(Received for publication, April 6, 1997, and in revised form, May 27, 1997)
From the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands
Murine/human ubiquitin-conjugating enzyme Ubc9 is
a functional homolog of Saccharomyces cerevisiae Ubc9 that
is essential for the viability of yeast cells with a specific role in
the G2-M transition of the cell cycle. The structure of
recombinant mammalian Ubc9 has been determined from two crystal forms
at 2.0 Å resolution. Like Arabidopsis thaliana Ubc1 and
S. cerevisiae Ubc4, murine/human Ubc9 was crystallized as a
monomer, suggesting that previously reported hetero- and
homo-interactions among Ubcs may be relatively weak or indirect.
Compared with the known crystal structures of Ubc1 and Ubc4, which
regulate different cellular processes, Ubc9 has a 5-residue
insertion that forms a very exposed tight -hairpin and a 2-residue
insertion that forms a bulge in a loop close to the active site.
Mammalian Ubc9 also possesses a distinct electrostatic potential
distribution that may provide possible clues to its remarkable ability
to interact with other proteins. The 2-residue insertion and other
sequence and structural heterogeneity observed at the catalytic site
suggest that different Ubcs may utilize catalytic mechanisms of varying
efficiency and substrate specificity.
Conjugation of ubiquitin to various eukaryotic cellular proteins
regulates their activities by controlling protein concentration through
ubiquitin-directed degradation (1-4) or by directly modifying protein
function through the attached ubiquitin molecules (5). The formation of
ubiquitin-protein conjugates proceeds via a cascade of reactions that
involve two, or often three, enzymes: the ubiquitin-activating enzyme
E1,1 the
ubiquitin-conjugating enzyme Ubc (E2), and the ubiquitin-ligating enzyme E3 (6-8). The first step is the ATP-assisted formation of a
high energy thioester bond between ubiquitin and E1. Ubiquitin is then
transferred to a conserved cysteine group of Ubc. In some ubiquitin
pathways, Ubc alone, or in cooperation with E3, attaches ubiquitin to
the -amino group of a lysine residue of a substrate via an
isopeptide bond. In others, Ubc first passes ubiquitin to a thiol group
of E3, and then E3 attaches it to the substrate. Repeated conjugation
of ubiquitin to lysine residues of previously bound ubiquitin moieties
is required for proteolysis of the substrates by the 26 S
proteasome (9). A large body of genetic and biochemical evidence
indicates that the Ubcs, together with E3s, are the primary determinants of the specificity of individual ubiquitin pathways (10).
Based on amino acid sequence comparison, Ubcs can be divided broadly into four classes (10). Class I enzymes consist of a relatively conserved catalytic core domain of about 150 residues showing at least 25% sequence identity. Class II and III enzymes have either extra C-terminal or extra N-terminal extensions attached to the core domain, respectively. Class IV enzymes have both C- and N-terminal extensions. Some of these extensions to the core domain confer a certain degree of specificity for enzyme-substrate recognition or provide a localization signal. However, both specificity and localization signals also reside within the core domain itself. Different subsets of Ubcs, comprising either a single member, or multiple members from the same or different classes, are involved in different cellular processes and thereby constitute distinct functional subfamilies (10).
Murine/human Ubc9 (18 kDa) has been cloned in yeast two hybrid assays as a class I ubiquitin-conjugating enzyme that interacts with a large variety of proteins, including the adenovirus E1A oncoprotein (11), the human Rad51 recombinase (12), the human papillomavirus type 16 E1 replication protein (13), the Saccharomyces cerevisiae centromere DNA-binding core complex (14), the negative regulatory domain of the Wilms' tumor gene product (WT1) (15), and the Fas antigen (CD95) (16). These interactions have been further confirmed in the cases of adenovirus E1A, WT1, and Fas by glutathione S-transferase (GST) pull-down assays, although their physiological relevance remains to be established. The amino acid sequence of Ubc9 is found to be 100% identical between mice and humans (11, 12). It is closely related to S. cerevisiae Ubc9 (17) (56% identity) and Schizosaccharomyces pombe Hus5 (18) (66% identity). Both yeast Ubc9 enzymes are essential for cell viability with a role in regulating cell cycle progression at the G2 or early M phase. S. cerevisiae Ubc9 has been shown to target the degradation of the M-phase cyclin Clb5, the S-phase cyclin Clb2 (17), as well as the G1 cyclins Cln1 and Cln2 (19). Mammalian Ubc9 can complement a S. cerevisiae Ubc9 temperature sensitive defect but not a similar mutation in the apparently more similar Hus5 of S. pombe (11-16). Here we describe the x-ray crystal structure of recombinant murine Ubc9 and compare it with the known crystal structures of Arabidopsis thaliana Ubc1 (20) and S. cerevisiae Ubc4 (21).
