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. 2012 Feb 10;287(7):4740-51.
doi: 10.1074/jbc.M111.321141. Epub 2011 Dec 22.

Determinants of small ubiquitin-like modifier 1 (SUMO1) protein specificity, E3 ligase, and SUMO-RanGAP1 binding activities of nucleoporin RanBP2

Jaclyn R Gareau  1 , David Reverter, Christopher D Lima
Affiliations

Determinants of small ubiquitin-like modifier 1 (SUMO1) protein specificity, E3 ligase, and SUMO-RanGAP1 binding activities of nucleoporin RanBP2

Jaclyn R Gareau et al. J Biol Chem. .

Abstract

The RanBP2 nucleoporin contains an internal repeat domain (IR1-M-IR2) that catalyzes E3 ligase activity and forms a stable complex with SUMO-modified RanGAP1 and UBC9 at the nuclear pore complex. RanBP2 exhibits specificity for SUMO1 as RanGAP1-SUMO1/UBC9 forms a more stable complex with RanBP2 compared with RanGAP1-SUMO2 that results in greater protection of RanGAP-SUMO1 from proteases. The IR1-M-IR2 SUMO E3 ligase activity also shows a similar preference for SUMO1. We utilized deletions and domain swap constructs in protease protection assays and automodification assays to define RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity. Our data suggest that elements in both IR1 and IR2 exhibit specificity for SUMO1. IR1 protects RanGAP1-SUMO1/UBC9 and functions as the primary E3 ligase of RanBP2, whereas IR2 retains the ability to interact with SUMO1 to promote SUMO1-specific E3 ligase activity. To determine the structural basis for SUMO1 specificity, a hybrid IR1 construct and IR1 were used to determine three new structures for complexes containing UBC9 with RanGAP1-SUMO1/2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2 and suggest that differences in SUMO specificity may be achieved through these subtle conformational differences.

