From the Departments of Biochemistry and
§ Chemistry, Emory University, Atlanta, Georgia 30322 and the
Department of Chemistry, Wayne State University,
Detroit, Michigan 48202-3489
Received for publication, October 23, 2002
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ABSTRACT |
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MBD4 is a member of the methyl-CpG-binding
protein family. It contains two DNA binding domains, an amino-proximal
methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific
glycosylase domain. Limited in vitro proteolysis of mouse
MBD4 yields two stable fragments: a 139-residue fragment including the
MBD, and the other 155-residue fragment including the glycosylase
domain. Here we show that the latter fragment is active as a
glycosylase on a DNA duplex containing a G:T mismatch within a CpG
sequence context. The crystal structure confirmed the C-terminal domain is a member of the helix-hairpin-helix DNA glycosylase superfamily. The
MBD4 active site is situated in a cleft that likely orients and binds
DNA. Modeling studies suggest the mismatched target nucleotide will be
flipped out into the active site where candidate residues for catalysis
and substrate specificity are present.
MBD4 is a mammalian DNA glycosylase that excises thymines from
G:T mispairs and contains both a methyl-CpG binding domain (MBD)1 and a domain found in
the Escherichia coli endonuclease III class of DNA
glycosylases (1). It has preference for G:T mismatches within a CpG
sequence context (1), and hence this enzyme can act upon G:T mismatches
that result from the deamination of 5-methylcytosines (5mC) at CpG
sites. The importance of this enzyme for mutation avoidance in mammals
is confirmed by an increase in 5mC to T mutations in
Mbd4 MBD4 is not the only DNA glycosylase reported to excise thymines from
G:T mismatches. Another enzyme, named thymine-DNA glycosylase (TDG),
was identified earlier to have this ability (5). However, TDG is
unrelated to MBD4 and belongs to the same structural superfamily as the
uracil-excising enzymes UDG (6) and SMUG1 (7). MBD4 also differs from
TDG in its substrate preference. Whereas the preferred substrates for
TDG are N4-ethenocytosine or uracil paired with
a G (8), MBD4 prefers thymine over
N4-ethenocytosine (9). Recombinant MBD4 can also
remove uracil, 5-fluorouracil, and 5mC at a low rate, particularly when
these bases are opposite a guanine within CpG dinucleotides (1, 9, 10).
The MBD domain of MBD4 is similar to domains within four other
mammalian proteins, MeCP2, MBD1, MBD2, and MBD3 (reviewed in Refs. 11
and 12). The latter proteins are involved in suppressing transcription
in regions of heavy CpG methylation, but no such role has been ascribed
to MBD4. Whereas the NMR structures of the MBD domains from MBD1 (13,
14) and MeCP2 (15) have been elucidated, no structural information
regarding the glycosylase domain of MBD4 is available.
Here we present the crystal structure of the C-terminal glycosylase
domain of MBD4, and we show that it belongs to the helix-hairpin-helix DNA glycosylase superfamily. The glycosylase domain alone is active on
DNA duplex containing a G:T mismatch within a CpG sequence context.
Overexpression and Purification--
The full-length mouse MBD4
was expressed as a His-tagged fusion protein in vector pET6H (16). The
four fragments of MBD4 (Fig. 1A), amino acids 49-187 (MBD
domain), 400-554 (glycosylase domain or
The full-length and
The MBD domain and the glycosylase domain were purified using nickel
chelate, HiTrap Q, and Superdex 75 columns. The proteins were stored in
a high salt buffer for crystallization (20 mM Tris-HCl, pH
7.5, 1 mM EDTA, 1 mM DTT, 5% glycerol, and 0.5 M NaCl) or in a low salt buffer for activity assay (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 50% glycerol, and 50 mM NaCl).
The GST- Limited Proteolysis of Full-length MBD4 Protein--
Protease V8
was added, as described previously (17), to MBD4 for 15 min incubation
at room temperature. Addition of the protease inhibitor
1-chloro-3-tosylamido-7-amino-2-heptanone stopped the reaction, and two
major MBD4 fragments were observed via SDS-PAGE. The mass of the two
fragments was determined by electrospray ionization mass spectrometry
to be 15,353.5 and 18,537.2 Da. The N-terminal sequences of the two
fragments were also determined. Combining these results allowed us to
deduce that the fragments represent residues 49-187 (the MBD domain)
and residues 400-554 (the C-terminal glycosylase domain) (Fig.
