Ub/Ubls are activated by E1 and transferred to E2 to form E2−Ub/Ubl-thioesters. Although competent for Ub/Ubl ligation to lysine -amino groups, E2s generally require E3 ligases to recognize substrate lysine residues specifically. Most E3s belong to either RING or HECT families, and whereas RING E3s recruit substrate and bind the E2−Ub/Ubl through a zinc domain to promote conjugation to lysine residues⁷, HECT E3s recruit E2−Ub/Ubl to generate E3−Ub/Ubl-thioesters for conjugation⁸. The SUMO E2 can directly bind the consensus sequence -K-X-D/E where is a hydrophobic amino acid, K is the substrate lysine, X is any amino acid and D or E is acidic⁹, although several SUMO E3s facilitate conjugation in vivo and in vitro; they include RING-type E3s^{10, 11}, Nup358/RanBP2 (ref. 12) and Pc2 (ref. 13). Nup358/RanBP2 and Pc2 seem unrelated to either RING or HECT E3 ligases.

One of the first discovered functions for SUMO-1 was its role in nucleocytoplasmic trafficking^{3, 4, 5}. SUMO conjugation localizes RanGAP1 to the nuclear pore complex (NPC) in a complex that includes the SUMO E2 Ubc9 and Nup358/RanBP2, a multi-domain 3,224-residue nucleoporin that also interacts with Ran and other nuclear transport factors^{3, 4, 5, 6, 14, 15}. The SUMO−RanGAP1−Ubc9−Nup358/RanBP2 complex does not dissociate on entry into mitosis and NPC disassembly, but redistributes to kinetochores and contributes to the stability of kinetochore−microtubule interactions¹⁶. A roughly 30-kDa Nup358/RanBP2 fragment named IR1-M-IR2 binds Ubc9 and promotes SUMO E3 activity in vitro and in vivo ^{12, 17, 18, 19}, although domains flanking IR1-M-IR2 also contribute to SUMO−RanGAP1 interactions⁶. IR1-M-IR2 was parsed into three elements: IR1 (residues 2,633−2,685), M (residues 2,686−2,710) and IR2 (residues 2,711−2,761). IR1 and IR2 were named for two internal sequence repeats. IR1-M-IR2, IR1-M and M-IR2 all promote SUMO-1 conjugation in vitro ^{18, 19}.

To determine the molecular details of this system, SUMO-1 was conjugated to RanGAP1, combined with Ubc9 and Nup358/RanBP2, purified by gel filtration, and crystallized. The model containing SUMO-1 (residues 20−97), RanGAP1 (residues 432−587), Ubc9 (residues 2−157) and Nup358/RanBP2 (residues 2,631−2,693) was refined to 3.0 Å (R = 24.7, R _free = 29.0; Table 1, Supplementary Table 1 and Supplementary Methods). The structure reveals Ubc9 as the central component of the quaternary complex, contacting SUMO, RanGAP1 and Nup358/RanBP2 (Fig. 1). SUMO contacts Ubc9 and Nup358/RanBP2, but contacts RanGAP1 only through the covalent bond between SUMO Gly 97 and Lys 524. Nup358/RanBP2 contacts SUMO and Ubc9 but does not contact RanGAP1.

Figure 1: Structure of SUMO−RanGAP1−Ubc9−Nup358/RanBP2 complex.

a, Ribbon and transparent-surface representation for the complex between SUMO-1 (yellow), Ubc9 (blue), RanGAP1 (pink) and Nup358/RanBP2 (magenta). Each protein is labelled. The SUMO C-terminal glycine (Gly 97) and RanGAP1 Lys 524 are represented by solid bonds located near the image centre. b, Orthogonal view of the complex. c, Orthogonal view of the complex to highlight the extended Nup358/RanBP2 structure. The N and C termini of Nup358/RanBP2 are indicated. Structural graphics were generated with Pymol (http://pymol.sourceforge.net).