The cloning of the mouse
UBC9 gene was as described previously (11). Ubc9 was expressed and
purified using a GST fusion system (22). The complete UBC9 gene was
subcloned into the pGEX-2T expression vector (Pharmacia Biotech Inc.)
and transformed into Escherichia coli strain DH5 (Life
Technologies Inc.). This expression system allows Ubc9 to be expressed
as the C-terminal part of a GST fusion protein with a thrombin cleavage
site in the linker region. Upon cleavage of the fusion protein with
thrombin, the recombinant Ubc9 acquires eight extra residues at the N
terminus: GSPGISLN. Transformed E. coli cells were grown to
an A600 of 0.8-1.2 in LB medium in the presence
of 34 µg/liter carbenicillin. Protein expression was then induced by
the addition of 0.5 mM
isopropyl-
-D-thiogalactopyranoside at 30 °C. Cells
were harvested 4-5 h after induction. Lysis of the cell pellets was
carried out by sonication in lysis buffer (100 mM NaCl,
0.5% Nonidet P-40, 5 mM EDTA, 1 mM EGTA, 60 mM 1,4-dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 25 mM Tris-Cl buffer, pH
8.0). The fusion protein was found in the soluble part of the lysate and was first purified over a glutathione-Sepharose 4B column (Pharmacia) by extensive washing with dissociation buffer (23) (3 mM ATP, 1 mM dithiothreitol, 10 mM
MgSO4, 150 mM NaCl, 10 mM Tris-Cl
buffer, pH 8.0) prewarmed at 37 °C. Ubc9 was then separated from GST
by bovine thrombin cleavage on the column at room temperature (22 °C). The cleavage solution was 1.5 units/ml bovine thrombin (Boehringer Mannheim) in cleavage buffer (2.5 mM
CaCl2, 150 mM NaCl, 20 mM Tris-Cl,
pH 8.5). Upon elution from the glutathione-Sepharose 4B column, Ubc9
was passed through a desalting column to exchange into 50 mM Bis-Tris buffer, pH 6.5. The protein was further
purified over a cation exchange column (MonoS from Pharmacia) using a 0 to 1 M NaCl gradient. Ubc9 eluted between 200 and 310 mM NaCl. The protein was finally purified to apparent
homogeneity through a gel filtration column (preparation grade Superdex
75 from Pharmacia). The apparent molecular weight of Ubc9 is 25 kDa as
estimated by comparing its elution profile with those of protein
markers of known molecular masses, which is somewhat larger than the
calculated molecular mass of 18 kDa for a monomer, but well short of
the size predicted for a dimer. The final yield of Ubc9 was 3 mg for each liter of LB culture, as estimated by measuring the UV absorbance of the purified protein at 280 nm and using the molar extinction coefficient (
M = 29400 M
1
cm
1) calculated with the GCG package (24).
