REVIEW ARTICLE Protein interactions in the sumoylation cascade – lessons from X-ray structures Zhongshu Tang1,*, Christina M Hecker2, , Astrid Scheschonka1 and Heinrich Betz1 Department of Neurochemistry, Max-Planck-Institute for Brain Research, Frankfurt, Germany Department of Biochemistry II, Johann-Wolfgang-Goethe-University, University Hospital, Frankfurt, Germany Keywords Aos1-uba2; neddylation; Pc2; PIAS proteins; RanBP2; RanGAP1; SUMO; sumoylation; ubc9; ubiquitination Correspondence H Betz, Department of Neurochemistry, Max-Planck-Institute for Brain Research, Deutschordenstrasse 46, 60528 Frankfurt, Germany Fax: +49 69 96769 441 Tel: +49 69 96769 220 E-mail: neurochemie@mpih-frankfurt.mpg.de Sumoylation is a multi-step protein modification reaction in which SUMO (small ubiquitin-like modifier) proteins are covalently attached to lysine residues of substrate proteins Here, we compare the sequences and structures of modifiers and enzymes involved in sumoylation with those of the related ubiquitination and neddylation cascades By using available structural data on modifier ⁄ enzyme ⁄ substrate interactions, we discuss and model sumoylation complexes that include SUMO-1 and the E1 and E2 enzymes Aos1uba2 and ubc9, or SUMO-1 and E2 together with the E3 ligase RanBP2 and its substrate RanGAP1 Their comparison provides insight into the protein interactions underlying sumoylation, and suggests how SUMO proteins may be translocated between enzymes during the various steps of the protein modification reaction Present addresses *NIH ⁄ NEI, Bethesda, MD, USA  Department of Systemic Cell Biology, MaxPlanck-Institute for Molecular Physiology, Dortmund, Germany (Received 21 November 2007, revised 20 March 2008, accepted 11 April 2008) doi:10.1111/j.1742-4658.2008.06459.x Introduction Sumoylation is a post-translational modification in which a SUMO (small ubiquitin-like modifier) protein is conjugated to the e-amino group of a lysine residue of the substrate protein [1,2] SUMO attachment has been shown to occur for a large number of proteins with important roles in many basic cellular processes, and to be highly regulated in both the nucleus and other cellular compartments [1–6] Sumoylation is mechanistically related to ubiquitination, a more extensively studied protein modification reaction [7] Although the process of ubiquitination has been recognized for about 20 years and sumoylation for 10 years, Abbreviations APPBP1, APP binding protein 1; CtBP, C-terminus binding protein; CTD, C-terminal domain; E1, activating enzyme; E2, conjugating enzyme; E3, ligase; HECT, homologous to E6AP C-terminus; IR, internal repeat; MIF-2, migration inhibiting factor 2; Nedd8, neural cell-expressed developmentally down-regulation protein 8; Pc2, polycomb protein 2; PIAS, protein inhibitor of activated STAT; RanBP2, Ran binding protein 2; RanGAP1, Ran GTPase-activating protein 1; RING, really interesting new gene; SAP, scaffold-associating region ⁄ Acinus ⁄ PIAS; SENP, SUMO-1 ⁄ sentrin-specific peptidase; SIM, SUMO interaction motif; SMT3, suppressor of MIF-2; SP-RING, Siz ⁄ PIAS-RING; STAT, signal transducer and activator of transcription; SUMO(A), SUMO non-covalently bound at the E1 adenylation site; SUMO(C), SUMO conjugated to a substrate; SUMO(T1), SUMO linked to the catalytic cysteine of activating enzyme E1 via a thioester; SUMO(T2), SUMO linked to the catalytic cysteine of conjugating enzyme E2 via a thioester; SUMO, small ubiquitin-like modifier; uba, ubiquitin activating enzyme; ubc ⁄ E2, ubiquitin-conjugating enzyme; UBL, ubiquitin-like modifier; UFD, ubiquitin-fold domain; Ulps, ubiquitin-like protein protease FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS 3003 Interactions between sumoylation proteins Z Tang et al understanding of the molecular mechanisms of these modification reactions began to emerge only in the last or years, when high-resolution structures of the proteins and protein complexes involved in ubiquitination, sumoylation and another ubiquitin-like modification, neddylation [conjugation of Nedd8 (for neural cell-expressed developmentally down-regulation protein 8)], were solved This review compares the available structures of modifiers and enzymes involved in the SUMO, ubiquitin and Nedd8 pathways (for references, see Table 1) and discusses how these proteins may interact during the sumoylation reaction For further details, on the function and cellular regulation of sumoylation, please refer to previous reviews [1–6] Sumoylation pathway The enzymatic machinery that adds and removes SUMO to and from substrate proteins resembles that involved in ubiquitination [1,4] Like ubiquitin, SUMO Table Protein structures referred to in the review All structures except for those labeled by asterisks (by NMR) were resolved by X-ray diffraction All proteins are of human except for 1EUV (yeast) 1Z7L (mouse), 