High resolution image and legend (44K)

Table 1: X-ray statistics

Full table

The human SUMO-1−RanGAP1−Ubc9 complex is similar to our previously described complex between mouse RanGAP1 and human Ubc9 in that most interactions to the RanGAP1 -K-X-D/E SUMO motif (L-K-S-E) are preserved⁹ (Fig. 2). Despite covalent attachment to SUMO Gly 97 via an isopeptide bond, Lys 524 remains within a groove created by Ubc9 Asp 127, Pro 128, Ala 129 and Tyr 87. SUMO-1 Gln 29 and Arg 63 and the C-terminal tail (Gln 92, Gln 94, Thr 95, Gly 96 and Gly 97) contact Ubc9 helix B and Ubc9 active-site residues, respectively (Fig. 2), burying only 650 Å² of total surface area²⁰. For comparison, Sae1/Sae2−SUMO-1 and Senp2−SUMO-1 bury 1,650 and 1,800 Å², respectively²¹. Residues in the interface between SUMO-1 and Ubc9 are conserved among SUMO and Ubc9 family members. The small interface between SUMO and Ubc9 is consistent with the complexes being primed to dissociate after conjugation. It is also consistent with the specificities of Ubc9−SUMO being achieved by the SUMO E1 activating enzyme, which brings SUMO and Ubc9 together to form the thioester adduct²¹.

Figure 2: E2 active site in complex with RanGAP1−SUMO-1.

Stereo view of the E2 active site in complex with SUMO-1−RanGAP1 in ribbon and solid-bond representation. Residues are labelled, and hydrogen-bonding interactions are indicated by dashed lines. SUMO-1, RanGAP1 and Ubc9 are coloured yellow, pink and blue as in Fig. 1.

High resolution image and legend (67K)

Several observations indicate that the Ub/Ubl−E2-thioester might adopt a similar configuration to that observed in our complex before conjugation. First, NMR chemical shift perturbations observed for a steady-state Ub−Ubc1-thioester were consistent with contacts between Ub, E2 helix B and an E2 channel that coordinates the Ub Gly-Gly (ref. 22). Second, Ub/Ubls and E2s are structurally conserved. Third, a rotation about Ubc9 Cys 93 Chi1 brings the Cys sulphur atom to within 2 Å of the SUMO Gly 97 carbonyl carbon (Fig. 2). We previously indicated that E2s might coordinate the lysine near the labile E2−Ub/Ubl-thioester to promote chemistry⁹. The absence of other E2 catalytic residues led to recent studies that implicated the conserved E2 His-Pro-Asn amino acid motif (HPN) in catalysis, namely that Asn N contributes to formation of an oxyanion hole that stabilizes the transition state²³. Our structure is consistent with this proposal in that as Asn 85 N moves away from the Ubc9 main chain it approaches the C-terminal Gly 97 carbonyl oxygen, although definitive evidence for the catalytic role of Asn 85 is precluded by the resolution limits of our structure (3.0 Å) and because our complex includes a conjugated product rather than a E2−SUMO-thioester substrate complex.

Nup358/RanBP2 IR1-M adopts a non-globular and extended structure in the complex (Fig. 3). IR1-M elements were divided into motifs I−V based on interactions in the complex (Fig. 3a). Motif I forms an antiparallel -strand with SUMO -strand 2. In addition to main-chain contacts, motif I includes two acidic and five hydrophobic residues that contribute ionic and van der Waals contacts to SUMO-1 residues, respectively (Fig. 3c). The C-terminal end of IR1 -helix A contacts the Ubc9 amino-terminal helix in addition to Pro 69, Pro 105 and Ala 106. Ubc9 Arg 8 bridges four main-chain carbonyl oxygen atoms, two from Ubc9 and two from Nup358/RanBP2 (Fig. 3d). Motif II forms a coil that packs hydrophobic residues into the interface formed by the Ubc9 N-terminal helix and residues between 1 and 2 of SUMO-1 (Fig. 3d). Motif III acidic residues bridge the Ubc9 N-terminal helix and contact Ubc9 Arg 13, Arg 17 and Lys 30 (Fig. 3e). IR1 then forms -helix B, packing motif IV hydrophobic residues onto Ubc9 1−3 (Fig. 3f) before contacting Ubc9 2, 3 and Phe 22 through Leu 2688-Tyr 2689-Leu 2690 in motif V (Fig. 3g). The extended structure buries 1,460 Å² in the SUMO interface and 830 Å² in the Ubc9 interface. On the basis of our structure, new sequence alignments between IR1 and IR2 were used to generate IR1*, IR2* and IR1-M-IR2* (Fig. 3a).

