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

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UNIPA

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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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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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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[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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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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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 UBC92.9 A from Sumo

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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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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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