Crystal structures of the human SUMO-2 protein at 1.6 A ˚ and 1.2 A ˚ resolution Implication on the functional differences of SUMO proteins Wen-Chen Huang 1,2 , Tzu-Ping Ko 1 , Steven S L Li 3 and Andrew H J. Wang 1 1 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 2 Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaoshiung, Taiwan; 3 Department of Biotechnology, College of Life Sciences, Kaoshiung Medical University, Taiwan The S UMO proteins are a class of small ubiquitin-like modifiers. SUMO is attached to a s pecific lysine side chain on the target protein via an isopeptide bond with its C-terminal glycine. There are at least four SUMO proteins in humans, wh ich are involved in protein trafficking and tar- geting. A truncated human SUMO-2 protein that contains residues 9–93 was expressed i n Escherichia c oli and crystal- lized in two d ifferent unit cells, w ith dimensions of a ¼ b ¼ 75.25 A ˚ ,c¼ 29.17 A ˚ and a ¼ b ¼ 74.96 A ˚ ,c¼ 33.23 A ˚ , both b elonging to the rhombohedral space group R3. They diffracted X-rays to 1.6 A ˚ and 1.2 A ˚ resolution, respectively. The structures were determined by molecular re placement using the yeast SMT3 protein as a search m odel. Subsequent refinements yielded R/R free values of 0 .169/0.190 and 0.119/ 0.185, at 1.6 A ˚ and 1 .2 A ˚ , r espectively. The peptide fo lding of SU MO-2 consis ts of a h alf-open b-barrel a nd two flank- ing a-helices with secondary structural elements arran ged as bbabbab in the sequence, identical to t hose of ubiquitin, SMT3 and SUMO-1. Comparison of SUMO-2 with SUMO-1 showed a surface region near the C terminus with significantly different charge distributions. This may explain their distinct intracellular locations. In addition, crystal- packing a nalysis s uggests a possible trimeric assembly of the SUMO-2 protein, of which the biological significance remains t o be determined. Keywords: homology m odeling; m olecular interactio ns; protein mod ification; surface charge distributions; synchro- tron radiations. Control of protein expression and regulation of protein activities are central to the cellular processes in an organism. Many proteins are rather short lived, and are eventually targeted to proteosomes f or degradation via conjugation with ubiquitin [1]. However, the functions of various proteins are not only a matter of time but also a matter of place. T hus, n ewly synthesized proteins must be directed toward specific subcellular compartments. SUMO is the acronym for small ubiquitin-like modifier and named after its three-dimensional structural similarity to ubiquitin. Both SUMO and ubiquitin a re attached to target proteins by forming an isopeptide bond between the C-terminal glycine and a specific lysine side chain o n the target [2]. The extra amino acids beyond the l ast g lycine–glycine m otif o f n ative SUMO proteins are proteolytically removed in vivo.In mammals, there are at least four different SUMO proteins, SUMO-1, -2, -3 and -4. The h uman hSMT3 cDNA encoding the SUMO-2 protein was first reported by Mannen et al.[3].SUMO-2andSUMO-3share87% sequence identity with each other, but they have only 47% identity with SUMO-1 [4]. The novel SUMO-4 associated with diabetes is also more similar in sequence to SUMO-2 than to SUMO-1 [5]. The first three-dimensional structure of SUMO-1 deter- mined by NMR showed that the SUMO proteins are remarkably similar in protein fold to ubiquitin despite the amino acid sequence identity of only 18% [6]. Recently, a high-resolution NMR structure of SUMO-1 was deter- mined by using heteronuclear resonance [ 7], in which the overall conformation was slightly different from the previ- ous model. On the other hand, the yeast SMT3 (SUMO) protein is 40–45% identical to human SUMO proteins in amino acid sequence a nd the human