Beginning at the end with SUMO

NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 12 NUMBER 7 JULY 2005 565

Beginning at the end with SUMOMichael J Matunis & Cecile M Pickart

The crystal structure of a four-protein complex comprising a SUMO ligase (E3), a SUMOylated protein substrate, and the cognate SUMO-conjugating enzyme sheds new light on catalysis, specificity and SUMO-protein interactions.

Ubiquitin-like protein modifiers (Ubls) regu-late a host of processes in eukaryotic cells, ranging from protein stability and localiza-tion to diverse stress responses1–3. Ubls share a homologous fold and a common biochemi-cal mechanism for attachment to substrates. Each family member is conjugated through the carboxyl group of its terminal glycine to lysine residues of specific target proteins, producing isopeptide conjugates. Individual Ubls are con-jugated to their target proteins through paral-lel enzymatic cascades (Fig. 1a), with the key final step of isopeptide bond formation usually involving a ligase (E3)1–3. Studies of ubiqui-tylation indicate that the E3 has two roles: it selects the target protein by interacting with a specific substrate-based signal and it facili-tates the transfer of the Ubl from the upstream E2-Ubl thiol ester to the substrate lysine. The molecular mechanisms used by E3s to carry out the second role are rather mysterious3. Now structural studies by Reverter and Lima, pub-lished recently in Nature4, have shed new light on both the mechanisms by which SUMO-1 can affect protein-protein interactions and the mechanism used by E3s to catalyze the transfer of Ubls to target proteins.

Ubiquitin, the original Ubl, signals proteo-lysis and diverse non-proteolytic outcomes3. Functional breadth is also characteristic of SUMO (small ubiquitin-like modifier)2. Dozens, perhaps hundreds, of proteins in eukaryotic cells are modified by SUMO, but compared to ubiquitylation, mechanisms for translating SUMOylation into specific downstream conse-quences are poorly understood. The situation is further complicated by the existence of three SUMO isoforms in higher eukaryotes (SUMO-1, SUMO-2 and SUMO-3).

SUMOylation was discovered because it is required to localize RanGAP1, a regulator of nucleocytoplasmic transport, to nuclear pore complexes (NPCs)5,6. Studies of the SUMOylation of RanGAP1 and other substrates have revealed two distinguishing features of the

process2,3. First, SUMOylation, unlike ubiquity-lation, is frequently site specific. RanGAP1 and many other substrates are modified at a lysine in the consensus sequence ΦKxD/E, where Φ is a hydrophobic residue. Second, the SUMOylation of RanGAP1 and (to a lesser extent) other substrates is frequently observ-able with just E1 and Ubc9 (the SUMO E2), whereas ubiquitylation proceeds inefficiently in the absence of an E3. These features can be explained by Ubc9’s ability to bind the con-sensus SUMOylation sequence7 in a manner that places the target lysine residue adjacent to the active site cysteine of Ubc9 (ref. 8). Known SUMO E3s apparently have little in common with one another2. Interestingly, although the enzymatic requirements for RanGAP1 SUMOylation in vivo remain unclear, the bind-ing partner of RanGAP1–SUMO-1 at the NPC, called Nup358/RanBP2 (Fig. 1b), can function as a SUMO E3 in vitro9. A small inverted repeat region of this protein, called IR1-M-IR2, binds

Ubc9 and sumoylates several substrates9–11. The shorter IR1-M, M-IR2 and IR1 polypep-tides also show SUMO E3 activity10,11.