1A).
Crystallography--
Crystals of the MBD4 glycosylase domain
stored in the high salt buffer were obtained in hanging drops under the
conditions of 22-27% polyethylene glycol 2000 monomethyl ether,
190-230 mM ammonium sulfate, 10-15% ethylene glycol, 100 mM sodium citrate, pH 5.26. The initial drops were set up
at 16 °C and moved to 4 °C after overnight incubation. Two
crystal forms were observed, good looking diamond-shaped crystals
appeared earlier at 16 °C but diffracted x-rays poorly. Rather
unpromising colorless crystals appeared after 2-3 weeks only at
4 °C, but these diffracted x-rays strongly and belonged to space
group P3121, based on the systematic absence of reflections
along the z axis. There is one molecule per asymmetric unit,
and unit cell dimensions were a = b = 48.58 Å and c = 146.57 Å.
Selenomethionine-containing glycosylase domain Preparation of E. coli Extract--
The His-tagged full-length
MBD4, Preparation of the Labeled DNA Substrate--
The following
oligonucleotides were obtained from Invitrogen:
T-oligo, 5'-GACTGGCTGCTCCTGGGCGAAGTGCC-3';
G-oligo, 5'-GGGCACTTCGCCCGGGAGCAGCCAGTC-3'.
The oligonucleotides were gel-purified prior to their use. The T-oligo
was labeled at the 5' end using T4 polynucleotide kinase (New England
Biolabs) in the presence of [ DNA Glycosylase Assay--
Twenty nM labeled duplex
was equilibrated with nicking buffer (10 mM Tris-HCl, pH
8.0, 5 mM EDTA, 1 mM DTT, and 0.1 mg/ml bovine
serum albumin), and the reactions were initiated by adding 100 ng of
purified MBD4 variants (Fig. 5A) or 2 µg of cell-free extract (Fig. 5B). Following incubation at 37 °C for
1 h, the reaction was stopped by heating to 95 °C for 7 min in
the presence of 0.1 M NaOH. Subsequently, 8 µl of gel
loading dye (80% formamide, 10 mM EDTA, 1 mg/ml each of
xylene cyanol and bromphenol blue) was added to the samples which were
then heated to 95 °C and electrophoresed in 20% sequencing gel. The
gel was exposed to a PhosphorImager screen (Amersham Biosciences), and
the reaction products were quantified using ImageQuant software.
MBD4 Glycosylase Domain Structure--
The overall structure of
MBD4 glycosylase domain consists of 11 helices ( Model of the MBD4-DNA Complex--
The high degree of structural
similarity among HhH glycosylases allowed us to create a model of the
MBD4 glycosylase domain bound to DNA. By using the coordinates of the
AlkA-DNA (26) or hOGG1-DNA (27) complexes, we superimposed the protein
components, and then the DNA was positioned over the surface of MBD4
with the cleft. Previous modeling studies of other HhH glycosylases MutY and EndoIII suggested that they bind to DNA in a manner similar to
that of AlkA (26). Our modeling suggests that the MBD4 glycosylase domain also binds DNA similarly to AlkA and hOGG1, which bind DNA via
the minor groove and bend it ~70° at the damaged base (26, 27).
The residues that contact the DNA backbone in the hOGG1 and AlkA
structures occupy similar positions in the free MBD4 structure (Fig.
3C), and the MBD4 glycosylase domain could contact bent DNA
without major physical distortion of the protein component (Fig.
3D). Two important DNA-binding loops are superimposed, the loop between helices Mechanisms for Recognition of Flipped Bases and
Catalysis--
First, where is the active site? In analogy to the
AlkA-DNA (26) and hOGG1-DNA (27) complexes, the MBD4 cleft defines the
location of the active site (Fig.