SUMO proteins have an insertion between the strands b1andb2, as shown in Fig. 1. The crystal structure o f yeast SMT3 was d etermined in complex with Ulp1 protease [8]. Significant deviation between the crystal structur e and solution s tructure of yeast SMT3 was also observed using high-resolution hetero- nuclear NMR spectroscopy [9]. In addition to the N-terminal extensions, the m ost significant difference between SUMO and ubiquitin are their surface charge distributions [6]. SUMO-1, - 2 and -3 proteins were shown to localize on nuclear membrane, in nuclear bodies and in the cytoplasm, respectively [10]. Presumably, the different locations are due to their Correspondence to S. S L. Li, Department of Biotechnology, College of Life Sciences, Kaoshiung Medical University, Kaoshiung 807, Taiwan. Fax: +886 7 312 5339, Tel.: +886 7 313 5162, E-mail: lissl@kmu.edu.tw and A. H J. Wang, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan. Fax: +886 2 2788 2043, Tel.: +886 2 2788 1981, E-mail: ahjwang@gate.sinica.edu.tw Abbreviations: CHES, 2-(cyclohexylamino)ethanesulfonic acid; IPTG, isopropyl thio-b- D -galactoside. (Received 2 1 May 2004, revised 14 July 2004, accepted 31 August 2004) Eur. J. Biochem. 271, 4114–4122 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04349.x functions in protein targeting. A rrangement of side chains confers t he protein w ith unique surface properties. Thus, comparison of SUMO-1, - 2 and -3 surface p roperties by modelling provides an approach to understanding the relationship b etween structure and function. To date, no crystal structure of mammalian SUMO proteins has been determined. In order to obtain more structural information, especially about the protein side chains, we tried to determine a three-dimensional structure of human SUMO at high resolution by X-ray crystallography. In this paper we present the crystal structure of a truncated SUMO-2. To facilitate crystallization, our strat- egy was to reduce the length of N-terminal arm while preserving the sequence of Val10–Lys11–Thr12–Glu13, as well as the C-terminal Gly92–Gly93 for conjugation via an isopepti de bond. The VKTE s equence in SUMO-2 is consistent with the S UMOylation consensus YKXE w here Y represents a hydrophobic amino acid and X means a ny amino acid in target proteins, and this consensus sequence is functional for possible polymerization [11]. Furthermore, the truncated SUMO-2 cDNA encoding sequence 9–93 was fusedtoaHis 10 tag at the N terminus with a Factor Xa cleavage site for efficient purificaion. Materials and methods Cloning, expression and purification The full-length cDNA encoding human SUMO-2 protein [3] was first cloned into the pET28a expression vector, and the cDNA sequence of truncated SUMO-2 was ampli- fied by PCR using the SUMO-2/pET28a as a template. The PCR was carried out for 25 cycles of 30 s at 95 °C, 30 s at 55 °Cand30sat72°C, using two primers 5¢-GGAATTCCATATGGGAGTCAAGACTGA GAA CAAC-3¢ and 5¢-CCGCTCGAGTCAACCTCCCGTCT G-3¢. The DNA products were checked on 1.5% agarose gels stained with e thidium bromide and t hen digested with restriction enzymes. The truncated SUMO-2 with an N-terminal His 10 tag was then expressed using pET16b (Novagen) in Escherichia coli BL21 (DE3) at 37 °C, induced by adding 1 m M isopropyl thio-b- D -galactoside (IPTG) at D 600 ¼ 0.8. Bacterial cells were harvested after 4 h of induction by centrifuging at 8983 g for 30 min using Avan tiÒ J-20XP (Beckman). Cells w ere lyse d i n a buffer c ontaining 25 m M Tris-base a nd 150 m M NaCl (pH 8.0) with a French Press (Cell Disruption, Constant- systems) at 206 843 kPa twice and centrifuged (18 592 g, 20 min ) for supernatant collection. The S UMO-2 protein was purified using a column packed with Ni–NTA HisBindÒ resin ( Novagen) in two steps. In the first