Post-translational modification by members of the small ubiquitin-related modifier (SUMO)3
protein family regulates multiple cellular processes, including nuclear transport, transcription, chromosome segregation, and DNA repair (1
). Conjugation of SUMO to substrates occurs through an enzymatic cascade composed of an E1-activating enzyme (SAE1/UBA2), an E2-conjugating enzyme (UBC9), and E3 protein ligases that results in formation of an isopeptide bond between the C terminus of SUMO and the ϵ-amino group of a substrate lysine residue (3
). The SUMO E2, UBC9, is able to specifically recognize and conjugate SUMO to substrates containing a SUMO consensus motif (ΨKX
D/E), although E3s often aid in conjugation through (i) recruitment of the E2~SUMO (where ~ indicates a thioester linkage) and substrate into a complex to promote specificity and (ii) stimulation of SUMO discharge from the E2 to the substrate (4
SUMO E3 protein ligases include members of the Siz/PIAS family which contain an SP-RING domain and are thought to function like ubiquitin RING E3s by binding the substrate and by binding and activating E2~SUMO for SUMO discharge (5
). A second and distinct class of SUMO E3 is represented singularly by RanBP2 (also termed Nup358), a component of the cytoplasmic fibrils of the nuclear pore complex (NPC). RanBP2 is required to localize SUMO1-modified RanGAP1 and UBC9 in a complex at the NPC to facilitate nucleocytoplasmic trafficking by promoting the Ran GTPase cycle (8
). In addition to its role in binding RanGAP1-SUMO1/UBC9, the RanBP2 IR1-M-IR2 domain can promote SUMO conjugation in vitro
and in vivo
. Furthermore, the internal repeats IR1 and IR2 each possess E3 activity in vitro
Previous structural work demonstrated that IR1 contains five motifs that are required to bind RanGAP1-SUMO1/UBC9 (14
). Motif I is a SUMO-interacting motif (SIM) that forms an anti-parallel β-strand with SUMO β-strand 2, establishing contacts to SUMO β-strand 2 and SUMO α-helix 1. Motif II is composed of an α-helix and coil that makes contacts to both SUMO and UBC9, whereas motifs III–V wrap around and form additional interactions with UBC9. In the absence of RanGAP1-SUMO1/UBC9, IR1 can bind an E2~SUMO thioester and coordinate it in an optimal orientation to promote catalysis.
RanGAP1 was the first SUMO substrate to be identified and is preferentially modified by SUMO1 in vivo
). This is not because SUMO2 does not modify RanGAP1, but rather because RanGAP1-SUMO1 and UBC9 form a more stable complex with RanBP2 that is better protected from the deconjugating activities of SUMO proteases (18
). The preference for SUMO1 is also observed in its E3 ligase activity, as previous studies demonstrated that the E3 ligase domains of RanBP2 exhibit higher ligase activity with SUMO1 compared with SUMO2 under single and multiple turnover conditions with a number of model substrates (11
Although it is clear that the E3 ligase domain of RanBP2 displays a preference for SUMO1, it is not yet known how this specificity is achieved. In addition, it is unclear whether IR1 and IR2 function together or separately in E3 ligase and binding activities of RanBP2. To define better the RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity, we utilized IR1-M-IR2 deletions and domain swap constructs in protease protection assays and automodification assays. Our results support a model in which SUMO1 specificity is achieved by both internal repeats of RanBP2. 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. Domain swaps further suggest that a hybrid IR1 containing IR2 motif II can promote SUMO1-specific modification of a model substrate. To determine the structural basis for SUMO1 specificity, IR1 and a hybrid IR1 construct containing IR2 motif II were used to crystallize and determine three new structures for complexes containing UBC9 with either RanGAP1-SUMO1 or RanGAP1-SUMO2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2.