1U9A (mouse) and RanGAP1 (mouse) in 1KPS Protein or components of the protein complex APPBP1-uba3 APPBP1-uba3, Nedd8 APPBP1-uba3, ubc12N26 APPBP1-uba3, Nedd8T, Nedd8A, Mg2+-ATP, ubc12 Cbl, ubch7 E6AP, ubcH7 Nedd8 Nedd8, Den1 Sae1-Sae2, SUMO-1, Mg2+-ATP (renamed as Aos1-uba2, SUMO-1 in the text) SAP* Senp2, SUMO-1 SMT3 SUMO-1* SUMO-1, ubc9, RanBP2, RanGAP1 SUMO-2 SUMO-3 C47S* SUMO-1, SIM of PIASX* uba1 ubc12, UFD of uba3 ubc9 ubc9, RanGAP1 ubc9, uba2 (Cys domain)* Ubiquitin 3004 PDB No Reference 1YOV 1R4N 1TT5 2NVU 21 21 29 24 1FBV 1C4Z 1NDD 1XT9 1Y8R 31 32 10 65 15 1V66 1TGZ 1EUV 1A5R 1Z5S 40 66 67 11 19 1WM3 1U4A 2ASQ 1Z7L 1Y8X 1U9A 1KPS 2PX9 1UBQ 12 13 20 23 30 27 28 36 14 proteins are expressed as precursors that need to be proteolytically processed by C-terminal hydrolases to expose a C-terminal Gly-Gly motif that is required for conjugation This processing is called SUMO maturation (Fig 1A) Two forms of Ulps (ubiquitin-likeprotein proteases) in Saccharomyces cerevisiae and several forms of SENP (SUMO-1 ⁄ sentrin-specific peptidase) proteins in human have been found to process SUMO precursors [1,2,8] The attachment of SUMO to substrate proteins requires three enzymes, which catalyze distinct steps of the conjugation reaction: SUMO-activating enzyme (E1), SUMO-conjugating enzyme (E2) and SUMO ligase (E3) [1,4] (Fig 1A) The first step, called activation, includes two processes: adenylation and transfer of SUMO to a cysteine within E1 As a result, a thioester bond is formed between the carboxyl group of the C-terminal glycine of SUMO and the E1 cysteine residue; this step requires Mg2+-ATP In the second step, SUMO is transferred from the E1 active site to another cysteine in the E2 enzyme, forming a SUMO– E2 thioester intermediate Finally, SUMO is attached to the amino group of a lysine residue within the substrate protein This ligation reaction is assisted by an E3 enzyme SUMO conjugation is a reversible process The isopeptide bond between SUMO and the substrate can be cleaved by members of the Ulp ⁄ SENP family [8] This process is called de-sumoylation (Fig 1A) Both maturation and de-sumoylation produce free mature SUMO [1,2] Components of the sumoylation machinery Sumoylation, neddylation and ubiquitination are all mediated by three-enzyme cascades as shown in Fig 1B–D For ubiquitination, a single ubiquitin gene and approximately 10 E1, 100 E2 and 1000 E3 enzymes have been found in mammals [7,9] In contrast, four SUMO isoforms (SUMO-1 to -4) have been identified in humans, and conjugation involves single E1 (Aos1-uba2) and E2 (ubc9) enzymes plus a growing list of about 10 E3 candidates SUMO The structures of yeast SUMO [called SMT3, suppressor of migration inhibiting factor (MIF-2)], human SUMO-1 ⁄ -2 ⁄ -3, human ubiquitin and human Nedd8 have all been determined [10–14] Basically, all these modifier polypeptides have a compact globular structure with a characteristic bbabbab fold and N- and FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS Z Tang et al Interactions between sumoylation proteins A DE-SUMOYLATION Ulp Nedd8 ubiquitin Aos1-uba2 APPBP1-uba3 uba1 ubc9 ubc12 E1 AMP + PPi S E1 E2 CONJUGATION SUMO D ATP ACTIVATION SUMO C SUMO-1/-2/-3/-4 + SH SUMO SUMO MATURATION B S ubc7H, - E2 Rbx1 PIAS-1, - E3 Cbl, - LIGATION substrate substrate substrate substrate SUMO Fig Overview of sumoylation, neddylation and ubiquitination reactions (A) The sumoylation pathway Free SUMO is generated by either proteolytic maturation of a SUMO precursor or de-sumoylation of a sumoylated substrate by Ulp ⁄ SENPs For conjugation, SUMO is firstly activated in an ATP-dependent reaction to form a thioester bond with the activating enzyme E1, then transferred to the conjugation enzyme E2, and finally conjugated to a substrate that is recruited by a ligase E3 (B–D) Comparison of the enzyme cascades involved in sumoylation, neddylation and ubiquitination reactions (A) and (B) are modified from [1], and (C) and (D) from [29] and [7], respectively The consensus lysine sites of SMT3 and SUMO-2 ⁄ 3, and the C-terminal GG motif are stringently conserved [13] Regions of low alignment are found along the a2 and b3 regions, which are located close to the N- and C-termini, respectively (Fig 2B,C) In addition, the C-termini, the a2 helix and the loop between a1 and b3 differ between modifiers (Fig 2B) Eleven residues of SUMO-1 have been shown to have directly contact uba2, a subunit of the sumoyla- C-termini extending to opposite sides (Fig 2A,B) Superposition of the SUMO isoforms with ubiquitin and Nedd8 shows that the peptide backbones are well conserved (Fig 2B,C) Many sumoylation sites lie in a consensus motif FKXE ⁄ D, where F is a large hydrophobic amino acid, K is lysine, X is any amino acid, and E ⁄ D is glutamate or aspartate Yeast SMT3 and mammalian SUMO-2 ⁄ ⁄ all contain such consensus motifs at their N-termini, whereas