purification, major protein was eluted using an i midazole gradient of 0–250 m M and the collec ted fractions were analysed by SDS/PAGE. The SUMO-2 protein in peak fractions was pooled and dialysed three times against 25 m M Tris-base, 150 m M NaCl (pH 8.0) and incubated for 26 h at room temperature in the presence of Factor Xa (Novagen). This step removes the His 10 tag to generate the truncated SUMO-2 protein (9–93 amino acids). The protein solution was then purified a second time, in which the flow-through was collected using a wash buffer that contained 20 m M inidazole, and dialysed t hree times i n 25 m M Tris-base, 20 m M NaCl, 1 m M dithiothreitol (pH 8.0). Molecular mass o f the truncated SUMO-2 was determined to be 9950 Da by ESI-MS, exactly as calculated from the amino acid sequence. The purified protein was concentrated to 60 mgÆmL )1 by ultrafiltration using 3 kDa Jumbosep TM membrane (Pall Corporation, MI). Fig. 1. Structure-based sequence alignment of SUMO proteins from human (Homo sapiens; h_SUMO-2/-3/-4/-1) and yeast (Sacchromyces cerevisiae; y_SMT3) SUMO, and human ubiquitin (h_Ubiquitin). Secondary st ructu re elements of SU MO-2 are shown ab ove the s equenc es with a-helices and b-strands depicted as red cylinders and green arrows, respectively, and t he N-terminal arm a s a line. Identical r esidues conserved in five or more sequences are shaded in yellow and gaps are denoted by dots. The residues of human SUMO-1 that interact with Ubc9 are coloured orange, those of yeast SMT3 that interact with Ulp1 are in cyan, and the overlapping regions are shown in magenta. The target proteins are a ttached directly to the C-terminal glycine of ubiquitin, whereas SUMO requires additional proc essing to remove the C-terminal tail. The C-terminal Gly-Gly motifs in the mature proteins are s hown in green. Ó FEBS 2004 Structure and function of human SUMO-2 (Eur. J. Biochem. 271) 4115 Crystallization and data collection Crystallization was achieved by the hanging-drop vapour diffusion method at room temperature using the CryoII screen kits (Emerald Biostructures). After optimization, two different crystal forms of the truncated SUMO-2 protein (9–93 amino acids) were obtained. One crystal form having a triangular plate shape ( type I, Fig. 2A ) grew in 40% (w/v) PEG-600, 0.1 M 2-(cyclohexylamino)ethanesulfonic acid (CHES) and 0.1 M Tris/HCl pH 8.0, and diffracted to 1.6 A ˚ . The other one, of rectangular p olyhedron shape (type II, Fig. 2B), grew in 40% (w/v) PEG-600, 0.1 M CHES, 0.1 M sodium HEPES pH 8.0, and diffracted well to a resolution of 1.2 A ˚ . Two data sets were collected using MSC R-AXIS IV++ image plate detectors and processed using the software package of HKL [12]. The first one was carried out using the triangular plate crystal form (type I) at Institute of Biological Chemistry, Academia Sinica, using an MSC MicroMax 002 X-ray generator. The second data set o f the polyhedral crystal form (type II) was c ollected at t he National Synchrotron Radiation R esearch Center, Hsinchu, Taiwan, using beam line 17B2 as an X-ray source. Crystallographic computing and modelling Most calculations for molecular replacement, electron density maps a nd structural refinem ents were c arried out using t he program CNS [13]. F or type II crystal, r efinements and map calculations also used SHELX -97 [14]. Substitution of side chains, addition of water molecules, manual adjustment of the protein models and rebuilding of the N- and C-terminal segments were performed using the program O [15]. For h omology modelling of SUMO-1 a nd -3, t he refined SUMO-2 model at 1.6 A ˚ resolution of type I crystal was used as a template. After substituting the side chains, their conformations were adjusted with reference t o the NMR structure of SUMO-1 and the crystal