2–25K and the Ub region of UBB+1 are shown in green, pale green, and navy, respectively. The red dashed line indicates the C-terminal tail region of UBB+1, which was not observed in the electrodensity map. Lys-14, Lys-48, and Cys-92 are depicted as space-filling models. B, shown is magnification of the E2–25K and UBB+1 interface with specific binding residues displayed. E2–25K and UBB+1 of the UBA domain are in pale green and navy, respectively. Hydrogen bonds are indicated by red dash lines. C, the overlapped structures of the E2–25K/Ub and E2–25K/UBB+1 complexes are displayed. The two were superimposed on the UBA domain using backbone atoms, and the interacting residues that affect the orientation of the protein are represented. D, a GST pulldown assay for GST-tagged UBB+1 with E2–25K and its mutants is presented. Proteins were resolved by 15% SDS-PAGE and visualized by Western blotting with an anti-E2–25K antibody.
FIGURE 5.
FIGURE 5.
NMR mapping of E2–25K/UBB+1 interactions in solution. A, 15N-labeled E2–25K was titrated with UBB+. The bar diagram shows the chemical shift changes at a E2–25K:UBB+1 molar ratio of 1:5. The residues showing chemical shift changes above 0.08 ppm (Δδ) are labeled and classified as green (0.08 ppm ≤ Δδ < 0.18 ppm), blue (0.18 ppm ≤ Δδ < 0.3 ppm), and brown (0.3 ppm ≤ Δδ < 0.5 ppm). B, UBB+1 binding sites are indicated by different colors based on chemical shift changes (Δδ) as described in A. In the magnification of the UBA domain, residues interacting with UBB+1 are colored based on the chemical shift changes and labeled based on the surface charge model in the inset box. C, 15N-labeled UBB+1 was titrated with E2–25K. The bar diagram shows the chemical shift changes at a UBB+1:E2–25K molar ratio of 1:5. The residues showing chemical shift changes greater than 0.1 ppm (Δδ) are labeled and classified as green (0.12 ppm ≤ Δδ < 0.24 ppm), blue (0.24 ppm ≤ Δδ < 0.48 ppm), and red (0.48 ppm ≤ Δδ < 1 ppm). Red stars indicate the residues lost due to peak broadening. D, E2–25K-binding residues are presented in the surface model of UBB+1. The different colors are defined as in C.
FIGURE 6.
FIGURE 6.
Proteasome inhibition and neurotoxicity assays. A, polyubiquitylation reactions using Ub and UBB+1 mixtures were run with Ub (50 μm), UBB+1 (50 μm), E1 (0.1 μm), and E2–25K wild type or its mutants (1 μm) (with ATP (5 mm) for 4 h at 37 °C). PolyUb chains anchored by UBB+1 were analyzed by Western blotting using a rat polyclonal anti-UBB+1 antibody. B, the relative GFPu levels in B103 cells transfected with empty vector, E2–25K, or an E2–25K mutant were determined by fluorescence microscopy. Bars depict the means ± S.D. (n = 4). C, B103 cells were transfected with empty vector (pcDNA3), wild-type E2–25K, and the most effective E2–25K mutants (M172A/F174A/L198A triple mutant and V190A/T194A double mutant), after which GFPu accumulation was assessed by examining the cells under a fluorescence microscope. GFPu and DsRed were used to detect intracellular proteasome activity. Red and green indicate basal protein expression and GFPu accumulation in the cytosol, respectively. Yellow indicates overlap of the red and green signals. D, B103 cells were transfected with empty vector (pcDNA3), E2–25K, or one of the E2–25K mutants for 48 h, after which cell viability was assessed based on the morphology of DsRed+ cells examined under a fluorescence microscope. Bars depict means ± S.D. (n = 4).
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