4A). The target nucleotide is
likely to be flipped out from the DNA helix into the active-site cleft
of the enzyme, in a similar manner to AlkA or hOGG1. The structural
superimposition of the HhH glycosylase-DNA complexes and the unbound
MBD4 reveals several informative features. Interestingly, the flipped
base can only be docked into the active site by stacking the base
between the side chains of Leu440 of
A second question is where the key catalytic residues are located.
Asp534, the last residue prior to helix
A third question regarding the MBD4 action is how it distinguishes an
A:T pair from a G:T. Although it is possible that the protein
distinguishes G:T from an A:T because of their differing geometries, it
is also possible that it may make specific contacts with the guanine in
a manner similar to E. coli MUG (31) or hOGG1 (27);
Arg486 of MBD4 is in the same position as
Arg204 of hOGG1 that forms hydrogen bonds in the minor
groove side with the G on the opposite strand of the flipped
nucleotide. A detailed answer to this question must await the
availability of a MBD4-DNA co-crystal structure.
Thymine and Uracil--
How does the flipped base specifically
bound in the active site? In MutY the adenine soaked into the crystal
are recognized by Glu37 and Gln182 (29) (Fig.
4D). Structural superimposition between MutY and MBD4 (Fig.
3A) indicates the side chains of Gln-423 and
Tyr514 of MBD4 are in the vicinity of the adenine-specific
interacting side chains of MutY (Fig. 4E).
In MBD4, the two polar residues (Gln423 of
Interestingly, Glu, Gln, or Tyr are often found in the active site of
the HhH glycosylases. A glutamate is found in MIG (Glu42;
Ref. 28) and TAG (Glu38; Ref. 32) in the equivalent
position as Glu37 of MutY; the corresponding main chain
position in MBD4 is Thr437 (helix DNA Glycosylase Activity of MBD4 N-terminal Truncations--
Among
the known HhH enzymes, MBD4 has the longest N-terminal sequence before
the glycosylase domain (for examples, see Fig. 1B). Zhu
et al. (10) analyzed a series of N-terminal deletion mutants
of human MBD4, and the results are consistent with our glycosylase
domain structure presented here. In that study, N-terminal deletions of
up to 65% of the total length of MBD4 retain the DNA glycosylase
activity. The smallest fragment that retained activity,
We used a DNA duplex containing a G:T within a CpG sequence context as
the substrate to test the glycosylase activities of purified
full-length MBD4 and several of its deletion derivatives. The
T-containing strand was radiolabeled, and the excision of this base was
monitored by gel electrophoresis. Typical results are presented in Fig.
5A and show that in addition
to the full-length MBD4, the
Petronzelli et al. (33) have reported that a deletion of the
first 454 amino acids of the human MBD4 still retained its enzymatic
activity. The murine MBD4 equivalent to this deletion would be missing
428 N-terminal residues (Fig. 1B), which include helix
To resolve these discrepancies, we attempted to duplicate the result of
Petronzelli et al. (33) by making the equivalent murine MBD4
truncation (
The activity of the We have described the crystallographic structure of the
glycosylase domain of the methyl-CpG-binding protein MBD4. The
structure reveals that the MBD4 glycosylase domain belongs to the HhH
DNA glycosylase superfamily. Modeling studies suggest that MBD4
glycosylase domain, similar to that of AlkA and hOGG1 HhH glycosylases,
binds DNA from the minor groove side (Fig. 3D).
Unlike other HhH glycosylases, MBD4 contains an additional DNA binding
domain, the MBD, near its N terminus. An NMR solution structure of the
MBD domain from human MBD1, in complex with methylated DNA, revealed
that the MBD domain contacts both methyl groups of methyl-CpG site via
the major groove of B-form DNA (13) (Fig. 3E). This is
consistent with the observation that of the DNA sequence tested, only
the fully methylated CpG or the methylated mismatch 5mCpG/TpG (both
contain two methyl groups in the major groove) is bound by the MBD of
MBD4 (1). Because all structurally characterized HhH glycosylases in
complex with DNA appear to bind DNA exclusively via the minor groove,
it is attractive to think that the MBD and the glycosylase domains of
MBD4 would come together at 5mCpG/TpG mismatches to engulf DNA from
opposite directions (28). However, because the MBD domain does not bend
DNA (13), whereas all HhH glycosylases appear to significantly bend DNA
and flip the target, it is not clear how DNA would be bent when both
domains bind together. Alternatively, perhaps the two domains separated
by ~200 residues bind DNA at adjacent but non-overlapping sites. The
function of the MBD domain in MBD4 may be to target the glycosylase
activity to regions of heavily methylated DNA as methyl-CpG
dinucleotides tend to occur in clusters (reviewed in Ref. 34), so the
tethered glycosylase domain could sample nearby sites for G:T
mismatches. This would raise the local concentration of glycosylase
activity in regions where methylated mismatch 5mCpG/TpG is most likely to occur.
