From the Biochemistry Department, University of Utah,
Salt Lake City, Utah 84132 and the ¶ Sealy Center for Molecular
Science, University of Texas Medical Branch, Medical Research Building,
Galveston Texas 77555
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ABSTRACT |
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The Saccharomyces cerevisiae
ubiquitin-conjugating enzyme (UBC) Rad6 is required for several
functions, including the repair of UV damaged DNA, damage-induced
mutagenesis, sporulation, and the degradation of cellular proteins that
possess destabilizing N-terminal residues. Rad6 mediates its role in
N-end rule-dependent protein degradation via interaction
with the ubiquitin-protein ligase Ubr1 and in DNA repair via
interactions with the DNA binding protein Rad18. We report here the
crystal structure of Rad6 refined at 2.6 Å resolution to an
R factor of 21.3%. The protein adopts an /
fold that
is very similar to other UBC structures. An apparent difference at the
functionally important first helix, however, has prompted a
reassessment of previously reported structures. The active site
cysteine lies in a cleft formed by a coil region that includes the
310 helix and a loop that is in different conformations for
the three molecules in the asymmetric unit. Residues important for Rad6
interaction with Ubr1 and Rad18 are on the opposite side of the
structure from the active site, indicating that this part of the UBC
surface participates in protein-protein interactions that define Rad6
substrate specificity.
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INTRODUCTION |
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Ubiquitin is a highly conserved 76-amino acid eukaryotic protein
that is covalently attached through its C terminus to the -amino
group of lysine side chains in acceptor proteins (reviewed in Refs.
1-4). The best known role for ubiquitination is to target substrate
proteins for degradation by the 26 S protease, an activity that plays a
key role in a number of cellular processes including cell cycle
progression, signaling pathways, stress responses, removal of damaged
or misfolded proteins, and the production of antigenic peptides.
Ubiquitination of substrates is performed by the numerous ubiquitin-conjugating enzymes (UBCs1 or E2s). UBCs receive ubiquitin from an E1 ubiquitin-activating enzyme to form a thiol-ester intermediate in which the UBC active site cysteine is linked to the ubiquitin C terminus. The ubiquitin is then transferred from the UBC to form an isopeptide bond through its C terminus to a lysine side chain of the target substrate protein. In some cases this process proceeds directly, whereas in other cases a further level of substrate specificity is provided by a ubiquitin-protein ligase or E3.
The Saccharomyces cerevisiae ubiquitin-conjugating enzyme
Rad6 (also known as UBC2) is a 172-residue protein that is
predominantly localized to the nucleus (5-7). Rad6 has a 149-amino
acid core domain common to other E2s and a 23-residue C-terminal
"tail" that is comprised almost entirely of acidic residues. A
single cysteine, Cys-88, in the core domain serves as the point of
attachment of ubiquitin prior to transfer to cellular targets.
rad6 mutants are extremely sensitive to UV light and other
DNA damaging agents and exhibit a defect in post-replicative bypass of
UV-damaged DNA and in damage-induced mutagenesis. Furthermore,
mutations in RAD6 cause poor growth, a sporulation defect,
and an increase in the rate of retrotransposition of yeast Ty elements
(6, 8-12). The ubiquitin-conjugating activity is essential for the DNA
repair, mutagenesis, and other functions of Rad6, because rad6 mutants in which the active site cysteine has been
replaced with alanine or serine have a rad6 phenotype
(13, 14).
Rad6 substrate specificity is apparently determined, at least in part, through interactions with the ubiquitin-protein ligases or E3 proteins Ubr1 and Rad18. The single strand DNA-binding protein Rad18 appears to mediate the DNA repair functions of Rad6 (15), whereas a separate Rad6 activity, the so called N-end rule degradation pathway, depends upon formation of a specific Rad6 complex with Ubr1 (7, 16, 17). Although Rad6 exists in complex with both Rad18 and Ubr1 in vivo, attempts to detect a Rad6-Rad18-Ubr1 ternary complex have failed, indicating that Rad6 associates with these proteins in separate complexes. This is consistent with roles for Rad18 and Ubr1 in defining distinct sets of Rad6 substrates.
To further the understanding of how Rad6 functions in diverse biological processes, we have determined the structure of S. cerevisiae Rad6 by x-ray crystallography. The structure closely resembles that of other UBC enzymes, although our analysis reveals a "frame shift" error in a functionally important part of previously reported structures. Residues that are required for binding the Rad6-specific E3 proteins Ubr1 and Rad18 are on the opposite side of the molecule from the active site.