Figure 3: Nup358/RanBP2 sequence alignment and E2−E3−SUMO-1 structure.

a, IR1-M-IR2 elements. Secondary structure is shown above the alignment. Single-letter amino-acid code is coloured for SUMO (yellow) and Ubc9 (blue) contacts. Nup358/RanBP2 IR constructs are indicated by bars. Mutational analysis¹⁸ is shown below IR1-M: double dagger, no defect; plus sign, impaired activity; minus sign, no activity. Asterisks above IR2 indicate identical amino-acid positions between IR1 and IR2. b, Nup358/RanBP2 motifs I−V (magenta), SUMO-1 (yellow) and Ubc9 (blue). c, Nup358/RanBP2 motif I. d, Nup358/RanBP2 motif II. e, Nup358/RanBP2 motif III. f, Nup358/RanBP2 motif IV. g, Nup358/RanBP2 motif V. Residues are labelled, and hydrogen-bonding interactions are indicated by dashed lines.

High resolution image and legend (101K)

RanGAP1 is easily conjugated and interacts strongly with Ubc9 through surfaces adjacent to the conjugation site⁹. It therefore remains unclear whether Nup358/RanBP2 E3 activity is required for RanGAP1 SUMO conjugation, or whether it is required merely to maintain a stable complex at the nuclear pore. The stable interactions observed between RanGAP1 and E2 and between E3, SUMO and E2, and the covalent interaction in RanGAP1−SUMO, indicated that our structure might represent a trapped product complex. To test this, conjugation assays with RanGAP1 under multiple-turnover conditions with and without Nup358/RanBP2 IR1* showed, in contrast to other substrates^{12, 18, 19}, that IR1* inhibited SUMO conjugation. SUMO−RanGAP1 accumulated to near-stoichiometric ratios of product, E2 and E3 (Fig. 4a). The addition of SUMO−RanGAP1 also inhibited SUMO conjugation to p53 in an IR1*-dependent manner (Fig. 4b). Again, stoichiometric ratios of SUMO-1−RanGAP1 to E2 seemed sufficient to sequester E2 and prevent further p53 conjugation. Partial disruption of the stable interface between RanGAP1 and Ubc9 alleviated IR1*-dependent inhibition in multiple-turnover assays and rendered RanGAP1 a substrate for Nup358/RanBP2 E3 activity under single-turnover conditions (Supplementary Fig. 1).

Figure 4: Nup358/RanBP2 activities.

a, SUMO-1 RanGAP1 conjugation under multiple turnover with (open circles) or without (filled circles) IR1*; gel insets are shown. b, Inhibition of SUMO-1 p53 conjugation by SUMO-1−RanGAP1, with (filled circles) or without (open circles) IR1*; gel insets are shown. c, Multiple-turnover SUMO-1 conjugation rates for p53 tetramerization domain (left), IB (middle) and p53 peptide (right). d, Gel insets for c with p53. e, Rates (pM s^-1) and relative rates for Nup358/RanBP2 constructs in c. f, Gel insets for g using p53. g, Single-turnover rates for p53 tetramerization domain (left) and IB (right) using Nup358/RanBP2 constructs or IR1-M−RanGAP1−Ubc9−SUMO−Nup358/RanBP2 (yellow). h, Rates (pM s^-1) for g. i, Single-turnover rates for p53 without (red) or with (black) IR1*. Note the different scales in the three panels. Results in graphs are means s.d.