The active-site cleft of the glycosylase domain suggests a base
flipping mechanism for accessing the damaged or mispaired base
(reviewed in Ref. 35), the mismatched base should be swung completely
out of the DNA helix by torsional rotation of its flanking sugar-phosphate backbones so as to occupy the active-site cleft of
MBD4. The structure also reveals candidate residues for catalysis (Asp534), for thymine (or uracil)-specific recognition
hydrogen bonding (Tyr514, Gln423, and
Val422), for the methyl group of thymine
(Ile449 and Gly445), and for the stacking
stabilization of the flipped base (Leu440 and
Lys536). With this information, our structure provides
useful starting points for more detailed studies of this interesting enzyme.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Big Blue mouse and by increased
occurrence of colon carcinoma in Mbd4
/
ApcMin/+ mice (2). Additionally, studies of MBD4
(also called MED1) using the yeast two-hybrid system have shown that it
interacts with MLH1 (a protein implicated in mismatch repair) and
suggest a role for this enzyme in maintaining genome stability (3). Consistent with this observation, it is found that MBD4 is mutated in
26-43% of human colorectal tumors that show microsatellite instability (4).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
399), 49-554 (
48), and
429-554 (
428), were cloned into a modified pET28b (Novagen) vector,
which contains an N-terminal tag of MGHHHHHH and accepts an
NdeI-EcoRI insert. The
428 fragment was also
expressed as a GST fusion in pGEX2T vector (Amersham Biosciences).
E. coli strain BL21(DE3) carrying respective plasmid was
grown in LB media supplemented with appropriate antibiotics at 37 °C
to A600 = 0.6, shifted to 22 °C, and
induced with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside overnight at
22 °C, except that the full-length MBD4 was induced at 37 °C for
1 h.
48 proteins were purified from cleared lysates
using three successive chromatography steps as follows: a nickel
chelate column, a HiTrap heparin column, and Superdex 200 (Amersham
Biosciences). The proteins were stored in 20 mM potassium
phosphate, pH 7.5, 1 mM EDTA, 0.1% 2-mercaptoethanol, and
0.2 M NaCl.
428 was purified using glutathione-Sepharose 4B (Amersham
Biosciences) and HiTrap Q column. The protein was stored in the low
salt buffer.
399 was expressed in
a methionine auxotrophic E. coli strain B834(DE3) grown in
LeMaster medium (18) supplemented with 25 µg/ml Se-methionine, and the protein was purified similarly to the native protein. X-ray
diffraction data for the native and the single-site selenomethionine substitution crystals were collected (Table
I), respectively, by an RAXIS-IV imaging
plate detector equipped with a Rigaku rotating anode generator (50 kV,
100 mA) and a Brandeis B2 (1 × 1) CCD-based detector at the
National Synchrotron Light Source beamline X12-C. The resulting images
were processed using HKL (19). One surface selenium site
(SeMet447 of
C) was determined by SOLVE (20). RESOLVE
(21) was then used to modify the electron density map at 2.5-Å
resolution (overall figure of merit 0.54). The modified map was of
excellent quality to place amino acids 411-554 of MBD4 into the
recognizable densities by using the graphic program O (22).
Electron density was not observed for the first 11 residues (400-410
of the full-length protein). The resulting model was refined to 2.09 Å resolution using X-PLOR (23) (Table I), with a final crystallographic R-factor of 0.213 and R-free of 0.263 (for 9% of total 21,552 reflections).