SUMO-1 does not CA B N- SUMO-3 SMT3 N- ubiquitin SUMO-2 –5 Nedd8 SUMO-1 CC β1 β2 α1 β3 β4 α2 β5 1A5R/SUMO-1 1U4A/SUMO-3 1ABQ/ubiquiti 1NDD/Nedd8 1WM3/SUMO-2 1EUV/SMT3 Fig SUMO-1 structure and comparison with the other SUMO paralogues, ubiquitin and Nedd8, by the NCBI vector alignment search tool (A) Structure of SUMO-1 showing the typical bbabbab fold (determined in [11]) (B) Superposition of the backbones of various SUMO proteins with ubiquitin and Nedd8, all shown in various colors Regions conserved among all proteins are shown in red, non-conserved aligned regions in light blue The structure of the C-terminus of SUMO-1 changes upon protein binding (C) Sequences of proteins aligned in (B) Residues in capitals are aligned among all modifiers, including the strictly conserved residues in red and other aligned residues in light blue Residues indicated in lower case could not be aligned Residues in light gray outside the boxed area are not included in the superposition shown in (B) Secondary structure elements (a-helices and b-sheets) corresponding to SUMO-1 are indicated above the alignment The )5 residues that confer selectivity for the corresponding E1 enzyme are highlighted by a yellow background (see also Fig 3) Sequences are from the NCBI Protein Data Bank, with accession numbers given on the left FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS 3005 Interactions between sumoylation proteins Z Tang et al tion E1 complex; seven of these residues lie in the very C-terminal tail [15] An exposed region formed by the first two b-sheets and the first a-helix is the binding site for enzymes and specific substrates, such as DNA glycosylase, Ube2–25k, isopeptidase SENP2, protein inhibitor of activated STAT (PIAS) and Ran binding protein (RanBP2) [16–20] The N-terminal loops of SUMO proteins are not homologous to those of ubiquitin and Nedd8, and their functions are still unknown E1 E1 activating enzymes facilitate the conjugation of ubiquitin-like modifiers to substrate proteins through adenylation, thioester formation within E1, and thioester transfer from E1 to E2 [15] Unlike the ubiquitin E1 enzyme uba1, which is a monomer, the E1 enzymes involved in the SUMO and Nedd8 modification pathways are all heterodimers SUMO E1 is composed of Aos1 and uba2 (also called Sae1 and Sae2, respectively), and Nedd8 E1 is composed of APPBP1 (APP binding protein 1) and uba3 The domain structures of Aos1 and APPBP1 resemble the N-termini, and those of uba2 and uba3 resemble the C-termini, of the ubiquitin E1 enzyme (Fig 3A) SUMO-1 is recognized exclusively by residues of uba2; no direct interactions have been observed between SUMO-1 and the Aos1 subunit [15] Aos1 contains only a single domain, which participates in adenylation of SUMO Uba2 includes three domains: the catalytic cysteine domain, the adenylation domain and the ubiquitin-fold domain (UFD), which is structurally similar to ubiquitin and other ubiquitin-like modifiers The adenylation domain of uba2 has a typical Gly-X-Gly-X-X-Gly ATP-binding motif [21], which is conserved among the ubiquitin-, SUMO- and Nedd8activating enzymes The UFD of uba2 shows strong interaction with E2, which is essential for recruiting E2 to E1 [15] The C-terminal extension of uba2 contains a unique nuclear localization signal that may be important for enrichment of the SUMO machinery in the nucleus [22] APPBP1 has two domains: an adenylation domain, which resembles Aos1, and part of a cysteine domain, which resembles that of uba2 Both subunits of SUMO E1 are conserved from yeast to human [2] The crystal structures of human Aos1-uba2, human APPBP1-uba3, and of the partly resolved mouse uba1, are closely related [15,21,23] (Fig 3C) From the side facing the ATP-binding motif, Aos1-uba2 resembles a U-shaped complex (Fig 3B) It forms a large groove, with the adenylation domain at the base, and the UFD and cysteine domains on both sides The catalytic cysteine residue lies in a long loop between the cysteine domain and the adenylation domain [15] Of the 986 residues of Aos1-uba2, 663 can be precisely aligned with APPBP1-uba3, and 203 residues are conserved between both proteins (Fig 3D,E) Two APPBP1-uba3 structures have been published: with and without Nedd8 bound at the catalytic site [24] They are largely identical, except for the UFD [24] Here, comparative structure analysis revealed a turn of the UFD of approximately 120° upon binding of Nedd8 to the cysteine domain [21,24] This conformational change of E1 may be important for passage of the modifiers through the conjugation machinery [24] Despite the similarities between the structures of all ubiquitin-like modifiers and their corresponding E1s, binding of the modifiers to their activating enzymes is highly specific This specificity is attributed to the selective pairing of amino acid side chains upon interaction of the