High resolution image and legend (96K)

To assess the importance of contacts between Nup358/RanBP2, SUMO and E2, assays were conducted at near-saturating substrate concentrations under multiple-turnover and single-turnover conditions to quantify rate velocities by using E3 constructs and substrates that included the p53 tetramerization domain, IB, and a peptide containing a SUMO consensus motif (Fig. 3a; see Methods). IR1* and IR1-M-IR2* enhanced conjugation rates up to 80-fold in multiple-turnover assays (Fig. 4c−e), and IR2* enhanced conjugation 4−16-fold. IR1T included only motifs I and II but catalysed E3 activity at a rate similar to that of IR2*, indicating that motifs III−V might be somewhat dispensable for activity. Deleting motif I (TIR1 and TIR2) from either IR1 or IR2 resulted in almost no E3 activity. Similar rate enhancements were observed under single-turnover conditions using isolated E2−SUMO-thioester (see Methods) (Fig. 4f−h).

Consistent with observations for motif I in our structure was the recent use of NMR to identify Nup358/RanBP2 IR1 amino acids (motif I) that interact with SUMO; although E3 activity was not assessed, several mutations within motif I diminished binding to SUMO-1−RanGAP1 (Fig. 3c)²⁴. SUMO-1 interactions with Nup358/RanBP2 motif I might be recapitulated by other proteins that interact non-covalently with SUMO²⁴ and Smt3 (ref. 25). Mutational analysis revealed the importance of Nup358/RanBP2 motif II residues Leu 2651, Leu 2653, Phe 2657 and Phe 2658 in Ubc9 binding and E3 activity¹⁸, residues that contact Ubc9 in our structure (Fig. 3a, d). This and another study identified Ubc9 mutations that partly diminished both E3 activity and binding to Nup358/RanBP2 (refs 18, 19). Most detrimental Ubc9 mutations face Nup358/RanBP2 motifs IV and V in our structure. The latter study also indicated a possible mechanism for SUMO paralogue selection by various Nup358/RanBP2 IR1-M-IR2 domains¹⁹. To confirm that SUMO-2/SUMO-3−E2 is activated in an analogous manner to that proposed for SUMO-1−E2, we assayed SUMO-2 and SUMO-3 under single-turnover conditions. These data revealed rate enhancements for IR1-M-IR2* and IR1*, and a strict dependence on motif I for activity (Supplementary Fig. 2).

If Nup358/RanBP2 binds E2−SUMO-thioester in a manner similar to that observed in our complex, E3 activity is achieved independently of contacts to the substrate or E2's active site. So how does Nup358/RanBP2 work? Conjugation rates could be increased by an allosteric mechanism that alters the E2 active site indirectly to enhance catalysis¹⁸, possibly activating the thioester, leaving group, or alters E1's ability to charge E2. However, thioester reactivity was not altered when E2−SUMO-thioester was incubated with 0, 1 or 10 mM dithiothreitol (DTT) with or without E3. Differences were also not observed in E1-mediated E2−SUMO-thioester formation with or without E3, although 10:1 or 100:1 E3:E2 molar ratios inhibited the formation of E1−SUMO and E2−SUMO-thioester.