structure of yeast A CD B Fig. 2. Photographs and electron density maps of the SUMO-2 crystals. Shown in (A) and (B) are two different crystal forms I and II obtaine d under slightly different conditions. The sizes of crystals are 0.25 · 0.25 · 0.05 mm 3 in (A) and 0.35 · 0.15 · 0.1 mm 3 in (B). In (C) and (D) are representative electron density maps superimposed on the refined models of the two crystal forms I a nd II, respectively. B oth were contoured at 2.0 r levels using 2Fo–Fc maps phased by the refined mo dels. The side chain of Lys21 lacks well-defined density, presumably because i t is flexible. 4116 W C. Huang et al. (Eur. J. Biochem. 271) Ó FEBS 2004 SMT3. The models were then subjected to molecular dynamics and energy minimization using CNS , while the backbone atoms were restrained w ith t he original model coordinates. For structural comparisons with ubiquitin, yeast SMT3 and human SUMO-1, models directly from the Protein Data Base (PDB) entries 1UBQ, 1 EUV (chain B) and 1A5R (model 1), respectively, were used. Figure 1 was produced using the program ALSCRIPT [16]. The r ibbon diagrams and the electron density m aps in Figs 2, 3 and 5 were drawn using MOLSCRIPT [17], BOBSCRIPT [18] and RASTER 3 D [19]. The molecular surface properties were examined using GRASP [20], w hich was a lso used t o generate Fig. 4. Model geometry and crystal contacts were analysed using the programs PROCHECK and AREAIMOL of the CCP4 package [21]. Results and Discussion Structure determination and refinement Analysis of the diffraction patterns suggested that both type I and type II SUMO-2 crystals belong to the rhombohedral space group R3. Statistics for the two data sets are shown in Table 1 . Although t he unit cell dimensions are similar in the a-andb-axes, the s ignificant difference in the c-axes i mplies that the crystals are not entirely isomorphous. Using synchrotron, type I crystals also d iffracted to a h igher resolution than 1.6 A ˚ , but not as good as type II crystals. With one SUMO-2 molecule in an asymmetric unit, the specific volumes (or Matthews coefficients [22]) are 1.60 and 1.81 A ˚ 3 ÆDa )1 , suggesting solvent contents of 23.0% Fig. 3. Tertiary structure of SUMO-2 and comparison with other proteins. (A) A ribbon r epresent ation of t he protein fold. (B) A topo logy diagram with well-defined backbone hydrogen bonds. The helices (a1, a2) and strands (b1–b5) are coloured in magenta, blue, green, yellow a nd red f rom N to C t erminus. The hydrogen bond d istances, with a c rite rion of less t han 3.2 A ˚ , are observed in the r efined model at 1.2 A ˚ , with o ne exception betweenAsp16andArg36,whichisseeninthe1.6A ˚ model. The amino acids are shaded in red, gree n and blue for acidic, neutral an d basic polar residues, and in yellow for prolines and glycines. In (C) the polypeptide tracings of two SUMO-2 models from type I (12–89) and type II (17–88) crystals, shown in green and red, are superimposed with th at of human ubiquitin (1–76), shown in blue. In (D ) the yeast SMT3 crystal structure (20–98) and human SUMO-1 NMR structure ()2–101), coloured yellow and cyan, respectively, are compared with the SUMO-2 structure (type I crystal), shown in red. Ó FEBS 2004 Structure and function of human SUMO-2 (Eur. J. Biochem. 