modifier and E1 [21] The fifth-last ()5) residue of the mature modifier proteins has been shown to be particularly crucial, i.e Glu93 in SUMO-1, Gln89 ⁄ 88 ⁄ 89 in SUMO-2 ⁄ ⁄ 4, Ala72 in Nedd8, and Arg72 in ubiquitin (highlighted in yellow in Fig 2C) Matching E1 residues are Thr149 in uba2, Arg190 in uba3 and Gln608 in uba1 (highlighted in purple in Fig 3E) Interestingly, Ala72 of Nedd8 has been found to be also essential for recognition by the Nedd8-specific protease NEDP1 [25] This suggests that a common mechanism may be used to recruit both activating and processing enzymes to ubiquitin-like modifiers Fig Domain organization, structures and alignment of various E1 activating enzymes (A) Schematic representation of domains in human Aos1-uba2, APPBP1-uba3 and uba1 (B) Structure of the Aos1-uba2 complex [15] The protein complex is sub-divided into three domains (indicated by dashed lines) The ATP-binding motif is highlighted by a dashed black circle, and the position of the catalytic cysteine residue is shown in yellow (C) Superposition of the polypeptide backbones of Aos1-uba2 and APPBP1-uba3 Residues conserved among all proteins are shown in red and other aligned residues are shown in light blue Divergent regions are shown in green for Aos1-uba2 and in pink for APPBP1-uba3 The position of the ATP-binding motifs and the catalytic cysteine residues are indicated as in (B) (D,E) Alignment of the sequences of Aos1 with APPBP1, and of uba2 with uba3, respectively Residues in capital letters are aligned, including conserved residues in red and other aligned residues in light blue Lower-case residues indicate non-aligned sequences Residues shown in light gray are not shown in (C) A green background delineates the activation motif Modifier-recognizing residues are shown on a purple background, and the catalytic cysteine residues on a yellow background 3006 FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS Z Tang et al Interactions between sumoylation proteins A Adenylation domain 346 Cys domain UFD domain 640 Cys173 Aos1-uba2 uba2 Aos1 529 1 NLS Cys216 442 APPBP1-uba3 APPBP1 uba3 Cys632 1058 uba1 B C D E FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS 3007 Interactions between sumoylation proteins Z Tang et al E2 After activation, the SUMO molecule is transferred to the conjugating enzyme E2 All SUMO proteins share a common E2 enzyme, ubc9 It is highly conserved from yeast to human [2] Mouse ⁄ human ubc9 displays high similarity to the human Nedd8 and ubiquitin E2 enzymes: 84% of its residues can be aligned with and superpositioned to the Nedd8 E2 ubc12 and the ubiquitin E2 ubcH7 (Fig 4B,C) Common to all E2 enzymes is a conserved 150-residue abbbbb(bb)aaa motif named the ubc superfold; differences are found only in their N- or C-terminal extensions [26–28] The four a-helices and six b-strands of ubc9 can be modeled into a cubicle (Fig 4A), in which a1, a2 and a3 ⁄ are each located on one side, and the anti-parallel b-sheet formed by the b1–4 strands on another side The two remaining surfaces are covered by loose peptide strands In the sumoylation cascade, ubc9 functions as a core component that interacts with nearly all other proteins involved in the modification reaction It possesses at least five protein interaction sites (a) The a1 region contains the a1 helix and surrounding residues of the b1b2 loop (Fig 5A) A similar region in the neddylation E2 enzyme ubc12 binds the UFD of uba3 [29,30] and thereby enables E2 recruitment Similarly, the UFD of uba2 has also been shown to strongly A interact with ubc9 [15] Although structural data are not available, it is likely that ubc9 and the UFD of uba2 interact at the a1 region (b) The region below the a1 helix consists mainly of the N-terminal extension of a1 and loops between the b2 and b3 strands and the b6 strand and the a2 helix, respectively (Fig 5B) In ubiquitin-conjugating enzymes, this is the region that interacts with both the HECT (homologous to E6AP C-terminus) and RING (really interesting new gene) domains of ubiquitin E3 ligases [31,32] As discussed later, most sumoylation E3 ligases contain RING domains Thus, this region may be a common site for E2–E3 interactions (c) In the SUMO-1–ubc9–RanBP2–RanGAP1 (Ran GTPaseactivating protein 1) complex, the four-stranded b-sheet domain of ubc9 provides the binding site for the IR1 (internal repeat 1) domain of RanBP2, another suspected E3 ligase, in the sumoylation pathway (Fig 5C) [33] (d) The a3 helix of the C-terminal a3a4 helical region (Fig 5D) binds RanGAP1, a special substrate that is known to have a second binding site for ubc9 [19,28,34] (e) The ‘catalytic groove’ contains the active site residue Cys93, and holds the SUMO-1 C-terminus [35] (Fig 5E) This region also includes the a3 helix of E2 which mediates E2–UBL (ubiquitin-like modifier) interactions in both the ubc9– SUMO-1 and ubc12–Nedd8 