The protein was
concentrated to 11 mg/ml in storage buffer (150 mM NaCl,
0.5 mM DTT, 10 mM Hepes buffer, pH 7.5) before
crystallization trials. Crystals of two different forms were grown from
hanging drops by the technique of vapor diffusion at room temperature. Both types of crystals have the shape of elongated parallelepiped rods
but belong to different space groups: I222 (a = 35.4 Å, b = 93.9 Å, c = 115.9 Å) for
crystal form I, and P21 (a = 52.0 Å, b = 35.2 Å, c = 58.1 Å, = 111.2°) for crystal form II. From crystallization drops with an
initial volume of 10 µl (5 µl of protein solution plus 5 µl of
precipitant solution), type I crystals grew to a full size of 1.6 mm × 0.1 mm × 0.06 mm within 2 days in the presence of 23%
polyethylene glycol monomethyl ether 5000, 9% isopropanol, 0.1 M (NH4)2SO4, 0.1 M MES buffer, pH 6.5; type II crystals grew up to a size of
1.8 mm × 0.06 mm × 0.04 mm over the period of one week in
the presence of 9% polyethylene glycol 4000, 9% isopropanol, 0.1 M Hepes buffer, pH 7.5. Diffraction data up to 2.2 Å resolution were initially recorded on a Macscience DIP2020 image-plate
system (Enraf-Nonius, Delft) at room temperature (22 °C). High
resolution data were collected using synchrotron radiation on a MAR
image-plate system (MAR-Research, Hamburg) at the EMBL outstation DESY,
Hamburg. Both types of crystals were cooled to 8 °C in cold air
streams and diffracted beyond 2.0 Å resolution (Table
I). All data were indexed with DENZO and
scaled with SCALEPACK (25).
|
Initial phase information for Ubc9 in the I222 crystal form was obtained by molecular replacement (MR) using AMoRe (26) from CCP4 (27). Atomic coordinates of plant Ubc1 (33) and yeast Ubc4 (34) were alternately used as search models against an initial 2.7 Å data set. The rotation and translation function searches performed with both search models yielded one distinct solution. The correlation coefficients of the MR solution were 0.31 for Ubc1, and 0.26 based on Ubc4, whereas the corresponding next highest peaks were 0.22 and 0.20, respectively. After rigid body fitting (AMoRe) of the MR solution using the Ubc1 model, the crystallographic R-factor was 48.2% with data in the range 8-3 Å. The correctness of the MR solution was further confirmed by the identification of a number of structural features unique to Ubc9 in a difference Fourier map. An initial structure solution was also found for the P21 crystal form by the MR method using the Ubc9 model that was already refined in the I222 space group. The Rcryst was 31.9% for this MR solution.
Refinement for the structures in both crystal forms followed similar
protocols. From each of the data sets used, 5% was set aside for the
Rfree (28) calculation, which was used to
monitor the progress of the refinement along with the
conventional Rcryst and stereochemical criteria.
Several rounds of refinement were carried out by subjecting the model
alternately to simulated annealing refinement with X-PLOR (29), and
manual adjustment based on 3Fo-2Fc and
Fo-Fc difference electron density
maps using O (30). Some electron density maps used for model building
were calculated with models generated using ARP (31) in combination
with PROLSQ (32). Waters were modeled and checked by ASIR (33) in
combination with TNT (34). Final rounds of refinement using a
conjugate-direction algorithm and bulk solvent correction in the TNT
program resulted in Rcryst of 18.5%
(Rfree = 25.2%) for Ubc9 in the I222 crystal
form and Rcryst of 16.0%
(Rfree = 25.5%) in the P21 crystal
form. Both Ubc9 models include the full 158 residues of the native
protein (Fig. 5). Of the 8-residue N-terminal extension introduced as a
cloning artifact, 1 (in I222) or 2 (P21) residues were
observed in electron density. Some modeled structural features,
including the 5-residue insertion that is unique to mammalian Ubc9 and
its yeast homologs, are better defined in the P21 crystal
form than in I222, or vice versa. The I222 model and the
P21 model include, respectively, 96 and 103 water molecules
with a few of them extending beyond the first water shell. Analysis
with PROCHECK (35) indicated that the final models for both types of
structure have good stereochemistry (Table I). General structure
analysis was carried out using WHATIF (36).
Sequence Analysis
Multiple sequence alignments of ubiquitin conjugating enzymes were constructed with CLUSTALW (37). Homology-derived structure prediction values of relative entropy of variability of amino acids (38) were calculated using the PHD server (39).
The structure of recombinant murine Ubc9
has been determined to 2.0 Å resolution in two crystal forms
(P21 and I222). Ubc9 forms a single domain +
structure that is typical of the Ubc core domain (21). The molecule is
asymmetric with overall dimensions of approximately 20 Å × 30 Å × 50 Å. The structure (Fig. 1) contains an
antiparallel
-sheet with four strands (
1 to
4, these and all
subsequent secondary structure assignments were carried out by the
program DSSP, Ref. 40) bound on one side and at both ends by four
-helices (
1 to
4).
