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 identi
Trang 1Crystal 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 Huang1,2, Tzu-Ping Ko1, Steven S.-L Li3and Andrew H.-J Wang1
1 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 2 Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaoshiung, Taiwan;3Department of Biotechnology, College of Life Sciences, Kaoshiung Medical University, Taiwan
The SUMO proteins are a class of small ubiquitin-like
modifiers SUMO is attached to a specific 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, which are involved in protein trafficking and
tar-geting A truncated human SUMO-2 protein that contains
residues 9–93 was expressed in Escherichia coli and
crystal-lized in two different unit cells, with dimensions of a¼ b ¼
75.25 A˚, c¼ 29.17 A˚ and a ¼ b ¼ 74.96 A˚, c ¼ 33.23 A˚,
both belonging 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 replacement
using the yeast SMT3 protein as a search model Subsequent
refinements yielded R/Rfreevalues of 0.169/0.190 and 0.119/
0.185, at 1.6 A˚ and 1.2 A˚, respectively The peptide folding
of SUMO-2 consists of a half-open b-barrel and two flank-ing a-helices with secondary structural elements arranged as bbabbab in the sequence, identical to those 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 analysis suggests a possible trimeric assembly of the SUMO-2 protein, of which the biological significance remains to be determined
Keywords: homology modeling; molecular interactions; protein modification; 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 for 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 Thus, newly 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 are attached to target proteins by
forming an isopeptide bond between the C-terminal glycine
and a specific lysine side chain on the target [2] The extra
amino acids beyond the last glycine–glycine motif of native
SUMO proteins are proteolytically removed in vivo In
mammals, there are at least four different SUMO proteins,
SUMO-1, -2, -3 and -4 The human hSMT3 cDNA encoding the SUMO-2 protein was first reported by Mannen et al [3] SUMO-2 and SUMO-3 share 87% 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 and the human SUMO proteins have
an insertion between the strands b1 and b2, as shown in Fig 1 The crystal structure of yeast SMT3 was determined
in complex with Ulp1 protease [8] Significant deviation between the crystal structure and solution structure of yeast SMT3 was also observed using high-resolution hetero-nuclear NMR spectroscopy [9]
In addition to the N-terminal extensions, the most 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 21 May 2004, revised 14 July 2004,
accepted 31 August 2004)
Trang 2functions in protein targeting Arrangement of side chains
confers the protein with unique surface properties Thus,
comparison of SUMO-1, -2 and -3 surface properties by
modelling provides an approach to understanding the
relationship between 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 isopeptide bond The VKTE sequence in SUMO-2 is
consistent with the SUMOylation consensusYKXE where
Y represents a hydrophobic amino acid and X means any
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
fused to a His10tag 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 95C,
30 s at 55C and 30 s at 72 C, using two primers
5¢-GGAATTCCATATGGGAGTCAAGACTGAGAA CAAC-3¢ and 5¢-CCGCTCGAGTCAACCTCCCGTCT G-3¢ The DNA products were checked on 1.5% agarose gels stained with ethidium bromide and then digested with restriction enzymes The truncated SUMO-2 with an N-terminal His10 tag was then expressed using pET16b (Novagen) in Escherichia coli BL21 (DE3) at 37C, induced by adding 1 mM isopropyl thio-b-D-galactoside (IPTG) at D600¼ 0.8 Bacterial cells were harvested after
4 h of induction by centrifuging at 8983 g for 30 min using Avanti J-20XP (Beckman) Cells were lysed in a buffer containing 25 mM Tris-base and 150 mM 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 SUMO-2 protein was purified using a column packed with Ni–NTA HisBind resin (Novagen) in two steps In the first purification, major protein was eluted using an imidazole gradient of 0–250 mMand the collected fractions were analysed by SDS/PAGE The SUMO-2 protein in peak fractions was pooled and dialysed three times against 25 mMTris-base, 150 mMNaCl (pH 8.0) and incubated for 26 h at room temperature in the presence of Factor Xa (Novagen) This step removes the His10tag 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 mMinidazole, and dialysed three times in
25 mM Tris-base, 20 mM NaCl, 1 mM dithiothreitol (pH 8.0) Molecular mass of 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)1by ultrafiltration using 3 kDa JumbosepTMmembrane (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 structure elements of SUMO-2 are shown above the sequences with a-helices and b-strands depicted as red cylinders and green arrows, respectively, and the N-terminal arm as a line Identical residues 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 attached directly to the C-terminal glycine of ubiquitin, whereas SUMO requires additional processing to remove the C-terminal tail The C-terminal Gly-Gly motifs in the mature proteins are shown in green.