271) 4117 and 31.9% for the type I and type II crystal forms, respectively. The NMR model of human SUMO-1 (PDB code 1A5R) contains full-length protein, whereas the N- and C-terminal regions are fl exible. Molecular replacement search u sing the NMR model did not yield a correct solution for the crystal structure of SUMO-2, even with omission of the terminal segments. Instead, i t was solved using yeast SMT3 (PDB code 1EUV) as a search model. The initial R value for the type I crystal was 0.465 after rigid-body refinement at 3.0 A ˚ resolution. The final m odel c ontains amino acid residues 12–89 and 67 water molecules, with R and R free values of 0.169 and 0.190, respectively. The R value for the type II crystal based on the refined type I model was 0.409 at 1.5 A ˚ . After refinement, the model contains amino acid residues 17–88 and 127 water molecules, with R and R free of 0.119 and 0.185, respectively. Statistics are shown in Table 1. Details of the refinement procedures are summarized in Table 2 . The atomic coordinates a nd structure f actors of type I and type II crystals have been deposited in the R CSB Protein D ata B ank, with accession cod es 1WM2 and 1WM3, respectively. Quality of the model and structure comparison The coordinate errors in the refined SUMO-2 models are between 0.15 A ˚ and 0.20 A ˚ as estimated by Luzzati plots [23]. The electron density maps in a representative region are shown in F ig. 2C and D. At 1.2 A ˚ resolution, individual atoms b egin to appear as discrete spheres. An overall r ibbon diagram i s shown i n F ig. 3A. The peptide folding of SUMO-2 protein c onsists of a h alf-open b-barrel and two fl anking a-helices, w ith secondary structure elements arranged as bbabbab in th e s equence (Fig. 1), identical to those of ubiquitin, SMT3 and SUMO-1. Fig. 3B shows a topology diagram of S UMO-2. The 39 w ell-defined back- bone hydrogen bonds include not only those for the b-st rands and a-helices, but also three bonds for turns and two for tertiary interactions. The protein models of SUMO-2 type I and type II crystals superimpose with a n r .m.s.d. of 0.544 A ˚ for 288 backbone atoms and 1.201 A ˚ for all 584 atoms. Larger deviations of Ca coordinates t han 1.0 A ˚ occur in the residues 17, 26, 27, 5 6 a nd 88. A lthough type I I crystal diffracts to higher resolution, its visible N terminus is shorter than that o f type I crystal by five residues. As shown in Fig. 3A, this segment extends away from t he protein core and should be flexible because of exposure to the bulk solvent. The smaller unit-cell dimension of type I crystal allows the N terminus to be docked onto a neighbouring molecule, specifically, near the region of Phe60–Thr70, and thus stabilizes the extended conformation. Also shown in F ig. 3C, the model of human ubiquitin (PDB code 1UBQ) is superimposed on the S UMO-2 models of type I and II crystals, with an r.m.s.d. of 0.952 A ˚ and 1.135 A ˚ for 55 and 65 Ca atoms, Fig. 4. Surface properties of SUMO proteins. The molecular surface of SUMO-2 (type I crystal)isshownin(A)and(C);thatofthe SUMO-1modelisshownin(B)and(D).The charge potentials in ( A) (C) and (D) a re cal- culated using GRASP with a r ange of )10 to +10 k B T, in which k B is Boltzmann constant and T is Kelvin temperature, and coloured from red t o blue. Neutral a reas are shown in white. In (B) the conserved regions that interact with Ubc9 and Ulp1 are highlighted andcolouredinorange,cyanandmagenta,as in Fig. 1. In (E) and (F) the c orresponding amino acids for different surface charges on SUMO-2 and SUMO-1 are shown. Positively charged, negative charge d and neutral polar residues are coloured blue, r ed and magenta, respectively, a nd nonpolar residues are shown in green. The views in (C–F) are similar to that of Fig. 3A and those of (A) and (B) a re rota- ted 180° about the horizontal axis. 4118 W C. Huang et al. (Eur. J. Biochem. 