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Figures

FIGURE 1.
FIGURE 1.
RanBP2 constructs. A, amino acid alignment of the internal repeat domain of RanBP2. A schematic representation of IR1 (magenta), M (gray), and IR2 (pink) is displayed below the amino acid sequence of RanBP2. Regions corresponding to the motif I SIM, motif II, and motifs III–V are shown above the IR1 sequence. Asterisks indicating identical amino acid positions between IR1 and IR2 are displayed above the IR2 sequence. B, IR1-M-IR2 constructs. Schematics of IR1-M-IR2 constructs used in this study are color-coded as above. C, IR constructs. Schematics of individual internal repeat domain constructs are shown.
FIGURE 2.
FIGURE 2.
Activities of RanBP2 IR1-M-IR2. A and B, bar graphs indicating the concentration of SENP1 required to deconjugate 50% of RanGAP-SUMO in the presence of UBC9 and indicated RanBP2 constructs for RanGAP-SUMO1 (A) and RanGAP-SUMO2 (B) as interpolated using data obtained from three independent experiments (supplemental Figs. 1 and 2). C, time course of IR1-M-IR2 automodification with SUMO1 (upper) and p53 modification with SUMO1 in the presence of IR1-M-IR2 (lower). Reactions were performed in the presence of the specified concentrations of UBC9 and RanGAP-SUMO/UBC9. Automodification was detected by Western blotting with SUMO1 antibody. p53 conjugation was detected by SYPRO staining. D, same as C, except that reactions were performed with SUMO2 and automodification of IR1-M-IR2 with SUMO2 was detected by Western blotting with SUMO2 antibody.
FIGURE 3.
FIGURE 3.
Activities of RanBP2 IR1 and IR2. A and B, bar graphs indicating the concentration of SENP1 required to deconjugate 50% of RanGAP-SUMO in the presence of UBC9 and indicated RanBP2 constructs for RanGAP-SUMO1 (A) and RanGAP-SUMO2 (B) as interpolated using data obtained from three independent experiments (supplemental Figs. 1 and 2). C, time course of SUMO1 conjugation to p53 with the specified RanBP2 construct. D, same as C, except that conjugation assays were performed with SUMO2. E, bar chart displaying quantitation of the conjugation assays shown in C and D. Assays were performed in triplicate. Error bars represent ± 1 S.D.
FIGURE 4.
FIGURE 4.
Structures of RanGAP1-SUMO/UBC9/RanBP2 complexes. Schematics represent complexes containing RanGAP1-SUMO1/UBC9/IR1 (A; PDB 1Z5S), RanGAP1-SUMO2/UBC9/IR1 (B), RanGAP1-SUMO1/UBC9/IR1SIM-IR2II-IR1III–V (C), and RanGAP1-SUMO2/UBC9/IR1SIM-IR2II-IR1III–V (D). SUMO1, SUMO2, RanGAP1, UBC9, IR1, and IR2 motif II are displayed in yellow, green, light pink, blue, magenta, and pink, respectively. Lys524 of RanGAP1 and the SUMO C-terminal glycine (Gly97 for SUMO1, Gly93 for SUMO2) and are in stick representation. A rotated view of each complex is shown below each panel to highlight the position of SUMO and the IR1 SIM with respect to UBC9. Structural figures were generated with PyMOL (38).
FIGURE 5.
FIGURE 5.
Active site of the RanGAP1-SUMO1/UBC9/IR1SIM-IR2II-IR1III–V complex. A, stereo view of the E2 active site in complex with RanGAP1-SUMO1, shown in schematic and stick representation. Residues are labeled, and potential hydrogen bonds are indicated by dashed lines. SUMO, RanGAP1, and UBC9 are colored yellow, light pink, and blue, respectively. B, alternate view of the isopeptide linkage with simulated annealing omit map contoured to 2.0 σ at 2.3 Å.
FIGURE 6.
FIGURE 6.
SUMO/SIM interactions in RanGAP1-SUMO/UBC9/RanBP2 complexes. A–D, ribbon and stick representation of interactions between RanBP2 SIM and motif II with SUMO and UBC9 in RanGAP1-SUMO1/UBC9/IR1 (A; PDB 1Z5S), RanGAP1-SUMO2/UBC9/IR1 (B), RanGAP1-SUMO1/UBC9/IR1SIM-IR2II-IR1III–V (C), and RanGAP1-SUMO2/UBC9/IR1SIM-IR2II-IR1III–V (D). SUMO β-strand 2 and SUMO α-helix 1 are colored yellow and green for SUMO1 and SUMO2, respectively. The IR1 SIM and motif II, IR2 motif II, and UBC9 are colored magenta, pink, and blue, respectively. Hydrogen bonding interactions are indicated by dashed lines. E and F, close-up view of hydrogen bonds between SUMO and the IR1 SIM for RanGAP1-SUMO1/UBC9/IR1SIM-IR2II-IR1III–V (E) and RanGAP1-SUMO2/UBC9/IR1SIM-IR2II-IR1III–V (F).
FIGURE 7.
FIGURE 7.
SIM contributions to IR1-M-IR2 activities. A and B, bar graphs indicating concentration of SENP1 required to deconjugate 50% of RanGAP-SUMO in the presence of UBC9 and indicated RanBP2 constructs for RanGAP-SUMO1 (A) and RanGAP-SUMO2 (B) as interpolated using data obtained from three independent experiments (supplemental Figs. 1 and 2). C, time course of automodification with SUMO1 (upper) and p53 modification with SUMO1 (lower) with the indicated IR1-M-IR2 constructs. Reactions were performed in the presence of the specified concentrations of UBC9 and RanGAP-SUMO/UBC9. Automodification was detected by Western blotting with SUMO1 antibody. p53 conjugation was detected by SYPRO staining. D, same as C, except that reactions were performed with SUMO2, and automodification was detected by Western blotting with SUMO2 antibody.
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