The active site residue Cys93 is situated close to the
middle of a long extended stretch of 31 residues (78-108) found
between the fourth -strand and the second
-helix. This
polypeptide segment contains five tight turns and one turn of
310 helix. Part of this long loop (residues 85-102) and
another loop between the
2 and the
3 helix (residues 122-130)
form a crevice with the active site cysteine in between. The structure
contains two cis-prolines: Pro69 (Fig.
2), and Pro79.
The N terminus is situated at one end of the long axis of the molecule, whereas the C terminus is located opposite to the catalytic site (Fig. 1). The observed portion of the artificial N-terminal extension indicates that it extends away from the core domain, and does not appear to affect the folding of the rest of the protein.
Comparison between the Two Ubc9 ModelsThe two Ubc9 models
from crystal form I (I222) and crystal form II (P21) are
essentially identical except for some minor differences (Fig.
3A). The root mean square
difference for all 158 C atoms corresponding to the
native protein is 0.44 Å after superposition. Protein atoms displaying
the largest differences include those of the first three N-terminal
residues, an exposed 5-residue insertion (32PDGTM36) which is barely visible in the I222
crystal form, and a large segment from residue Asn121 to
Lys146, including the active crevice forming loop between
the
2 and the
3 helix (residues 122-130). Compared with the rest
of the protein, this segment rotates by 3°, resulting in a slight
widening of the active crevice in the I222 model compared with the
P21 model.
Genetic and biochemical data indicate that some Ubcs interact with one another in homo- and heterocomplexes (41). In some cases these interactions may play an important functional role (42, 43). We and others have also observed the self-association of Ubc9 in yeast two-hybrid assays (12, 14). However, our gel filtration experiments did not show Ubc9 as dimers, even in high concentration (10 mg/ml) at close to physiological ionic strength (150 mM NaCl, 10 mM Tris-Cl, pH 7.5). Crystal packing analysis of the two crystal forms of Ubc9 revealed only a small set of residues involved in the formation of intermolecular contacts that are conserved in both crystal forms but they are not more extensive than can be expected from normal crystal packing (~150 Å2). They certainly do not represent a general mechanism for dimer formation among Ubcs because they have no equivalents in the crystal packing of plant Ubc1 or in yeast Ubc4, which were both crystallized as monomers as well (21, 44). It appears that the widely observed interactions among Ubcs may be either relatively weak or indirect.
Comparison with the Structures of Ubc1 and Ubc4Arabidopsis Ubc1 is highly similar to S. cerevisiae Ubc2 (Rad6) involved in DNA repair (45), whereas
S. cerevisiae Ubc4 is involved in the degradation of
abnormal and short lived proteins, especially in stress conditions
(46). Murine/human Ubc9 shares 39% sequence identity with
Arabidopsis Ubc1 and 35% identity with S. cerevisiae Ubc4. Despite their involvement in distinct functional pathways and the limited sequence similarity for the three Ubcs, the
tertiary structure of Ubc9 is similar to those of Ubc1 and Ubc4 (Fig.
3B). Murine/human Ubc9 has 6 more residues than
Arabidopsis Ubc1 and 10 more than S. cerevisiae
Ubc4. These differences in amino acid sequence are primarily
accommodated by two insertions in the structure of Ubc9. The first
insertion occurs at residues 32-36 and these 5 residues form most of a
very exposed -hairpin that connects strand
1 and
2. The second
insertion occurs at residues 100-101, and forms a bulge in a loop
(residues 94-102) close to Cys93. Overall, the root mean
square differences are 2.4 Å for 150 equivalent C
atoms
between Ubc9 and Ubc1, and 2.0 Å for 148 equivalent C
atoms between Ubc9 and Ubc4.