271) Ó FEBS 2004 respectively. This is based on a distance criterion o f le ss than 2.0 A ˚ , which excluded t he residues 45–58 in the former model and 40 , 49 and 55–58 in the latter model of SUMO-2 and the equivalents of ubiquitin. Although the sequences have only 18% identity, the protein folds of SUMO-2 and ubiquitin are very similar, even without insertion (Fig. 1). Yet th ese t wo classes of proteins h ave very different functions, which m ay be explained by the disparate surface charge distributions [6]. Significant difference between the yeast SMT3 crystal structure and the human SUMO-1 NMR s tructure has been observed by Mossessova and Lima [8]. In Fig. 3D the SUMO-2 model is superimposed with those of SMT3 (1EUV) and S UMO-1 (1A5R). Based on a distance criterion of 2 .0 A ˚ , the r.m.s.d. is 1.096 A ˚ between 43 pairs of Ca atoms in S UMO-1 ( NMR) and SUMO-2 (type I crystal). Under the same condition, the r msd is 0.918 A ˚ between 67 Ca pairs in SUMO-2 a nd SMT3, and it is 0.470 A ˚ for 40 matched pairs with a distance criterion of 1.0 A ˚ . Therefore, the crystal structure of human SUMO-2 is more similar to that of yeast SMT3 than to the NMR structure of SUMO-1. The difference between SUMO-2 Table 1. X-ray data statistics for S UM O-2 crystals. Numbers in parentheses are for the highest resol ution shells. Crystal form Type I Type II Data collection Space group R3 (hexagonal indexing) R3 (hexagonal indexing) Unit cell (A ˚ )a¼ b ¼ 75.25, c ¼ 29.17 a ¼ b ¼ 74.96, c ¼ 33.23 X-ray source MicroMax 002 NSRRC BL17B2 Wavelength (A ˚ ) 1.5418 1.0717 Detector RAXIS-IV++ RAXIS-IV++ Crystal-to-film distance (mm) 100 83.4 Oscillation range (°) 1.0 1.5 Mosaicity (°) 0.614 0.292 Number of frames 186 145 Resolution range (A ˚ ) 50–1.6 (1.66–1.60) 20–1.2 (1.24–1.20) Number of observations 42023 (2120) 141402 (11874) Unique reflections 8015 (690) 21781 (2109) Completeness (%) 98.5 (85.1) 100.0 (99.8) Average I/r(I) 45.5 (5.3) 39.6 (2.6) R merge (%) 3.9 (24.6) 4.6 (55.9) Refinement Software CNS 1.1 SHELX -97 Total reflection used [F >0r(F)] 7868 (633) 20948 (1924) R for 95% working data set 0.169 (0.266) 0.119 (0.217) R free for 5% est data set 0.190 (0.273) 0.185 (0.239) rmsd from ideal bond lengths (A ˚ ) 0.017 0.013 rmsd from ideal bond angles (°) 1.8 2.3 rmsd from ideal dihedral angles (°)2726 rmsd from ideal improper angles (°) 1.3 1.8 Ramachandran plot: number of residues in most favored regions (%) 97.1 96.8 In additional allowed regions (%) 2.9 3.2 Average B-values/number of atoms for protein backbone (A ˚ 2 ) 17.7/312 18.4/288 For protein side chains (A ˚ 2 ) 22.8/322 27.6/297 For water molecules (A ˚ 2 ) 34.3/67 42.8/127 Table 2. Refinement procedures of the SUMO-2 crystals. Description of steps Protein Water Resolution R/R free Type I crystal, yeast SMT3 model 13–98 (SMT3) 3.0 A ˚ 0.464 Delete N- and C-termini, insert Asp26 16–88 (SUMO-2) 2.0 A ˚ 0.339/0.371 Add water molecules, B-value refinement 16–88 38 1.6 A ˚ 0.190/0.224 Extend the termini, add more waters 12–89 67 1.6 A ˚ 0.169/0.190 Type II crystal, type I model 12–89 67 1.5 A ˚ 0.409 Delete N- and C-termini, remove waters 16–87 0 1.5 A ˚ 0.375 Modify N-terminus, add water molecules 17–87 102 1.2 A ˚ 0.191/0.205 Use SHELX , anisotropic B-values 17–87 102 1.2 A ˚ 0.133/0.190 Extend C-terminus, add more waters 17–88 127 1.2 A ˚ 0.119/0.185 Ó FEBS 2004 Structure and function of human SUMO-2 (Eur. J. Biochem. 271) 4119 crystal structure and SUMO-1 NMR structure is partic- ularly evident in the regions of 28–43 and 71–83, that correspond to the strand b2, the N terminus of the h elix a1, the helix a2, and the connecting loop to the strand b5 (Fig. 3 A,D). SuchalargedifferencebetweentheNMRandcrystal structures may explain the fact t hat we were not able to solve our crystal structure by the molecular replacement method using SUMO-1 N MR structure as the starting model. The high-resolution NMR structure of SUMO-1 determined later using heteronucle ar NOE also showed difference from the s tructure of 1A5R [7 ]. Interestingly, this new SUMO-1 NMR structure is similar to the SMT3 NMR structure, whereas significant deviations betw een the crystal structure and solution structure of SMT3 were also observed [9]. Therefore, the deviations may be