complexes [19,24] Cys93 B C Fig Structure and alignment of ubc9 with ubc12 and ubcH7 (A) Structure of ubc9 showing the typical ubc superfold as determined in [27] Secondary structure elements (a-helices and b-sheets) are indicated (B) Superposition of the polypeptide backbones of ubc9, ubc12 and ubcH7 Residues conserved among all proteins are shown in red, non-conserved aligned residues are shown in light blue Non-aligned regions are shown in pink for ubc9, dark blue for ubcH9, and brown for ubc12 The positions of the catalytic cysteine residues are indicated in yellow (C) Corresponding sequence alignment Sequences are from the NCBI Protein Data Bank, with accession numbers indicated on the left Residues aligned among all modifiers are given in capital letters, with fully conserved residues in red and other aligned residues in light blue Residues in lower case are not aligned Secondary structure elements (a-helices and b-sheets) corresponding to ubc9 are indicated above and below the sequences Catalytic cysteine residues are indicated by black arrows 3008 FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS Z Tang et al Interactions between sumoylation proteins B A C D E F G H Fig Protein interactions involved in sumoylation and related pathways (A) E1–E2 interaction [29] Ubc12 (blue) is bound to the UFD (yellow) of uba3 (B) E2–E3 interaction UbcH7 (yellow) is bound to the RING domain (blue) of Cbl [31] An interface is formed between ubcH7 a1 + L3 + L6 and the Cbl RING L1 + L2 + a (C) E2–RanBP2 interaction Ubc9 (blue) and RanBP2 (IR1 domain, yellow) are shown in the SUMO-1–ubc9–RanBP2–RanGAP1 complex The C-terminal IR1 motif contacts loop and the b-sheet of ubc9 [19] (D) E2–substrate interaction [19] RanGAP1 binds ubc9 at its a3 region (E) SUMO-1–E2 interaction [19] Ubc9 (yellow) and SUMO-1 (blue), with the SUMO-1 b-sheet located near the ubc9 a2 helix Note that SUMO-1 is not conjugated to ubc9 in this complex, but a strong interaction between ubc9 and SUMO-1 has been characterized [19] (F) E1–SUMO-1 interaction at the adenylation site [15] The SUMO-1 (blue) C-terminus interacts close to an ATP (highlighted in red) which is bound at the adenylation motif (yellow) (G) E1–SUMO-1 interaction at the catalytic cysteine site [24] A covalent bond is formed between SUMO-1 (blue) and the catalytic cysteine (red) (H) RanBP2 (IR1 domain, yellow) interaction with SUMO-1 [19] The N-terminal IR1 domain binds SUMO-1 in a cleft between b2 and a1 is located close to the center of a long extended stretch between the fourth b-strand and the second a-helix [27] Chemical shift perturbation experiments have shown that the a3 helix of ubc9 displays affinity to the Cys domain of uba2 [36] This interaction, although weak, seems to be important for the transfer of SUMO from E1 to E2 [36] In addition to the covalent bond formed between SUMO and ubc9 during sumoylation, non-covalent interactions between these proteins have recently been characterized [37,38] They occur between the a–b1 loop of ubc9 and the b1,3,5 strands of SUMO This assignment is distant from the E2 active site and partially overlaps with the E1–UFD and RanBP1–IR1 FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS 3009 Interactions between sumoylation proteins Z Tang et al interaction sites on ubc9 Interestingly, it seems that the non-covalent interactions between SUMO and ubc9 promote SUMO chain formation, but seem not to be important for SUMO–ubc9 thioester formation [37] A E3 C The last step in sumoylation is the transfer of SUMO to a substrate protein that is recruited by an E3 ligase Unlike SUMO and the E1 and E2 enzymes, E3 ligases are quite diverse from yeast to mammals [1] Ubiquitin ligases can be broadly subdivided into two groups, based on the presence of either a RING or HECT domain While both domains bind E2 enzymes at the same position [31,32], HECT domain E3 ligases form a covalent ubiquitin–thioester complex prior to conjugation of ubiquitin to its target, but RING domain E3 enzymes not [7] The most carefully analyzed group of SUMO E3 ligases, the PIAS proteins, contain a modified RING domain and not form thioesters with SUMO [1,39] HECT domain-containing E3 ligases for SUMO have not been identified, but two other proteins have been found to act as SUMO ligases: the polycomb protein Pc2 and the nucleoporin RanBP2 [33] PIAS proteins PIAS proteins are characterized by a so-called SPRING (Siz ⁄ PIAS-RING) domain, and bind both ubc9 and substrate proteins However, they not form a covalent bond with the substrate but actually act as adaptors Within PIAS polypeptides, an N-terminal SAP (scaffold-associating region ⁄ Acinus ⁄ PIAS) domain is followed by the SP-RING domain, a SUMO interaction motif (SIM), and a highly divergent C-terminal domain (CTD) (Fig 6A) [39] The SAP domain is known to bind DNA and proteins such as tumor suppressor p53 [40] and lymphoid enhancer factor [41] The SIM domain has been implicated in directly binding SUMO [42] The SP-RING domain is crucial for the interaction with E2 [1,5,43] The CTD has been repeatedly found to bind sumoylation substrates; hence it is considered to be a substrate interaction domain [1,42,44,45] Except for the SP-RING domain, no homology has been found between PIAS proteins and single-chain ubiquitin E3 ligases NSE2a and TOPORS, two other proteins that contain RING domains at positions different from those in PIAS proteins, have recently been reported to function as SUMO ligases; however, little is known about their precise modes of action [46–48] Of the functional domains of PIAS proteins, only the human SAP domain has been analyzed at the 3010 B Fig Domain organization of E3 ligases (A) Typical PIAS proteins contain an N-terminal SAP domain, an SP-RING domain, a SUMO interaction motif (SIM) and a C-terminal domain (CTD) [39] (B) Pc2 contains an uncharacterized E3 domain, a ubc9 binding domain and a Pro-Ile-Asp-Leu-Ser motif involved in substrate binding [53] (C) RanBP2, modified from [56] RanBP2 harbors an N-terminal leucine-rich domain, four RanBP1 repeats (R1–R4), a region containing eight zinc-finger motifs and the SUMO interaction domain RanBP2DFG, which consists of two internal repeats (IR1 and IR2) and a linker (M) domain [56] structural level It is formed by a four-helical bundle and is thought to bind DNA [40] The structures of the SP-RING domain and of the CTDs, the two domains most important for E2 and substrate binding, have not yet been analyzed The only structural data presently available are from the RING domain of the ubiquitin E3 ligase Cbl which binds to the E2 enzyme ubcH7 [31,32] (Fig 5B); the latter was referred to above when considering E2–E3 interactions Pc2 Pc2 was identified as a SUMO E3 for the transcriptional co-repressors CtBP (C-terminal binding protein of adenovirus E1A) and CtBP2, both in vivo and in vitro [49,50] However, the enhancement of CtBP sumoylation by Pc2 in vitro is very modest, and PIAS1, PIASx and RanBP2 also can promote SUMO attachment to CtBP, suggesting that multiple factors may be involved in CtBP sumoylation [1,50] Pc2 is a member of the polycomb group of proteins, which were first identified in Drosophila as regulators of segment identity [51,52] Pc2 contains various domains The ubc9 binding domain is located in a CTD fragment (Fig 6B, amino acids 401–469) that neighbors a CtBP-binding fragment called the Pro-Ile-Asp-Leu-Ser (PIDLR or PIDLS) motif (Fig 6B, amino acids 469– 486) At present, no structural data are available for the ubc9-binding and substrate interaction regions A separate domain in the N-terminal domain has been reported to show E3 activity in vitro In vivo, both the FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS Z Tang et al N- and C-terminal domains appear to contribute to E3 activity [53] RanBP2 The nuclear pore complex protein RanBP2 has been found to catalytically enhance sumoylation of a nuclear body component [54] It directly interacts with ubc9 and strongly enhances SUMO-1 transfer from ubc9 to the SUMO-1 target Sp100 [54] RanBP2 is a multidomain protein (Fig 6C) with interaction sites for proteins including nuclear transport receptors, the GTPase Ran, ubc9, and sumoylated RanGAP1 [55] Its SUMO E3 ligase activity lies in a fragment called RanBP2DFG, which contains two approximately 50-residue internal repeats (IR1 and IR2) separated by a 25-residue spacer domain (M) [54,56] The IR1 is a UBL protein binding domain that promotes sumoylation of both SUMO-1 and SUMO-2 [33] The human IR1 sequence has been crystallized in a complex with human SUMO-1, human ubc9 and part of human RanGAP1 (residues 432–587) Its N-terminal region interacts with SUMO-1 (Fig 5H), but its C-terminal portion binds ubc9 (Fig 5C) Hence, it was proposed that the RanBP2DFG region acts to position the SUMO–E2 thioester in an optimal orientation, thereby enhancing conjugation [19] This shorter fragment does not display the substrate specificity seen with fulllength RanBP2, suggesting that sequences outside the ligase active site control substrate recognition, possibly by interacting with other proteins Approximately 100 proteins have been demonstrated to serve as sumoylation substrates Most were initially identified as binding partners of PIAS proteins and ⁄ or ubc9 by protein–protein interaction assays, such as yeast two-hybrid and ⁄ or GST pulldown, and confirmed later to be sumoylated For only four proteins, to our knowledge, has sumoylation been shown to be specifically enhanced by RanBP2 These are RanGAP1 [57,58], Sp100 [54], promyelocytic leukaemia protein [33] and histone deacetylase [59] However, RanBP2 does not directly interact with either RanGAP1 or Sp100 in pulldown assays [54,60], and a direct interaction between RanBP2 and the