Despite the overall similarity in the
folding of the three Ubcs, there are considerable differences in the
detailed features of the molecule, especially at the active site. There
are 10 residues in Ubc9 within 6 Å of the sulfhydryl group of the
ubiquitin-accepting cysteine, Cys93. Among these residues,
Asn85, Tyr87, Glu98,
Lys101, and Asp127 are the most likely to
mediate in the catalytic action as their side chains are orientated
toward the ubiquitin-accepting sulfhydryl group (Fig.
4). Only Asn85,
Leu94, and Pro128 are conserved compared with
both Ubc1 and Ubc4. In all three Ubc crystal structures, the carbonyl O
atom of Asn85 is hydrogen bonded to the backbone N atom of
Cys93, whereas the side chain of Asn85 makes
hydrogen bonds to the main chain of residues 124 and 127, keeping this
relatively mobile loop between the 2 and
3 helix in position.
Therefore it is not obvious that Asn85 can participate in
the catalytic mechanism unless this loop moves away upon binding of Ubc
to the E1 ubiquitin adduct. Otherwise the catalytic site displays
considerable sequence and structural heterogeneity among the three
Ubcs.
A major structural difference is created by the 2-residue insertion in Ubc9: Asp100 and Lys101. These 2 residues form a small protruding loop near the active site cysteine. Ubc3 (cdc34), another ubiquitin-conjugating enzyme with known cell cycle function, has a 12-residue insertion at the equivalent position. It appears that such inserted loops can provide additional binding sites for substrates without blocking access to the active site cysteine. Charge-to-alanine scanning mutagenesis indicated that charged residues of the 12-residue insertion in Ubc3 are important to its in vivo function without affecting its enzymatic competence with respect to unfacilitated (E3-independent) ubiquitination (47). The 2-residue insertion in Ubc9 could similarly contribute to Ubc9-specific functions.
There are two ordered water molecules found in the vicinity of Cys93: Wat15 and Wat93. It is possible that these active-site water molecules play some role in the catalytic mechanism for ubiquitin-conjugating enzymes. Wat15 is attached to the carbonyl oxygen of Cys93, and Wat93 is directly attached to the sulfhydryl group of the active site cysteine. Whether Ubc1 and Ubc4 also possess similarly positioned water molecules is not clear, since few or no water molecules were modeled due to the lower resolution limits of these two structures.
Structural differences at the catalytic site may provide some clues to why different subfamilies of Ubc's are involved in different functional pathways. In addition to substrate specificity and requirement for different E3s, recent discoveries of ubiquitin-like proteins (48, 49) suggest another possible reason for the variability of the catalytic machinery of the ubiquitin-conjugating system, that some ubiquitin-conjugating enzymes may be involved in conjugating the ubiquitin-like proteins rather than ubiquitin itself to substrates.
Surface Electrostatic PotentialsLarge variations are found
in the surface electrostatic potentials of the three Ubc structures
(Fig. 5), with the exception of a
negative patch surrounding the active site. While this negative patch
may be important in orienting common interaction partners such as E1 or
ubiquitin, the varied electrostatic features probably reflect the need
to recognize different E3s and substrates. Overall, Ubc9 possesses a
considerably stronger electrostatic dipole (541 Debye, calculated by
GRASP (50) at pH 7.0) than either Ubc1 (310 Debye) or Ubc4 (149 Debye).
Positive patches are scattered on the "back face" of Ubc9 (Fig. 5),
including the N-terminal region composed of a segment of basic residues
separated by nonpolar residues (13RKAWRK18)
that is highly conserved among Ubc9s (51). Notably, this conserved negative patch is close to the highly exposed -hairpin, another distinguishing structural feature of Ubc9. The spatial proximity of
these two "specifically" conserved structural features suggests that this region could be important for Ubc9 function. The lack of
these two structural features in Arabidopsis Ubc9 can be
explained because it belongs to another subfamily, yeast Ubc4 (52).
Sequence analysis of the known interaction partners of mammalian Ubc9
indicates that they mostly have a strong overall negative charge, or at
least possess a large region of about 100 residues with a particularly
low isoelectric point (pI). In particular, the region on adenovirus E1A
responsible for interacting with Ubc9 has been mapped to a polypeptide
segment of 70 residues with a predicted pI of 4.6 (11). These
observations suggest an important role for electrostatic attractions in
the liaison between mammalian Ubc9 and its multiple interaction
partners. However, these interactions must also have a relatively
strong hydrophobic component, as evidenced by the fact that the
association between Ubc9 and adenovirus E1A is sensitive to a single
LeuIle mutation in the transformation-relevant conserved region 2 of
E1A (11).