X-ray data collection and structural refinement statistics
48, MBD domain (amino acids 49-187), glycosylase domain
399, and
428 were all transformed into an E. coli
strain BH161, which carries ung
and a copy of
T7 RNA polymerase (24). Ten milliliters of culture was grown from each
clone in LB media supplemented with 100 µg/ml ampicillin (full-length
MBD4) or 50 µg/ml kanamycin (all MBD4 fragments) at 37 °C until
the A600 reached 0.6. The cultures were induced by adding isopropyl-1-thio-
-D-galactopyranoside
to a final concentration of 0.5 mM. After incubation at
37 °C for 3 h, the cells were recovered by centrifugation, and
the cell pellets were washed with a buffer containing 20 mM
Tris-HCl, pH 7.6, and 0.1 mM EDTA. Cells were resuspended
in 0.5 ml of extraction buffer (20 mM Tris-HCl, pH 7.8, 0.1 mM EDTA, 5 mM 2-mercaptoethanol) containing 1 mg/ml lysozyme and incubated on ice for 15 min. Finally, the cells were
broken by sonication on ice, and cell-free lysate was recovered
following centrifugation at 12,000 × g for 15 min at
4 °C. Protein concentration in the extracts was determined using the
Bradford Reagent (Bio-Rad).
-32P]ATP (specific
activity 6000 Ci/mmol, PerkinElmer Life Sciences). The reaction was
terminated by heating it to 65 °C for 20 min. The labeled T-oligo
was mixed with 3-fold molar excess of the unlabeled G-oligo in the STE
buffer (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). The mixture was heated to 95 °C for 3 min and then slowly cooled to room temperature over a period of 2-3 h
to promote duplex formation. The unincorporated
[
-32P]ATP was removed from the labeled duplex by
passage through a G-50 micro column (Amersham Biosciences).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A to
K) (Fig.
1B) forming a single domain
with a cleft in the middle (Fig. 2).
Structural comparison with other DNA glycosylases (Fig.
3, A and B) reveals
that the MBD4 glycosylase domain belongs to the
helix-hairpin-helix (HhH) DNA
glycosylase superfamily (25), named after a conserved structural motif
H-hairpin loop-
I (shown in red in Fig. 2A).
The six helices before the HhH motif (
B to
G in green) are highly
conserved structural elements among family members, forming the bottom
of the cleft in the orientation shown (Fig. 2A). Among the
known HhH enzymes, MBD4 has the shortest sequence following the HhH
motif (Fig. 1B). The C-terminal helices
J and
K, the
short N-terminal helix
A, and its 12-residue preceding loop, the HhH
motif, come together to form a hydrophobic core (Fig. 2C),
forming the top of the cleft.
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Fig. 1.
A, schematic representation of
mouse MBD4. Two stable fragments (shaded) were identified by
limited proteolysis. Below the full-length mouse MBD4 are the depicted
protein fragments ( 48, MBD, glycosylase domain
399, and
428)
used in this study. B, structure-guided sequence alignment
of three MBD4 glycosylase domains (mouse, AAC68878; human, AAC68879;
and Gallus, AAF68981) and five HhH glycosylases. We note that the
reported MBD4 homolog in chickens only contains the glycosylase domain
and has no consensus sequence for the N-terminal MBD (10). The
secondary structure of MBD4 is indicated above the sequence
and colored the same as in Fig. 2A. The dashed
lines indicate gaps introduced to optimize alignments, and MBD4
has the shortest loop prior to the HhH motif. The dots
indicate extra residues outside the glycosylase domain. The residues in
the active site, proposed to interact with the extrahelical target
nucleotide, are colored to match their associated region. The residues
marked by * above the sequence are discussed in the text.
The deletion mutants made in human MBD4 (10, 33) and mouse MBD4 (this
study) are indicated by arrows. The four differences between
mouse and human MBD4 sequences are boxed.
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Fig. 2.
Structure of MBD4 glycosylase domain.
A, stereo view of Ribbon (36) diagram. The protein is
colored according to Fig. 1B. The proposed thymine
recognition residues Gln423 (orange) and
Tyr514 (red), and the catalytic
Asp534 (cyan) are shown in
ball-and-stick model. B, two orthogonal views of
solvent-accessible Grasp (37) surface with charge distribution
(blue for positive charge, and red for negative
charge). Left, the protein is shown in a similar orientation
as in A. Right, the cleft predicted to hold the
active site is visible after rotation ~90° on the vertical axis.