The new crystal structure consists of a RanGAP1–SUMO-1 conjugate bound to Ubc9 and IR1-M. Although it is unclear whether Nup358/RanBP2 SUMOylates RanGAP1 (or other substrates) in vivo, the complex is repre-sentative of a trapped product complex because an analogous product-inhibited complex can be generated during a single turnover of IR1 with RanGAP1 (ref. 4). Given that IR1-M cata-lyzes substrate SUMOylation, an unexpected feature of the complex is that IR1-M does not contact the RanGAP1 ‘substrate’ (Fig. 1c). One simple explanation for this absence—that this is a product complex—seems unlikely. The Ubc9-RanGAP1 interface in the new structure is essentially identical to that seen in the binary Ubc9/RanGAP1 complex and SUMO-1’s ter-minal glycine is located only 2 Å from the Ubc9 cysteine thiol4,8, suggesting that RanGAP1 is

The authors are in the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.e-mail: cpickart@jhmi.edu

Figure 1 Nup358/RanBp2 and catalysis of RanGAP1 SUMOylation. (a) The SUMO conjugation pathway2. SUMO is activated by the E1 activating enzyme, a heterodimer consisting of Aos1 and Uba2. SUMO is subsequently transferred to the E2 enzyme, Ubc9, and conjugated to substrates in a reaction that is catalyzed by a variety of E3 enzymes, including Nup358/RanBP2. SUMO is removed from substrates in reactions catalyzed by a family of isopeptidases called SENPs. (b) Domain structure of Nup358/RanBP2. Domains involved in binding the RanGTPase, Ubc9 and SUMO-1-modified RanGAP1 are indicated. FG, FxFG, motifs involved in nuclear transport receptor binding; CHD, cyclophilin homology domain. (c) RanGAP1–SUMO-1–Ubc9–IR1-M complex (pink, RanGAP1; blue, Ubc9; red, IR1-M; yellow, SUMO-1).

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566 VOLUME 12 NUMBER 7 JULY 2005 NATURE STRUCTURAL & MOLECULAR BIOLOGY

not markedly repositioned after SUMOylation. Thus, the trapped product complex is also likely to provide insights into the upstream SUMOylation reaction. The idea that E3-substrate contacts may not be strictly neces-sary for catalysis is consistent with the ability of Ubc9 to SUMOylate many substrates in an E3-independent manner and with the ability of IR1-M and some other SUMO E3s to catalyze the SUMOylation of diverse substrates.

Even though the IR-M polypeptide does not contact RanGAP1, it engages in many other interactions that are relevant to catalysis (Fig. 1c). These include extensive, functionally important contacts with Ubc9 (refs. 4,10,11). Perhaps the most remarkable revelation of the complex is that the N-terminal segment of IR1, which was recently identified as a SUMO-interacting motif12, forms a strand that extends the β-sheet of SUMO-1 in an intermolecular manner. This interaction might immobilize SUMO-1 in the bound Ubc9–SUMO-1 thiol ester and could orient SUMO-1’s terminal gly-cine residue in a manner favorable for attack by the target lysine. Consistent with this idea, deleting this region of IR1 profoundly inhibits IR1-catalyzed SUMOylation4.

The reigning model for catalysis by E3s that use noncovalent mechanisms of cataly-sis is that these enzymes practice catalysis by proximity—that is, fixing the two reactants reduces the entropic cost of reaching the tran-sition state3. The previous Ubc9–RanGAP1 structure showed how the substrate lysine’s orientation is fixed through interactions with Ubc9 (ref. 8). The new structure shows for the first time how the terminal glycine of SUMO-1 could be fixed by an interaction with the E3 (ref. 4). Recent analyses of peptide bond for-mation catalyzed by the ribosome suggest that a reduction in entropy achieved by extensive noncovalent interactions with a carrier entity (in this case, tRNA) can bring about a 107-fold rate enhancement13. Although catalysis by IR1-related polypeptides is much less efficient (~200-fold rate enhancement relative to Ubc9 alone4), this is a minimal E3 domain and further interactions and catalysis could occur with full-length Nup358/RanBP2. The structure provides tantalizing evidence about a possible chemical mechanism of catalysis, too: the side chain of an asparagine residue of Ubc9 is seen in an orientation that could be consistent with a previously proposed oxyanion-binding function14.

What are the implications for ubiquity-lation? On the substrate side, catalysis through a reduction in entropy is generally more attrac-tive for SUMO, with its target lysine specific-ity, than for ubiquitin, which is typically not ligated to substrates in a site-specific manner.