The importance of motif I and motif II for IR1* and IR2* E3 activity and the small interface observed between Ubc9 and SUMO indicated a possible model in which Nup358/RanBP2 catalyses E3 activity by tethering SUMO and Ubc9 together to reduce conformational flexibility, to prevent non-productive E2−SUMO conformations, and to align the complex and thioester for Ubl transfer. If this is true, and contrary to previous observations^{12, 18, 19} or the lack of substrate contacts, both substrate binding and catalysis should be affected. To test this, initial rate velocities were calculated over various p53 concentrations under single-turnover conditions with and without IR1*. The kinetic data fitted well to an equation from which K _d and k ₂ (k _cat) were derived (Fig. 4i; see Methods). In the absence of IR1*, K _d and k ₂ for p53 conjugation were 23.1 4.8 µM and 4.27 0.37 pM s^-1, respectively ( s.d.). In the presence of IR1*, K _d and k ₂ were 3.04 0.47 µM and 104.7 4.3 pM s^-1, respectively. Thus, both K _d and k ₂ were affected by IR1* to increase K _d/k ₂ about 185-fold.

SUMO-conjugated RanGAP1, in stoichiometric quantities, sequesters Ubc9 and substantially diminishes its ability to promote conjugation of exogenous substrate in the presence of Nup358/RanBP2 (see above). Because IR1-M contacted only Ubc9 and SUMO in our complex, we predicted that an additional equivalent of Ubc9−SUMO-thioester should compete with a preformed E2−E3−SUMO−RanGAP1 complex for IR1-M interaction. In fact, single-turnover assays indicated that IR1-M dissociated from the complex, enhancing Ubc9−SUMO-1-thioester conjugation 11-fold (Fig 4f−h). These data indicate that although Nup358/RanBP2 might form a stable complex with SUMO−RanGAP1, it might still be available to promote E3 activity by binding additional E2−SUMO-thioesters, a model that has cellular implications for Nup358/RanBP2 E3 activity if E2−SUMO concentrations are sufficient to compete for Nup358/RanBP2 at the NPC.

The SUMO−RanGAP1−Ubc9−Nup358/RanBP2 structure provides data for several interactions that are crucial for E3-assisted E2 conjugation, including the model that Nup358/RanBP2 enhances conjugation by coordinating the E2−SUMO-thioester optimally for substrate binding and catalysis. Comparison between Nup358/RanBP2−Ubc9−SUMO−RanGAP1 and two other E2−E3 structures^{26, 27}, E6AP−UbcH7 and c-Cbl−UbcH7, reveal distinct but overlapping E2 surfaces used in each complex. By aligning respective E2s, SUMO was placed into the other E2−E3 complexes. The RING domain and other E3 surfaces are well positioned to recognize Ub within the modelled E2−Ub-thioester complexes (Supplementary Fig. 3).

The indirect mechanism used by Nup358/RanBP2 to enhance SUMO conjugation might shed some light on 'allosteric' activation observed in other Ub/Ubl pathways such as the RING-finger and Nedd8-induced activation of the Cullin SCF complexes⁷, Apc11 induced activation of the APC complex²⁷, all of which might coordinate E2−Ub in a complex similar to that observed in our structure. Mechanisms proposed here might also provide insights, although less clear ones, into activities that promote E2−Ub/Ubl-thioester activation during polyubiquitin chain formation²⁸.

Acknowledgments

We thank M. J. Matunis for the original clone containing Nup358/RanBP2 (residues 2,596−2,836), and K. R. Rajashankar and A. Yunus for discussion and for reagents that contributed to this work. Use of the Advanced Photon Source (APS) is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences. Use of the SGX Collaborative Access Team beamline facilities at Sector 31 of the APS was provided by Structural GenomiX, Inc., which constructed and operates the facility. D.R. and C.D.L. were supported in part by a National Institutes of Health grant. C.D.L. acknowledges support from the Rita Allen Foundation.

Competing interests statement:

The authors declared no competing interests.

Supplementary information accompanies this paper.

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1. Structural Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA

Correspondence to: Christopher D. Lima¹ Correspondence and requests for materials should be addressed to C.D.L. (Email: limac@mskcc.org).
Coordinates have been deposited with the Protein Data Bank under accession number 1Z5S.

Received 11 January 2005; Accepted 31 March 2005

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