C, stereo view of the hydrophobic core formed by the
N-terminal loop and helix A (orange), helix
I of HhH
motif (red), and the C-terminal helices
K and
J
(cyan). The hydrophobic side chains are shown in
ball-and-stick model, colored to match their associated
region. D, stereo view of difference electron density
(blue) contoured at 3.0
above the mean, superimposed with
Val422, Gln423, and Tyr514, and
three associated water molecules.
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Fig. 3.
Structural comparison of HhH glycosylases.
A, superimposition of MBD4 glycosylase domain (colored
according to Fig. 1B) and MutY (gray; Protein
Data Bank code 1MUD). MutY (29), as well as EndoIII (30) and MIG (28),
contains a C-terminal [4Fe-4S] cluster (shown in space-filling
model). B, superimposition of MBD4 glycosylase domain and
AlkA (gray; Protein Data Bank code 1DIZ). AlkA (26) and
hOGG1 (27) contain an additional N-terminal -sheet domain, and a 13- or 15-residue insertion between
C and
D (see Fig. 1B).
C, superimposition of two DNA binding loops between MBD4
(red and green) and AlkA (gray). The
Gly-rich hairpin loop of HhH motif is indicated by conserved
Gly510 and Gly512 of MBD4. The minor groove
wedge (Leu125 in AlkA), which assists in base flipping,
superimposed on Arg442 of MBD4 in the loop between helices
B and
C (see Fig. 1B). D, based on the
superimposition shown in C, the MBD4 glycosylase domain is
docked to DNA from the minor groove side. E, the MBD domain
of MBD4 has not yet been structurally characterized; however, the NMR
solution structure of the MBD domain of MBD1 was shown to bind DNA from
the major groove side (13).
B and
C and the Gly-rich hairpin loop of HhH
motif (Fig. 3C). Arg442 of MBD4, as well as
Arg47 of MIG (28), is in the same position as
Leu125 of AlkA (or Asn149 of hOGG1) that fills
the space in the DNA duplex vacated by the flipped nucleotide.
Thr443 of MBD4 is in the same position as
Asn150 of hOGG1 that makes main chain contacts to the
phosphate groups 3' to the flipped nucleotide. Ser444 of
MBD4 is in the position of Asn151 of hOGG1 that forms
hydrogen bonds with the base 5' immediate to the flipped nucleotide. It
seems that the loop between helices
B and
C contains residues
(Arg442-Thr443-Ser444) important
for DNA binding and base flipping.
B and
Lys536 of
K (Fig. 4B). Although these
residues are not conserved in HhH glycosylases, similar stacking
appears to be conserved: in hOGG1 8-oxoguanine is between
Cys253 and Phe319 (27) and in MutY adenine
soaked into the crystal lies between Leu40 and
Met185 (29). Leu440 of MBD4 corresponds to
Leu40 of MutY (Figs. 1B and 4E),
whereas MutY Met185 corresponds to Phe319 of
hOGG1.
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Fig. 4.
The active-site cleft and where the flipped,
mismatched thymine will bind. A, close-up view of the cleft,
where Asp534 is colored in red;
Tyr514, Gln423, and Val422 are
colored in green, and Ile449,
Gly445, and Leu440 are in yellow. B,
the thymine (yellow) is docked in between Leu440
and Lys536. C, the Watson-Crick pairing edge of
thymine (O-2, N-3, and O-4) is aligned with three ordered water
molecules (green) that make hydrogen bonds with
Tyr514, Gln423, and Val422. The C-5
methyl group would make van der Waals contact with Ile449
and Gly445. The C1' of the target sugar would be in a
direct contact with the carboxylate group of Asp534. The
atom spheres are colored in red (oxygen), blue
(nitrogen), and gray (carbon); the chemical bonds are
colored in gray for the protein residues and yellow for
thymine. D, schematic drawing of adenine-specific
interactions in MutY (29). E, stereo view of superimposition
of active site residues of MutY (gray) and the proposed MBD4
active site residues (colored according to Fig. 1B).