other two substrates has not been shown either In conclusion, RanBP2 works in a way that is different from that of PIAS proteins or Pc2 Sumoylation complexes and mechanism An intriguing question is how the individual components of the sumoylation machinery interact during the Interactions between sumoylation proteins various steps of the modification cycle Above, we describe interactions involving E2 enzymes (Fig 5A– E) Structural analysis suggests that other interactions are also crucial for sumoylation to proceed SUMO–E1 interaction at the adenylation domain (Fig 5F) No covalent bond exists between E1 and SUMO1 at this site Rather, the C-terminal Gly-Gly motif is modified here by an ATP residing in the adenylation domain, resulting in SUMO activation [15] SUMO–E1 interaction at the catalytic domain Here, the first thioester bond is formed No structural data are available yet for SUMO bound to Aos1-uba2 at its catalytic cysteine residue However, the corresponding complex has been resolved for the neddylation pathway [24] In the Nedd8–APPBP1–uba3–ubc12 complex, one Nedd8 molecule is bound covalently to the cysteine site of E1 [24] (Fig 5G) SUMO–E3 interaction For RanBP2, the interaction with SUMO-1 has been analyzed (Fig 5H) and shown to require the N-terminal region of this E3 ligase, i.e the IR1 domain [19] Competitive binding experiments have disclosed that, in the ubiquitination and neddylation cascades, the E2 enzymes must dissociate from E1 prior to E3-catalyzed transfer of the modifier to the substrate [61,62] This implies that distinct protein complexes must form during the activation, conjugation and ligation reactions Consistent with this view, two large UBL complexes have been crystallized and structurally resolved: a neddylation E1 modifier E2 complex comprising APPBP1– uba3–Nedd8(A)–Nedd8(T)–Mg-ATP–ubc12(C111A) (Protein Data Bank number 2NVU), and a sumoylation E2–E3–substrate complex containing ubc9, the IR1 and M domains of RanBP2, SUMO-1 and a RanGAP1 fragment containing the sumoylation site (Protein Data Bank number 1Z5S) [19,24] We have used the 2NVU template to model the corresponding sumoylation E1–E2 complex (Z T., unpublished data); the result is shown together with the published structure of the E2–E3–substrate complex in Fig 7B In the modelled E1–E2–SUMO complex (Fig 7B, bottom), the E1 enzyme Aos1-uba2 forms the core structure The E2 enzyme ubc9 is positioned on top of E1, with its a1 area attached to the E1 UFD SUMO-1 is predicted to be associated with the E1–E2 complex via at least three distinct sites, two of which have been localized in the corresponding neddylation complex structure (2NVU) At the adenylation domain of E1, adenylated SUMO-1 [SUMO(A)] is formed, and covalent attachment to Cys173 at the catalytic domain generates E1-conjugated ‘thiolated’ SUMO-1 [SUMO(T1)] (Fig 7B) Both domains are located on uba2 In addition, SUMO-1 must interact with ubc9 to form FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS 3011 Interactions between sumoylation proteins Z Tang et al A B C E2-E3-substrate complex Fig Structures of sumoylation complexes (A) Various orientations of SUMO-1 that correspond to its positions at the various SUMO-1 binding sites of the sumoylation complexes (B) Model of the E1–E2 and structure of the E2–E3–substrate sumoylation complexes, with SUMO-1 molecules bound ⁄ attached at various sites [orientations as shown in (A); the other components are colour-coded as shown in (C)] The positions of the ATP-binding motif of E1 (dashed red circle), the catalytic cysteine residues of E1 (filled red rectangle) and E2 (black arrows) and the conjugated lysine residue of RanGAP1 (green arrow) are indicated The E1–E2 model was compiled from published structures using WINCOOT [63] and PYMOL [64] (C) Individual sumoylation components: E1 Aos1-uba2 (yellow), E2 ubc9 (cyan), the IR1–M domain fragment of the E3 RanBP2 (magenta) and substrate RanGAP1 (residues 435–587; brown) The complex structures displayed in (B) indicate the following sequence of SUMO-1 interactions Bottom: a free SUMO molecule (violet) enters the adenylation site of E1, where its C-terminal GG motif accesses an ATP molecule (firebrick red, within the dashed red circle) bound at the ATP-binding motif of E1 (dashed red circle), ˚ and thus becomes adenylated SUMO(A) is then translocated (distance approximately 30 A, 90° turn) to the E1 catalytic site and attached to its reactive cysteine (red rectangle) to generate SUMO(T1) Thereafter, SUMO(T1) is transferred to the reactive cysteine of the catalytic site of E1-bound E2 to form SUMO(T2); a structure showing SUMO-1 conjugated to Cys93 of E2 is not yet available Conjugation results in detachment of E2–SUMO-1 from E1, which renders E2 accessible for interaction with E3 and substrates In the resulting SUMO-1–E2–E3– substrate complex (top), SUMO-1 is covalently