Trang 3Crystallization 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, 0.1M 2-(cyclohexylamino)ethanesulfonic acid
(CHES) and 0.1M Tris/HCl pH 8.0, and diffracted to
1.6 A˚ The other one, of rectangular polyhedron shape (type
II, Fig 2B), grew in 40% (w/v) PEG-600, 0.1M CHES,
0.1M 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 of the polyhedral crystal form (type II) was collected
at the National Synchrotron Radiation Research Center, Hsinchu, Taiwan, using beam line 17B2 as an X-ray source
Crystallographic computing and modelling Most calculations for molecular replacement, electron density maps and structural refinements were carried out using the programCNS[13] For type II crystal, refinements and map calculations also usedSHELX-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 programO[15]
For homology modelling of SUMO-1 and -3, the 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 to the NMR structure of SUMO-1 and the crystal structure of yeast
A
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 obtained 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 and II, respectively Both were contoured at 2.0 r levels using 2Fo–Fc maps phased by the refined models The side chain of Lys21 lacks well-defined density, presumably because it is flexible.
Trang 4SMT3 The models were then subjected to molecular
dynamics and energy minimization using CNS, while the
backbone atoms were restrained with the original model
coordinates For structural comparisons with ubiquitin,
yeast SMT3 and human SUMO-1, models directly from the
Protein Data Base (PDB) entries 1UBQ, 1EUV (chain B)
and 1A5R (model 1), respectively, were used
Figure 1 was produced using the programALSCRIPT[16]
The ribbon diagrams and the electron density maps in
Figs 2, 3 and 5 were drawn usingMOLSCRIPT[17],BOBSCRIPT
[18] andRASTER3D[19] The molecular surface properties
were examined usingGRASP[20], which was also used to
generate Fig 4 Model geometry and crystal contacts were
analysed using the programsPROCHECKandAREAIMOLof
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 the unit cell dimensions are similar in the a- and b-axes, the significant difference in the c-axes implies that the crystals are not entirely isomorphous Using synchrotron, type I crystals also diffracted to a higher 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˚3ÆDa)1, suggesting solvent contents of 23.0%
Fig 3 Tertiary structure of SUMO-2 and comparison with other proteins (A) A ribbon representation of the protein fold (B) A topology diagram with well-defined backbone hydrogen bonds The helices (a1, a2) and strands (b1–b5) are coloured in magenta, blue, green, yellow and red from N
to C terminus The hydrogen bond distances, with a criterion of less than 3.2 A˚, are observed in the refined model at 1.2 A˚, with one exception between Asp16 and Arg36, which is seen in the 1.6 A˚ model The amino acids are shaded in red, green and blue for acidic, neutral and 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 that 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.
Trang 5and 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 flexible Molecular replacement search using the
NMR model did not yield a correct solution for the crystal
structure of SUMO-2, even with omission of the terminal
segments Instead, it 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 model contains amino acid residues
12–89 and 67 water molecules, with R and Rfreevalues 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 Rfreeof 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 and structure factors of
type I and type II crystals have been deposited in the RCSB
Protein Data Bank, with accession codes 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 Fig 2C and D At 1.2 A˚ resolution, individual atoms begin to appear as discrete spheres An overall ribbon diagram is shown in Fig 3A The peptide folding of SUMO-2 protein consists of a half-open b-barrel and two flanking a-helices, with secondary structure elements arranged as bbabbab in the sequence (Fig 1), identical to those of ubiquitin, SMT3 and SUMO-1 Fig 3B shows a topology diagram of SUMO-2 The 39 well-defined back-bone hydrogen bonds include not only those for the b-strands 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 an 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 than 1.0 A˚ occur in the residues 17, 26, 27, 56 and 88 Although type II crystal diffracts to higher resolution, its visible N terminus is shorter than that of type I crystal by five residues As shown
in Fig 3A, this segment extends away from the 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 Fig 3C, the model of human ubiquitin (PDB code 1UBQ) is superimposed on the SUMO-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) is shown in (A) and (C); that of the SUMO-1 model is shown in (B) and (D) The charge potentials in (A) (C) and (D) are cal-culated using GRASP with a range of )10 to +10 k B T, in which k B is Boltzmann constant and T is Kelvin temperature, and coloured from red to blue Neutral areas are shown in white In (B) the conserved regions that interact with Ubc9 and Ulp1 are highlighted and coloured in orange, cyan and magenta, as
in Fig 1 In (E) and (F) the corresponding amino acids for different surface charges on SUMO-2 and SUMO-1 are shown Positively charged, negative charged and neutral polar residues are coloured blue, red and magenta, respectively, and nonpolar residues are shown
in green The views in (C–F) are similar to that
of Fig 3A and those of (A) and (B) are rota-ted 180 about the horizontal axis.