F, schematic drawing of proposed thymine-specific
interactions in MBD4.
K (Fig.
1B), is in a position structurally equivalent to the
catalytically important Asp238 of AlkA (26),
Asp268 of hOGG1 (27), Asp138 of MutY (29), and
Asp138 of EndoIII (30). Two mechanisms have
been suggested for the function of this structurally conserved aspartic
acid in HhH glycosylases: (i) it activates a catalytic nucleophile,
which is either a water (29) or the
-amino group of a lysine (27),
for the attack on the deoxyribose C1' carbon atom of the target
nucleotide; or (ii) it directly assists base removal by protonating the
leaving group of the substrate sugar (26). In the docking model of
MBD4-thymine (Fig. 4C), the C1' position of a modeled
substrate is in direct contact (~3.0 Å) with the carboxylate of
Asp534, which would favor the second (protonation) mechanism.
A and
Tyr514 of
I) and three hydrophobic residues
(Val422 prior to
A, Gly445, and
Ile449 of
C) line in the cleft next to the catalytic
Asp534 (Fig. 4A). We suggest that these amino
acids are the major determinants of specificity after docking the
flipped thymine into the binding pocket. In the absence of the target
nucleotide, the active site is occupied by ordered water molecules
(Fig. 2D), which lie almost in a plane and directly interact
with Tyr514, Gln423, and Val422
(Fig. 4C). We docked a thymine with its Watson-Crick pairing edge (O-2, N-3, and O-4) occupying three water sites (Fig.
4C). The OH group of Tyr514 can make one
hydrogen bond with the O-2 atom, the side chain carbonyl C = O of
Gln423 can make a hydrogen bond to the protonated N-3-H. In
addition, the main chain N-H group of Val422 can make a
hydrogen bond to the O-4 atom. Gly445 and
Ile449 form a surface hydrophobic patch near the end of the
cleft, in a perfect position to accommodate the methyl group of
thymine. Of all contacts made to the thymine base (Fig. 4F),
the hydrophobic-methyl interaction will be absent for a uracil base.
B), whose side chain
points opposite as that of Glu37 in MutY (Fig.
4E) and makes a hydrogen bond with main chain carbonyl oxygen atom of Leu434. A tyrosine is proposed for
interacting target thymine in MIG (28) (Tyr126 occupying
equivalent position as Tyr514 of MBD4 in helix
I), for
interacting target 3-methyladenine in AlkA (26) (Tyr222
from an
I-equivalent helix, i.e. the second helix of HhH
motif), and in TAG (32) (Tyr16 of an N-terminal helix). A
glutamine is common to MutY (Gln182) and hOGG1
(Gln315) in recognizing their substrate base, adenine and
8-oxoguanine, respectively; both Gln182 of MutY and
Gln315 of hOGG1 are located in a C-terminal helix outside
of the structurally homologous regions among the HhH glycosylases shown
in Fig. 1B. Although MBD4 does not have an equivalent
C-terminal helix, the N-terminal and C-terminal regions of all
structurally characterized HhH glycosylases are folded together, above
the cleft as shown in Fig. 3; and in the case of MBD4,
Gln423 is from the N-terminal helix
A and its side chain
occupies a similar position as that of Gln from the C-terminal helix.
N433 (10),
is very similar in size to our glycosylase domain determined by
proteolysis (see Fig. 1B).
399 mutant used for crystallography is
an active thymine DNA glycosylase. A construct missing the first 48 amino acids of the full-length protein (
48) has less activity, but
the construct containing only the MBD segment of the protein (amino
acids 48-187) has no detectable activity (Fig. 5A). We also
measured the activity of all MBD4 constructs in crude cell extract by
expressing the proteins in a strain lacking the endogenous uracil
glycosylase gene (ung
) to minimize background.
Full-length MBD4,
48, and
399 all have detectable activity in
this assay (Fig. 5B).
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Fig. 5.