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EXPERIMENTAL PROCEDURES |
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Expression and Purification--
Rad6 was expressed from plasmid
pSCW242 in the S. cerevisiae strain CMY135 (11). Cells grown
to high density (A600 > 1.4) in culture medium
(6 mg/ml NaOH, 10 mg/ml succinic acid, 3.6 mg/ml ammonium sulfate, 36 mg/ml glucose, 2.9 mg/ml yeast nitrogen base (Difco), 2.6 mg/ml vitamin
assay casamino acids (Difco), 7.2 × 102 mg/ml each
of adenine and uracil) were harvested by centrifugation at 11,300 × g and then stored at
80 °C. Frozen cells were thawed and resuspended in lysis buffer (50 mM Tris, pH 7.5, 10 mM EDTA, 10% glycerol, 200 mM KCl, and 10 mM
-mercaptoethanol). Purification was by four column
chromatography steps with fractions containing Rad6 protein identified
by SDS-polyacrylamide gel electrophoresis. All purification steps were
performed at 4 °C. Cells were disrupted by two passages through a
French press at >15,000 p.s.i., and the lysate was clarified by
centrifugation at 100,000 × g for 90 min. The
supernatant was passed through a 0.4-µm syringe filter and loaded
onto a 100-ml fast flow DEAE-Sepharose column equilibrated with buffer
A (20 mM potassium phosphate, pH 7.4, 10 mM
-mercaptoethanol, 10% glycerol, 1 mM EDTA) plus 100 mM KCl. The column was washed thoroughly and a 400-ml
linear gradient (100 to 500 mM KCl) was used to elute Rad6
protein. Fractions containing Rad6 protein were pooled and dialyzed
against buffer A and then loaded onto a Hi-Load Q Sepharose column
(Pharmacia Biotech Inc.) equilibrated to Buffer A plus 200 mM KCl. Rad6 protein was eluted using a 600-ml linear
gradient from 200 to 500 mM KCl. Fractions containing Rad6 protein were pooled and concentrated to <3 ml and passed over a
preparation size Superdex 75 sizing column (Pharmacia) equilibrated to
50 mM Tris, pH 7.5, 10 mM
-mercaptoethanol,
1 mM EDTA, and 200 mM NaCl. Fractions
containing Rad6 were pooled and dialyzed against 50 mM
potassium phosphate, pH 6.8, and 10 mM
-mercaptoethanol. The protein was loaded onto a 25-ml hydroxyapatite column and eluted
with a 100-ml linear gradient from 50 to 300 mM potassium phosphate. Rad6 fractions were dialyzed against crystallization buffer
(10 mM HEPES, pH 7.0, 1 mM EDTA, 1 mM
-mercaptoethanol) and concentrated to ~30 mg/ml.
Purification of full-length Rad6 protein was confirmed by electrospray
mass spectrometry, which indicated a molecular mass of 19,704.5 ± 2.4 daltons (calculated molecular weight = 19,705.5).
Crystallization and Data Collection-- Rad6 was crystallized by vapor diffusion in sitting drops over 1 ml of well solution (12% (w/w) polyethylene glycol 8000, 50 mM MES, pH 5.0, and 1% (w/v) spermine tetrahydrochloride). The initial drop was 5 µl of well solution and 5 µl of protein solution. Crystals appeared after a few days and grew to maximum dimensions of 0.4 × 0.4 × 0.4 mm after 2 weeks. The thimerosal derivative was prepared by soaking crystals overnight in well solution plus 5 mM thimerosal. For data collection at 100 K, crystals were transferred gradually, in increments of increasing glycerol and polyethylene glycol 8000 concentration, to a final cryoprotectant solution consisting of 18% (w/w) polyethylene glycol 8000, 50 mM MES, pH 5.0, 1% (w/v) spermine tetrahydrochloride, and 20% (v/v) glycerol. The crystals were then suspended in a small rayon loop and flash-frozen by plunging into liquid nitrogen. The derivative data were collected using an RAXIS II imaging plate detector and an RU200 rotating anode x-ray source with a graphite monochromator. The native data were collected on a MAR image plate detector at beamline X12B of the Brookhaven National Synchrotron Light Source. Data were processed using DENZO and SCALEPACK (18) (Table I).