This is because the maximum benefit from reduced entropy will be realized if the react-ing lysine residue is forced into a catalytically favorable orientation. Simply binding the substrate at a different site, as many ubiqui-tin E3s seem to do, is unlikely to achieve the same degree of fixation. On the other hand, it is attractive to imagine that the E3 positions the E2-bound ubiquitin so as to maximize the ter-minal glycine’s reactivity. Modeling by Reverter and Lima4 suggests that existing structures of ubiquitin-dedicated E2–E3 complexes could accommodate such interactions. Addressing these possibilities is an important challenge for the future.

The new structure has further important implications. SUMO-1-modified RanGAP1 and Ubc9 both colocalize with Nup358/RanBP2 at the cytoplasmic filaments of the NPC in vivo and all four proteins copurify as a complex from both Xenopus laevis egg extracts and mammalian cell extracts9,15. Therefore, the complex character-ized by Reverter and Lima may also represent the E3–E2–RanGAP1–SUMO-1 complex as it exists at the NPC in vivo. In this context, the structure provides several important insights relevant to how SUMO modification may mediate interac-tions of different substrates with unique down-stream effectors. First, the structure indicates that SUMO-1 conjugation does not affect RanGAP1 conformation, with the only contact between SUMO-1 and RanGAP1 being the isopeptide bond. Because Nup358/RanBP2 interacts stably with SUMO-1-modified RanGAP1, but not with unmodified RanGAP1 or SUMO-1 alone, the absence of a conformational change implies that Nup358/RanBP2 interacts coopera-tively with a bipartite signal containing deter-minants from both RanGAP1 and SUMO-1. Consistent with this model, the Reverter and Lima4 structure indicates that the SUMO-interacting motif in the IR1 domain of Nup358/RanBP2 interacts directly with β-strand 2 of SUMO-1. Although the structure does not reveal direct contacts between Nup358/RanBP2 and RanGAP1, this is very likely because of the minimal Nup358/RanBP2 domain used in the study, as longer Nup358/RanBP2 fragments can bind the RanGAP1–SUMO-1 conjugate in the absence of Ubc9 (ref. 16). Taken together, the findings may provide an important paradigm for SUMO-mediated protein-protein interac-tions in which different substrates interact with unique downstream effector proteins (or protein complexes) that are defined by their abilities to bind simultaneously and cooperatively to both SUMO and the substrate. Such a model was originally proposed on the basis of NMR data that similarly found that SUMO-1 modification does not significantly alter RanGAP1 structure17.

A final unanswered question concerns the precise function of this E3 activity in vivo. It has been proposed that Nup358/RanBP2 facili-tates the SUMO modification of substrates as they are transported between the nucleus and the cytoplasm; however, bona fide substrates modified at the NPC, or by Nup358/RanBP2, remain to be identified. Biochemical studies by Reverter and Lima suggest that the minimal E3 domains of Nup358/RanBP2 could be deficient in catalyzing SUMOylation when complexed with Ubc9 and RanGAP1–SUMO-1 (ref. 4), but it is important to note that the IR1-M domain used in these studies represents only a small segment of a 358-kDa protein. Although the structure and biochemical data indicate that Ubc9 may be sequestered and inactive in the IR1-M complex, the full IR1-M-IR2 domain contains two Ubc9 binding sites10. Moreover, other domains within Nup358/RanBP2 may also affect E3 ligase activity. Flanking the IR domain of Nup358/RanBP2, for example, are binding sites for nuclear transport receptors as well as for RanGTP (Fig. 1b). Determining whether these domains aid in recruiting substrates and defining E3 ligase function and specificity is another important goal for the future.

ACKNOWLEDGMENTSWe thank C. Lima for providing Figure 1c and M. Hochstrasser for comments on the manuscript. Work in our laboratories is funded by grants from the US National Institutes of Health.

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