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J Biol Chem, Vol. 273, Issue 11, 6271-6276, March 13, 1998
From the 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 Ubiquitin is a highly conserved 76-amino acid eukaryotic protein
that is covalently attached through its C terminus to the 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 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.
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 × 10 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).
Crystal Structure of the Saccharomyces cerevisiae
Ubiquitin-conjugating Enzyme Rad6 at 2.6 Å Resolution*
§,
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
ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References
/
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.
INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References
-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.
phenotype
(13, 14).
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References
2 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).
Data statistics
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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Abstract
Introduction
Procedures
Results & Discussion
References
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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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-sheet with four strands, four -helices, and one short 310 helix (Fig.
2A). The -sheet is exposed
to solvent on one side and is flanked on the other side by -helices
1 and 2 and the 310 helix. The active site cysteine is
located in a shallow cleft formed by residues 89-95, which includes
the 310 helix, which makes a small promontory beneath the
cysteine, and residues 115-121, which form a loop above the cysteine.
Due to crystal packing interactions, residues 115-121 adopt different
conformations in the different molecules of the asymmetric unit. In two
of the molecules, the side chain of Asn-117 is hydrogen bonding to the
amide nitrogen of Ala-119. In the third molecule this side chain is
hydrogen bonded to a symmetry related molecule, resulting in
displacement of -carbon atoms by up to 3.9 Å for Pro-118. Because
these alternate loop conformations affect the exposure of the active
site cysteine, it is possible that this mobility is necessary for Rad6
function.
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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.
positions are near the N
termini, with significant divergence before residue 6 of Rad6, and in
the loop between -helix 2 and -helix 3 that comprises the roof of
the active site cleft (residues 115-121). There is also an apparent
register shift in the first helix of the superimposed structures with
respect to the sequence alignment of Fig.
3. Thus Rad6 residues 6-13 appear
structurally equivalent to UBC1 residues 7-14. The return to register
at Arg-15 of Rad6 occurs because the C-terminal portion of helix 1 (residues 14-16) in the UBC1 model is in a 310 helical
conformation. One effect of this N-terminal register shift is to place
Leu-9 and Phe-13 of the UBC1 structure at the positions of Arg-8 and
Asp-12 of Rad6. Although these two charged Rad6 side chains are buried, their hydrogen bonding potential is saturated. The two residues form a
salt bridge with each other, and each has hydrogen bonding interactions
with the phenolic hydroxyl of Tyr-63. In addition, the Asp-12 side
chain hydrogen bonds with the backbone nitrogen of Val-102, and Arg-8
hydrogen bonds with the carbonyl oxygens of Tyr-100 and Pro-98 (Fig.
1). Although hydrophobic residues, such as Leu-9 and Phe-13 of UBC1,
are commonly found in buried environments, in the context of the UBC
fold it appears that this arrangement would leave a number of potential
hydrogen bonds unsatisfied. It is noteworthy that Arg-8, Asp-12, and
Tyr-63 are invariant residues among Rad6 homologs (Fig. 3).
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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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1-9 protein are deficient in N-end rule degradation of model substrates (7). The observation that Rad61-9 forms a thiolester bond with ubiquitin in the presence of E1 and forms
a complex with Rad18 in vitro indicates that removal of residues 1-9 does not grossly alter the Rad6 fold (7, 15), although an
important caveat for all deletion studies is that propagation of subtle
conformational changes may impair activity at some, but not all,
distant active sites on a protein structure.
Rad6150-172, a mutant lacking the C-terminal 23 amino
acids, also has a reduced efficiency in N-end rule protein degradation
and does not form a stable complex with Ubr1 but maintains the ability
to form a thiolester bond with ubiquitin in the presence of E1 and to
ubiquitinate proteins in vitro (7, 32-34). Because Rad6154-172 shows an essentially wild type phenotype, residues 150-153 also appear to be important for Rad6-Ubr1 interaction (7). All of these residues (Glu-150, Asp-151, Asp-152, and Met-153) are
solvent exposed, although none are well conserved among the Rad6
homologs.
It has been shown, both in vivo and in vitro,
that Rad6 forms a tight complex with the single strand DNA binding
protein Rad18, which likely functions to target Rad6 to sites of DNA
damage (35). Rad18 is a member of the Rad6 DNA repair epistasis group
(6, 12), and rad18 mutant strains are equally sensitive to
UV radiation and are as defective in post-replicative bypass and
UV-induced mutagenesis as rad6 mutants, although
sporulation and N-end rule proteolysis are not affected (6, 12,
36).
Because Rad6-Rad18 complex formation occurs for the
Rad61-9 or Rad6150-172 mutants but not
with the Rad61-22 or Rad6142-172
mutants, it appears that residues 10-22 and 142-149 are important in
this interaction (15). Residues 10-19 are part of -helix 1, whereas
residues 20-22 are in an extended conformation. Exposed or partly
exposed residues in this N-terminal Rad18 binding determinant include Met-10, Arg-11, Phe-13, Lys-14, Arg-15, Lys-17, Glu-18, Asp-19, and
Pro-22. All of these except Arg-15, Lys-17, Glu-18, and Pro-22 are
invariant among Rad6 homologs, with just one Rad6 homolog having a
lysine at position 15 instead of arginine. The C-terminal Rad18 binding
determinant, residues 142-149, is contained within -helix 4. Exposed residues here include Lys-142, Glu-143, Glu-146, Lys-147, and
Trp-149. Of these, only Glu-146 is invariant among Rad6 homologs, with
position 149 always occupied by Trp or Phe. Thus, Glu-146 and
Trp/Phe-149, which are adjacent in space on the same exposed face of
-helix 4, may be the critical features of -helix 4 in the
Rad6-Rad18 binding interaction.
Because the Rad6142-172 mutant forms a thiolester bond
with ubiquitin in the presence of E1, it is unlikely that failure to
bind Rad18 is due to protein misfolding (15). However, the binding
study that failed to show complex formation between Rad61-22 and Rad18 utilized a
GST-Rad623-153 fusion protein, and there was no
independent check that this construct was correctly folded. Both
-helix 1 and -helix 4 are on the opposite side of the molecule
from the active site cysteine, which would allow binding of Rad18 to
Rad6 without hindering access of substrate to the active site of Rad6.
A 40-residue domain of Rad18 is sufficient for binding to Rad6 (15).
Because the distance between the centers-of-mass of Rad6 residues
10-22 and 142-149 is 28.5 Å and a simple model building exercise
suggests that at least 12 residues are required to span these two
determinants across the surface of Rad6, the 40-residue Rad18 domain
would have to be highly extended to contact both determinants.
In summary, the crystal structure of Rad6 that we have determined at
2.6 Å resolution can serve as a framework for the design and
interpretation of mutagenic studies. In particular, clarification of
the conformation and solvent accessibility of residues in the N-terminal helix is relevant to deciphering details of Rad6
interactions with Rad18 and Ubr1.
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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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Abstract
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References
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. Dashed white
lines indicate hydrogen bonding interactions of Asp-12, Arg-8, and
Tyr-63. The figure was made using the program O (