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Structure Determination and Refinement-- Rad6 crystals belong to space group I222 with cell parameters of a = 113.75 Å, b = 146.36 Å, and c = 109.88 Å. There are three Rad6 molecules in the asymmetric unit corresponding to 3.9 Å3/dalton (68% solvent). Most crystallographic calculations used programs from the CCP4 suite (19). The mercury atom positions were determined by inspection of difference Patterson and Fourier maps. Initial protein maps using the mercury phases were of poor quality, and although they show protein versus solvent regions, they were otherwise uninterpretable. Molecular replacement using the structure of the Rad6 homolog UBC1 from Arabidopsis thaliana as a search model (20) and the program AMoRe (21) readily located the three molecules in the asymmetric unit. The previously identified mercury positions corresponded to the highest peaks in a difference Fourier calculated with model derived phases. Rigid body refinement of the UBC1 molecular replacement solutions using X-PLOR (22) gave an R factor of 46.1% for 7.0-3.5 Å data. This solution was used to define noncrystallographic symmetry (NCS) operators and to construct a molecular mask (23). The single isomorphous replacement phases were then refined by 3-fold averaging, histogram shifting, and solvent flattening using the program DM (24). The resulting phases, which depended upon the molecular replacement solution only for NCS operators and the molecular mask, gave an electron density map that was readily interpretable (see Fig. 1). Rounds of automated positional and temperature factor refinement using XPLOR were interspersed with model building using the program O (25). 5% of the data were withheld from refinement to optimize weights and to monitor the refinement by cross-validation (26). NCS restraints on 1617 main chain atoms and 1695 side chain atoms (3312 atoms out of a total of 3699 protein atoms) were used during automated refinement. As a check of the propriety of the NCS restraints assignment, the coordinates of the structure late in the refinement were subjected to a 3000-degree simulated annealing refinement using torsion angle molecular dynamics and no NCS restraints (22). The resulting structure showed significant deviations between the superimposed monomers only in portions of the molecule where NCS restraints were not applied in the previous refinement cycles, namely residues 81-87 and residues 114-124. The average pairwise root mean square deviations in atomic positions of these superimposed monomers was 0.69 Å using all atoms, and 0.57 Å when residues 81-87 and 114-124 were omitted. Refinement statistics are given in Table II.
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RESULTS AND DISCUSSION |
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Rad6 Structure-- The crystal structure of Rad6 was determined using a combination of single isomorphous replacement, molecular replacement, and NCS averaging. The molecular replacement solution was used to generate NCS operators and for the calculation of a molecular mask but was not used to estimate phases directly. NCS averaging and other density modifications resulted in a high quality electron density map devoid of model bias (Fig. 1). A model built into this map has been refined to an Rfactor of 21.3% (Rfree of 24.6%) against 2.6 Å data. The refined structure has good geometry (27) (Table II). The N-terminal methionine and C-terminal 18 residues of each molecule are disordered and have been omitted from the model. Note that electrospray mass spectrometry demonstrated that the N-terminal methionine and 18 C-terminal residues were retained after purification, and SDS-polyacrylamide gel electrophoresis analysis of washed crystals was consistent with full-length protein. In addition, mobile side chains of four residues have been included with zero occupancy (Lys-14 of molecule A and Lys-131 of all three molecules in the asymmetric unit).
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Comparison with Other UBC Structures--
The Rad6 structure
closely resembles that of other published UBCs. Least squares overlap
of Rad6 on the known structure of the Rad6 homolog UBC1 from A. thaliana (20), which was used as the search model in molecular
replacement and shares 63% residue identity with Rad6, gave a root
mean square deviation of 1.44 Å over 149 C atoms.
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Possible E1, Ubr1, and Rad18 Binding Surfaces-- There are greater than 60 sequences of either confirmed or putative UBCs available from various organisms and 13 for S. cerevisiae alone. The level of conservation between these sequences indicates that they all have a similar fold for the conserved core domain. Because all 13 UBCs of S. cerevisiae interact with ubiquitin and with at least one of the two homologous ubiquitin-activating enzymes Uba1 and Uba2, it is expected that this binding surface(s) on the UBC proteins will be formed by conserved residues. On the other hand, because each UBC has a distinct group of substrates that are either recognized directly by the UBC-ubiquitin complex or via an E3, it is expected that E3 and substrate-binding surfaces will be comprised of residues that are not conserved between different UBCs. Residues that are conserved in an alignment of the 13 UBCs from S. cerevisiae are located predominantly on one face of the molecule in the vicinity of the active site cysteine (Fig. 4A). This surface is therefore likely to participate in binding E1, ubiquitin, or both. A similar observation has been reported by Cook et al. (28).
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ACKNOWLEDGEMENTS |
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We are grateful to Brian Carr for help with protein purification and Drs. Robert M. Sweet and Malcolm S. Capel for assistance with data collection and Drs. Daniel P. Bancroft, William J. Cook, and Arthur Haas and members of the Hill lab for advice and helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM50163 and GM19261 and American Cancer Society Grant JFRA B-74386.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (codes 1ayz and r1ayzsf) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
§ Supported by National Institutes of Health predoctoral training Grant 5-T32-GM08573.
To whom correspondence should be addressed. Fax: 801-581-7959;
E-mail: chris{at}msscc.med.utah.edu.
1 The abbreviations used are: UBC, ubiquitin-conjugating enzyme; MES, 2-(N-morpholino)ethanesulfonic acid; NCS, noncrystallographic symmetry.
2 W. J. Cook, personal communication.
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REFERENCES |
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