Spiegazione in aula dell 'articolo scientifico: "Determinants of Small Ubiquitin-like Modifier 1 (SUMO1) Protein Specificity, E3 Ligase, and SUMO-RanGAP1 Binding Activities of Nucleoporin RanBP2"

1. Determinants of Small Ubiquitin-like Modifier 1
(SUMO1) Protein Specificity, E3 Ligase, and SUMO-
RanGAP1 Binding Activities of Nucleoporin RanBP2
Jaclyn R. Gareau, David Reverter, and Christopher D. Lima
Prestigiacomo Valeria Seminario in aula- Valeria Prestigiacomo-
UNIPA

2. E1  enzyme that activates
( SAE1/UBA2 )
RanBP2
IR1-M-IR2
E2  enyme that conjugates
( UBC9 )
E3  protein ligase
( Siz/Piaz ,domain SpRing)
Interaction with
Sumo1
Protection/
Ligase E3
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3.  Motif I is a SUMO-interacting motif (SIM)
that forms an anti-parallel β-strand with
SUMO β-strand 2.
 Motif II is composed of an α-helix and coil
that makes contacts to both SUMO and
UBC9.
abrogated or greatly diminished
their E3 ligase activity.
- Vector pSmt3 BL21 cell of E.Coli
- Deletions of motif I and III-V
CONSTRUCTS IR
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4.  Protease protection assay
 Automodification assay
-7000-fold higher
-3000-fold higher
- 86 fold lower
Western Blotting
- antibodies against Sumo1 Boston Biochem
- antibodies against Sumo2 Sigma
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5. [SNP1] higher
RanBP2 protects
144-fold better
[SNP1] lower
IR2 interacts better with Sumo1 in the presence of motif II-V of IR1
Activities of RanBP2 IR1 and IR2
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6.  RanGAP1-Sumo1/UBC9/IR1
 RanGAP1-Sumo2/UBC9/IR1
 RanGAP1-Suno1/UBC9/IR1SIM-
IR2II-IR1III–V
 RanGAP1-Sumo2/UBC9/IR1SIM-
IR2II-IR1III–V
Structures of RanGAP1-SUMO/UBC9/RanBP2 complexes
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7. The complementary interaction include:
• SUMO C-terminal diglycine motif
• the UBC9 catalytic site
• RanGAP1 lysine side chain
Active site of the RanGAP1-SUMO1/UBC9/IR1SIM-IR2II-IR1III–V complex
- Oxygen of G97 Sumo  3.0 A from UBC9
- Oxygen of D127 UBC92.9 A from Sumo
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8. The absence of isoleucine in
SUMO1 creates a deeper
hydrophobic pocket that might
account for the ability of the IR1
SIM to approach SUMO1 more
closely compared with SUMO2
SUMO/SIM interactions in RanGAP1-SUMO/UBC9/RanBP2 complexes
SUMO1 SUMO2
Val 38 Ile 34
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9. CONCLUSIONS
 The RanBP2 IR1-M-IR2 domain is a SUMO E3 ligase and is responsible for localization of
RanGAP1-SUMO1/UBC9 at the NPC. both IR1 and IR2 exhibit specificity for SUMO1. Most
amino acid sequence differences between IR1 and IR2 lie in motifs III–V, and our results
suggest that the inability of IR2 to interact with UBC9 underlies its inability to protect
RanGAP1-SUMO or catalyze E3 ligase activity compared with IR1.
 Differences in SUMO specificity may be achieved through subtle conformational differences
observed for SUMO1 and SUMO2 in the RanGAP1-SUMO/UBC9/IR complexes.
 R1 functions as both the primary receptor for RanGAP1-SUMO1/UBC9 as well as the primary
E3 ligase within IR1-M-IR2.
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