We have extended the comparison of the
amino acid sequences to the currently available sequences of the
catalytic domain of the entire Ubc family (66 annotated sequences from
SWISS-PROT (53), March 1997). We have used the homology-derived
structure prediction program to analyze variability for amino acids
with equivalents in Ubc9. A histogram representation of these values shows that residues comprising the loops are better conserved than
those forming the regular secondary structure elements in general (Fig.
6A). This probably reflects
the importance of the "unstructured" stretch (from residue 78 to
108 in Ubc9) of Ubc structures in forming the active site, as well as
the important structural role of some other loops. Apart from the
Cys93 residue essential to the catalytic action, there are
15 residues which are particularly well conserved (Gly47,
Lys48, Gly56, Tyr68,
Pro69, Pro73, Phe77,
His83, Pro84, Asn85,
Gly90, Trp103, Pro105,
Leu120, and Pro128) with a relative entropy of
variability less than 15% of the value for the most variable residue
(Fig. 6A). These residues are clustered around both ends of
the long irregular loop containing the active-site (within 18 Å of
Cys93, Fig. 6B). However, with the exception of
Asn85, whose possible involvement in the catalytic reaction
has been discussed, and Pro128, they are outside the
immediate vicinity of the ubiquitin-accepting cysteine (>6 Å to the
sulfhydryl group of Cys93). Notably, 11 of the 15 highly
conserved residues are nonpolar residues. It is unlikely that any of
these is directly involved in the catalytic action but most are
positioned to maintain the special conformation of the active site. The
important structural role for such conserved residues is demonstrated
in the case of cis-Pro69 (Fig. 2). A
Pro69 Ser mutation has been shown to cause a
temperature sensitive defect in S. cerevisiae Ubc9 (17).
Loss of this proline makes Ubc9 sensitive to proteolysis by a
ubiquitin- and proteasome-dependent pathway at the
restrictive temperature (54), indicating that this mutation
destabilizes the protein fold.
Mapping of the amino acid variability values onto the surface of the three-dimensional structure of Ubc9 indicates that one side of the active site cysteine (the "front" side of Fig. 1) displays a higher degree of conservation than the other, as was shown in the comparison between the Ubc1 and the Ubc4 structure (21). Our analysis provides further support to the hypothesis by Cook et al. (21) that this better conserved side may contain possible binding sites for the E1-ubiquitin adduct, although the conserved regions do not appear to be as contiguous as when only two structures were compared.
The overall similarity of the high resolution mammalian Ubc9 structure to those of plant Ubc1 and yeast Ubc4 suggests that the folding of the catalytic domain of the family of Ubc enzymes is conserved in all eukaryotes. Mapping of amino acid variability onto the surface of the three-dimensional structure of Ubc9 shows a better conserved surface on one side of the ubiquitin-accepting cysteine that may serve as possible recognition surface regions on Ubcs for their common physiological partners, E1 and ubiquitin.
There is considerable structural heterogeneity observed in the catalytic crevice among the Ubcs with known crystal structures. Sequence consensus analysis for the entire Ubc family also shows a lack of conserved residues close to the active site cysteine. A variable catalytic machinery might account for some of the differences among Ubcs in their efficiency and in their requirement for E3s to ubiquitinate different sets of target proteins.
A number of features unique to mammalian Ubc9, such as a protruding surface loop and a strong overall electrostatic dipole, may have a role in conferring the distinctive property to Ubc9 for interacting with an exceptionally large variety of proteins. Understanding such interactions may also provide insight into the modus operandi for some of the biologically important interaction partners.
The atomic coordinates and structure factors for Ubc9 (1U9A and R1U9ASF for crystal form I; 1U9B and R1U9BSF for crystal form II) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
We thank Alex Teplyakov and Zbigniew Dauter for assistance during synchrotron data collection at beamlines BW7B and X11 of the EMBL/Hamburg outstation. We also thank Pim van Dijk for general assistance.