Glycosylase activities of MBD4 N-terminal
truncations. A, thymine excision activities of purified
full-length MBD4 and its deletion derivatives are shown. One hundred
nanograms of the His-tagged or GST-tagged ( 428) versions of the
proteins were used with 20 nM radiolabeled duplex
containing a G:T. The products of the reaction were separated on a
sequencing gel, and the gel was scanned with a PhosphorImager. The
PhosphorImager scan is shown. B, glycosylase activities in
cell extracts prepared from overproducers of MBD4 variants. Thymine
excision activities in the cell extracts of the overproducers of
full-length MBD4 and its deletion derivatives are shown. Cell extracts
containing 2 µg of total proteins were used with 20 nM
radiolabeled duplex containing a G:T. The products of the reaction were
separated on a sequencing gel, and the gel was scanned with a
PhosphorImager. The released product as a percentage of total labeled
DNA is shown.
A
and its preceding loop that provides part of the hydrophobic core above
the cleft (Trp412, Pro414, Pro415,
Pro418, and Phe419; Fig. 2C) and
Val422 and Gln423 that are proposed to contact
the target thymine (Fig. 4C). Thus the results reported by
Petronzelli et al. (33) are not compatible with the crystal
structure and are surprising.
428; Fig. 1) and fusing it to a six histidine tag or GST
tag. We were unable to detect any expression of the His-tagged
428
protein, either by Coomassie staining or anti-His tag antibody (data
not shown), whereas all other MBD4 fragments were expressed and soluble
under the same conditions. Not surprisingly, no glycosylase activity
was detected in the extract of
428 construct using the
ung
strain (Fig. 5B). The
GST-tagged
428 was expressed to high level, but most of the protein
was insoluble (data not shown). However, we did manage to partially
purify some GST-
428 fusion protein using a glutathione affinity
column and a HiTrap Q column. The protein was heavily associated with
Hsp60 (data not shown), an indication that the protein may not be
folded properly. When the GST-
428 protein was tested for glycosylase
activity, none was detected (Fig. 5A). The observation that
428 mutant does not fold properly is consistent with the important
structural roles of the missing residues. In addition, although
sequence similarity of MBD4 to other glycosylases starts at helix
B,
MutY, MIG, EndoIII, and TAG all have N-terminal extensions similar in
size to
399 of MBD4 (Fig. 1B). We do not know the origin
of the discrepancy between our data and that of Petronzelli et
al. (33), as the sequences of human
454 and mouse
428
deletions are almost 100% identical except 4 residues (see Fig.
1B). One possibility is that the pET28b vector (Novagen)
used for the human
454 construct would add at least 10 additional
residues besides the 6 histidines at the N terminus. These residues may
fortuitously substitute the natural MBD4 residues and allow folding and
enzymatic activity.
399 deletion was easiest to detect in the
extracts, whereas the full-length MBD4 and the
48 construct displayed relatively poor activity (Fig. 5B). The lower
activity of the full-length MBD4 in cell-free extracts was surprising
but reproducible. It is noted that the MBD domain of MBD4 binds DNA with G:T mismatches (1), and it is possible that both the MBD and the
glycosylase domains compete for the DNA substrate. Regardless, it is
clear from these data that the
399 construct of the murine MBD4,
which has almost the same N-terminal extension as the MutY, MIG,
EndoIII, and TAG, is a stable protein fragment with substantial glycosylase activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Adrian Bird (University of Edinburgh) for providing the constructs to overexpress mouse MBD4; Drs. Anand Saxena and Dieter Schneider (Brookhaven National Laboratory) and John R. Horton (Emory University) for help with x-ray data collection at beamlines X12-C and X26-C in the National Synchrotron Light Source; and Drs. Robert Blumenthal (Medical College of Ohio) and Paul Wade (Emory University) for critical comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM49245 (to X. C.) and GM57200 (to A. S. B) and the Georgia Research Alliance.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 the structure factors (code 1NGN) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ Both authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Biochemistry, Emory University, 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-8491; Fax: 404-727-3746; E-mail: xcheng@emory.edu.
Published, JBC Papers in Press, November 26, DOI 10.1074/jbc.M210884200
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ABBREVIATIONS |
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The abbreviations used are: MBD, methyl-CpG binding domain; 5mC, 5-methylcytosines; TDG, thymine-DNA glycosylase; GST, glutathione S-transferase; DTT, dithiothreitol.
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