Trang 6respectively This is based on a distance criterion of less
than 2.0 A˚, which excluded the 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 these two classes of proteins have
very different functions, which may be explained by the
disparate surface charge distributions [6]
Significant difference between the yeast SMT3 crystal
structure and the human SUMO-1 NMR structure has been
observed by Mossessova and Lima [8] In Fig 3D the SUMO-2 model is superimposed with those of SMT3 (1EUV) and SUMO-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 SUMO-1 (NMR) and SUMO-2 (type I crystal) Under the same condition, the rmsd is 0.918 A˚ between 67 Ca pairs in SUMO-2 and 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 SUMO-2 crystals Numbers in parentheses are for the highest resolution shells.
Crystal form
Data collection
Space group R3 (hexagonal indexing) R3 (hexagonal indexing) Unit cell (A˚) a ¼ b ¼ 75.25, c ¼ 29.17 a ¼ b ¼ 74.96, c ¼ 33.23
Resolution range (A˚) 50–1.6 (1.66–1.60) 20–1.2 (1.24–1.20)
Refinement
Total reflection used [F > 0 r(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)
Ramachandran plot: number of residues in most favored regions (%) 97.1 96.8
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
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
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
Extend C-terminus, add more waters 17–88 127 1.2 A˚ 0.119/0.185
Trang 7crystal 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 helix a1,
the helix a2, and the connecting loop to the strand b5
(Fig 3A,D)
Such a large difference between the NMR and crystal
structures may explain the fact that we were not able to
solve our crystal structure by the molecular replacement
method using SUMO-1 NMR structure as the starting
model The high-resolution NMR structure of SUMO-1
determined later using heteronuclear NOE also showed
difference from the structure of 1A5R [7] Interestingly, this
new SUMO-1 NMR structure is similar to the SMT3 NMR
structure, whereas significant deviations between 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 110 kDa, and there is
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 several subclasses [4] The enzymes Ulp1 and
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 in some aspects they are competitive [25] As first
proposed by studying the SUMO-1 NMR structure, the
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 charge potential In
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 and Ubc9 have been
studied by NMR chemical shift perturbation experiments
[26,27] Three major regions had the most significant
changes; these are indicated in the sequences of Fig 1 and
mapped on the surface of our SUMO-1 model in Fig 4B
The positively charged 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 Gly-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 conserved 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% identical, they are located in different cellular com-partments: SUMO-2 was found in nuclear bodies but SUMO-3 was located in the cytoplasm [10] The surface charge distribution of SUMO-2/-3 is even more similar When these two protein surfaces are compared, the only visible difference corresponds to residue 77, which is a negatively charged Glu in SUMO-2, but is a positively charged Arg 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 resulting difference in their surface properties can be attributed to at least 10 residues These include Glu33, Lys48, Glu49, Gln53, Asn60, Leu65, Arg70, Lys78, Gly81 and Glu93 in SUMO-1, whereas the 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 region shown in Fig 4C and D, which is flanked by 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 4E and F In particular, the concave surface is near the C terminus, and thus may serve as a potential site for discrimination between SUMO-1 and -2 in human cells 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 paper 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 crystal (1.6 A˚) and 2211 A˚2 in type II crystal (1.2 A˚), whereas the molecular surface areas of the SUMO-2 protein models, containing residues 12–89 and 17–88, are 5264 A˚2and 4866 A˚2, respectively
The first and most conserved interface is between molecules related by the crystallographic threefold axis The buried areas are 856 A˚2and 821 A˚2on each SUMO-2 monomer in the type I and type II crystals, respectively, corresponding to about quarter and more than one-third of the contact surfaces The 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 of Asp26 and Arg50* (Amino acid residues of the symmetry-related molecules are denoted by asterisks.) The latter is also hydrogen bonded 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
Trang 8other 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 in type I crystal These
include docking of the flexible N-terminal segment onto a
neighbouring molecule
Polymers of ubiquitin have been studied extensively since
they were discovered [28] The site of self-conjugation is
Lys48 This residue corresponds to Gln65 in SUMO-2 and
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 were identified in vitro due to the existence of
VKXE motif, a specific consensus SUMOylation site, in the
N-terminal arm The distance between Ca atoms of the
N-terminal Thr12 and 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 Gln88* 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 conformations
of ubiquitin polymer for efficient recognition by the 26S
proteosome, yet no conclusion has been reached due to the
inherently flexible intermolecular links [29] The SUMO-2
trimer in Fig 5 has a completely different arrangement
from those of ubiquitin polymers, and the sites of
conju-gation are also different It is uncertain whether the trimer is
an oligomerization motif for SUMO-2, and this possibility
is currently under investigation
Acknowledgements
We thank Drs Chia-Cheng Chou, Rey-Ting Guo and Cheng-Chung Lee for their assistance in data collection We also thank the National Synchrotron Radiation Research Center for beam time allocation This work was supported by grants from National Science Council (NSC 92–3112-B-110-001 and NSC 93-3112-B-110-001) to SSLL and from Academia Sinica to A.H.J.W.
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