attached to RanGAP1 (ligation area indicated by dashed black rectangle) the second thioester bond during conjugation; however, a template structure for E2-conjugated SUMO-1 [SUMO(T2)] is not yet available Within the E2–E3–substrate complex co-crystallized with SUMO-1 (Fig 7B, top), the fragment of RanGAP1, a well-characterized sumoylation substrate, binds to ubc9 at its a3 region The other subdomains of ubc9 extend freely, leaving sufficient space for binding of other interacting proteins The partial E3 sequence of RanBP2 interacts with both SUMO-1 and ubc9 via its IR1 domain Conjugation of SUMO-1 to the acceptor Lys524 of RanGAP1 generates the final product of the sumoylation cascade [SUMO(C)] The modeled and resolved structures discussed above allow rationalization of the protein interactions occurring during the various steps of the sumoylation reaction Accordingly, a free SUMO-1 molecule enters the adenylation site of E1 where its C-terminal GG motif accesses an ATP bound at the E1 ATP-binding motif to allow formation of SUMO(A) SUMO(A) is 3012 then transferred to the E1 catalytic cysteine; this ˚ implies that a distance of approximately 30 A between the adenylation and catalytic sites is bridged, with a 90° turn of the SUMO-1 molecule being required (Fig 7A,B) After thioester bond formation, SUMO(T1) must be further transferred to the catalytic cysteine of E2 The details of this second (and the first) transfer reaction are unknown; however, it is assumed that conformational changes accompanying the adenylation and thiolation steps allow molecular movement within the enzyme–substrate complex Successful conjugation is then thought to result in detachment of the E2–SUMO-1(T2) conjugate from E1, allowing interaction with specific E3 enzymes and their substrates Within the resulting E2–E3–substrate complexes, SUMO-1 is then ligated to the respective substrate protein Here, we referred to structural data obtained for the RanGAP1-containing E2–E3 complex; it should, however, be noted that RanGAP1 is unusual in that it can interact directly with the E2 ubc9 In FEBS Journal 275 (2008) 3003–3015 ª 2008 The Authors Journal compilation ª 2008 FEBS Z Tang et al general, substrates are recruited by E3 ligases, which, by docking onto ubc9, act as adaptors for distinct target proteins Perspectives The details of SUMO conjugation as outlined above have emerged from the structures and paired interactions characterized so far These include SUMO-1, the full-length E1 and E2 enzymes, as well as partial sequences of the E3 RanBP2 and the substrate RanGAP1 Interactions within the SUMO-1–E1–E2 complex have been inferred from data obtained on the related neddylation proteins [19,28,31], and require validation in future studies It should be noted that the presently available biochemical data suggest different roles for the RanBP2, Pc2 and PIAS proteins, although all these E3 enzymes have been reported to enhance sumoylation of specific substrates Clearly novel structural data will be required to better understand the mechanistic roles of these ligases Future studies should also address the question of how SUMO proteins are translocated between their various sites of interaction with the sumoylation enzymes First, SUMO must move from the adenylation domain to the catalytic site within E1 For the neddylation machinery, the distance between these sites ˚ has been calculated to be about 20 A, and is suggested to be reduced by conformational changes of the E1 enzyme [24] In the E1–E2–SUMO-1 complex modeled here, the distance between bound SUMO(A) and ˚ SUMO(T1) is predicted to be about 30 A, and the distance between the E1 and E2 reactive cysteines is ˚ deduced to be about 20 A As its C-terminal loop is flexible, even comparatively low extents of SUMO-1 re-orientation may suffice to allow thioester bond formation between these residues In the crystallized E2– E3–substrate complex, Lys524 of RanGAP1 lies only ˚ about 10 A away from the E2 cysteine; hence only a modest axial movement of SUMO(T2) might bring it into close proximity to the substrate’s acceptor residue Clearly structural studies that provide information about additional intermediates of the sumoylation cascade are required to fully understand the precise mechanisms of the activation, conjugation and ligation reactions In conclusion, the structural data summarized in this 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(Ran GTPaseactivating protein 1) complex, the. .. and mechanism An intriguing question is how the individual components of the sumoylation machinery interact during the Interactions between sumoylation proteins various steps of the modification... ligases: the polycomb protein Pc2 and the nucleoporin RanBP2 [33] PIAS proteins PIAS proteins are characterized by a so-called SPRING (Siz ⁄ PIAS-RING) domain, and bind both ubc9 and substrate proteins