due to different environments and different experimental tech- niques used in the structure determinations. Surface potential and functional difference The mechanisms of protein ubiquitination and SUMOyla- tion are similar, which involve the activating, conjugating, and ligation enzymes E1, E2 and E3. A peptidase is also required to remove the C-terminal peptide of a SUMO protein to render the mature form, which has the C-terminal Gly-Gly motif for conjugation with target proteins [4]. In yeast, an E1-specific for SUMO has been identified as a large heterodimeric Aos1/Uba2 of  11 0 kDa, and there i s a heterodimeric homologue SAE1/SAE2 in man. The E2 in both human and yeast is a highly conserved Ubc9 of 18 kDa, whereas the E3 proteins have a broader definition and comprise s everal s ubclasses [ 4]. The enzymes Ulp1 a nd Ulp2 in yeast are located in the nuclear pore complex and nucleoplasm, and they are the protease and isopeptidase for processing SUMO precursor and deSUMOylation of target proteins, whereas in mammals the Ulp1 family comprises several proteases with various localizations [24]. Despite the similar mechanism, ubiquitination and SUMOylation path- ways are different, involving two distinct sets of enzymes, and i n some aspects they are comp etitive [25]. As first proposed by studying the SUMO-1 NMR s tructure, t he functional difference is expressed in the surface charge distributions [6]. In Fig. 4A, the surface of SUMO-2 protein shows a region with strong negative c harge potential. I n contrast, the corresponding region of ubiquitin is mostly neutral (data not shown). Presumably this is the basis for them to interact differently with the various enzymes and other proteins. The interactions between SUMO-1 a nd Ubc9 have been studied by NMR chemical shift perturbation experiments [26,27]. Three major regions had the most significant changes; these a re indicated in t he sequences of Fig. 1 and mapped on the surface of our SUMO-1 model in Fig. 4B. The positively ch arged Lys25 ( Lys21 in SUMO-2) and a cluster of four negatively charged amino acids Glu83-Glu84- Glu85-Asp86 (Glu79-Asp80-Glu81-Asp82 in SUMO-2) are supposed to interact with Ubc9. These two regions are conserved among four human SUMO proteins as well as the yeast SMT3 protein (Fig. 1). In the crystal structure of yeast Ulp1–SMT3 complex, the Ulp1 protein makes direct contact not only with the C-terminal segment that contains the functional G ly-Gly motif, but also with the region Arg64–Arg71 (Fig. 1). These correspond to Arg59–Pro66 in SUMO-2 and , with an adjacent Arg61 substituting Leu66 in SMT3, the surface features in this region are also con served. However, interactions between SUMO and other proteins, including E3, may be established with other surface regions. Although the sequences of human SUMO-2 and -3 are 87% i dentical, they a re located in different c ellular com- partments: SUMO-2 was found in nuclear bodies but SUMO-3 was located in the cytoplasm [10]. The s urface charge distribution of SUMO-2/-3 is even more similar. When these two protei n surfaces a re compared, t he only visible difference corresponds to residue 77, which is a negatively charged Glu in SUMO-2, but is a positively charged A rg in SUMO-3. On the other hand, SUMO-1 is 47% identical to SUMO-2 in sequence, and has a longer N-terminal arm. The r esulting difference in their surface properties can be attributed to at least 10 residues. These include Glu33, Lys48, Glu49, Gln53, Asn60, Leu6 5, Arg70, Lys78, Gly81 and Glu93 in SUMO-1, whereas t he corres- ponding surface residues in SUMO-2 are Val29, Met44, Lys45, Glu49, Arg56, Arg61, Pro66, Ala74, Glu77 and Gln89, respectively. The most prominent is a concave re gion shown i n F ig. 4 C a nd D, which i s fl anked b y the helix a1 and the strands b3/b4 (Fig. 3A). This region is neutral in SUMO-2 but positively charged in SUMO-1, probably caused by the substitution of Met44 in SUMO-2 with Lys48 in SUMO-1, as shown in Fig. 4 E and F. In particular, the concave surface is near the C terminus, and thus may serve as a potential site for d iscrimination between SUMO-1 and -2 i n humancells. The flexible N-terminal arms of SUMO-1, -2 and -3 proteins, which have different lengths, may also be involved in the interactions with other proteins, whereas ubiquitin does not have such an equivalent. Crystal packing and oligomeric assembly The SUMO-2 structure presented in this p aper is the first high-resolution crystal structure of human SUMO protein. The two crystal forms of truncated SUMO-2 studied here are not isomorphous, but the crystal packing is similar. Each protein molecule is in lattice contact with 10 symmetry- related molecules via five types of contact interfaces. The total areas buried by the lattice contact interfaces are 3412 A ˚ 2 in type I c rystal (1.6 A ˚ ) a nd 2211 A ˚ 2 in type II crystal (1.2 A ˚ ), whereas the molecu lar s urface areas of t he SUMO-2 protein models, containing residues 12–89 and 17–88, are 5264 A ˚ 2 and 486 6 A ˚ 2 , respectively. The first and m ost conserved i nterface is between molecules related by the crystallographic threefold axis. The buried areas are 856 A ˚ 2 and 821 A ˚ 2 on each SUMO-2 monomer in the type I and type II crystals, respectively, corresponding to about o ne-quarter and more than one- third of the contact surfaces. T he interactions include two hydrogen bonds between backbone atoms of Gly27(O)– Lys33*(N) and Val29(N)–Gln31*(O), and a salt bridge between the side chains o f Asp26 and Arg50*. (Amino acid residues of t he symmetry-related molecules are denoted by asterisks.) The latter is also hydrogen b onded to Tyr47(OH) and Gln51(OE1). Such interactions, particularly those between the strands b2, may stabilize a possible trimeric assembly of SUMO-2 in solution, shown in Fig. 5. The 4120 W C. Huang et al. (Eur. J. Biochem. 271) Ó FEBS 2004 other four interfaces are not all conserved, whereas the buried surface areas are much larger in type I crystal than in type II. Because the c-axis is significantly shorter, more lattice interactions were observed i n type I crystal. These include docking of the flexible N-terminal segment onto a neighbouring molecule. Polymers of ubiquitin h ave been studied extensively since they were discovered [28]. The site of self-conjugation is Lys48. This residue corresponds to Gln65 in S UMO-2 a nd is conserved in SUMO-1 and -3 (Fig. 1). Consequently, SUMO does not form polymers in the same manner as ubiquitin. However, in a recent study [11], oligomers of SUMO-2/-3 w ere identified in vitro due to the existence of VKXE motif, a specific consensus SUMOylation site, in the N-terminal arm. The distance between C a atoms o f the N-terminal Thr12 a nd C-terminal Gln89* of neighbouring SUMO-2 molecules related by the triad axis is 20.6 A ˚ in type I crystal, and that between His17 and G ln88* in type II crystal is 20.7 A ˚ , comparable to the distance between Ca atoms separated by six peptide bonds in extended confor- mations. Thus, in the trimer, it is possible for the Lys11 of one SUMO-2 molecule to form an isopeptide bond with the Gly93 of another. The crystal structures of diubiquitin and tetraubiquitin showed some alternatives of the quaternary c onformations of ubiquitin polymer for e fficient r ecognition by the 26S proteosome, y et no conclusion has been reached due to the inherently flexible intermolecular links [29]. The SUMO-2 trimer in Fig. 5 has a completely d ifferent arrangement from those of ubiquitin polymers, and the sites of conju- gation are also different. 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