From the Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, EH9 3JR, United Kingdom and § Edinburgh Centre for Protein Technology, University of Edinburgh, Edinburgh, EH9 3JJ, United Kingdom
Received for publication, August 9, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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The methyl-CpG binding domain (MBD) of the
transcriptional repressor MeCP2 has been proposed to recognize a single
symmetrically methylated CpG base pair via hydrophobic patches on an
otherwise positively charged DNA binding surface. We have tested this
binding model by analysis of mutant derivatives of the MeCP2 MBD in
electrophoretic mobility shift assays complemented by NMR structural
analysis. Exposed arginine side chains on the binding face, in
particular Arg-111, were found to be critical for binding.
Arg-111 was found to interact with the conserved aspartate side chain
Asp-121, which is proposed to orientate the arginine side chain to
allow specific contacts with the DNA. The conformational flexibility of
the disordered B-C loop region, which forms part of the binding face,
was also shown to be important. In contrast, mutation of the exposed
hydrophobic side chains had a less severe effect on DNA binding. This
suggests that the Arg-111 side chain may contribute to
sequence-specific recognition of the CpG site rather than simply making
nonspecific contacts with the phosphate backbone. The majority of
missense mutations within the MBD found in the human genetic disorder
Rett syndrome were shown or predicted to affect folding of the domain rather than the DNA recognition event directly.
Methylation of cytosine residues in the dinucleotide sequence CpG
within mammalian DNA is associated with transcriptional repression. A
family of methyl-CpG binding proteins, which are thought to mediate the
biological consequences of DNA methylation, has been identified and
characterized (1), of which the founder member is the transcriptional
repressor MeCP2. These proteins contain a conserved
methyl-CpG binding domain
(MBD),1 which is 70-75 amino
acids in length and exhibits 50-70% similarity between the proteins.
The MBD of MeCP2 is necessary and sufficient for DNA binding in
vitro and allows MeCP2 to recognize a single symmetrically
methylated CpG dinucleotide in diverse sequence contexts (2). Of the
other MBD-containing proteins, MBD1 and MBD2 also bind to single
symmetrically methylated CpG dinucleotides in vitro and act
as transcriptional repressors (3, 4). Repression by MeCP2 can act at a
distance from a promoter and involves the targeting of histone
deacetylases via a C-terminal transcription repression domain, which
interacts with the corepressor mSin3 (5), although a
deacetylase-independent mechanism may also contribute to repression
(5-7). Targeting of histone deacetylases is probably also important in
transcriptional repression by MBD1 and MBD2 (3, 4). Mammalian MBD3 is
found in a histone deacetylase complex, the Mi-2 or NuRD complex (8),
but does not bind specifically to methylated DNA in vitro
and is not localized to methylated chromosomal regions in
vivo (1). MBD4 is probably a methyl-CpG-TpG mismatch-specific
glycosylase (9).
Recently, an indication of the role of MeCP2 in human development has
been provided by the linking of mutations in the MeCP2 gene
to the human neurodevelopmental disorder Rett syndrome (10). This
disorder is a childhood-onset regressive disease that causes loss of
speech and hand movement, coupled with autistic behavior, microencephaly, and growth retardation and affects one in every 10,000-15,000 live female births (11, 12). Mutations found in the DNA
of Rett syndrome patients include frameshift mutations generating
truncations within the MBD, the transcription repression domain, or the
C terminus of MeCP2, nonsense mutations preceding and immediately
downstream of the MBD or within the transcription repression domain, or
most interestingly, missense mutations within the MBD and transcription
repression domain domains (10, 13-18). It is of particular interest to
determine what effects this latter class of mutations might have on the
ability of the MeCP2 protein to carry out its functions of methylated
DNA binding and transcriptional repression.
We have recently solved the solution structure of the MeCP2 MBD by NMR
spectroscopy (19). The domain has a novel fold that forms a
wedge-shaped structure, comprising an N-terminal four-stranded anti-parallel In this study, we have employed rationally designed site-directed
mutant proteins in quantitative mobility shifts to test more thoroughly
the model for DNA recognition by the MBD domain of MeCP2. In
particular, we have defined the critical role of surface-exposed
residues and the large flexible loop on the DNA binding face of the
protein in recognition of methylated DNA. In contrast, we find that the
conserved hydrophobic side chains in the binding face play a less
crucial role in the interaction. We have also investigated the effects
that certain missense mutations within the MeCP2 MBD associated with
Rett syndrome have on the folding of the domain and on DNA binding and
have used our structural information on the MBD to account for the
phenotypic effects of a number of such mutations. This leads us to
propose that the majority of missense mutations within the MeCP2 MBD
associated with Rett syndrome affect the ability of the domain to
interact with methylated CpG dinucleotides via a disruption of the
domain fold rather than direct mutation of side chains involved in DNA recognition.
Cloning and Site-directed Mutagenesis--
The MeCP2 MBD
was amplified from the plasmid pET6HMBD (21) using the primers
5'-CTAGCAGCCATGGCCTCTGCTTCTCCCAAACA-3' and 5'-CTAGCAGGATCCTTACCCTCTCCCAGTTACAGTGA-3', and the resulting
amplification product was digested with NcoI and
BamHI and cloned into
NcoI-BamHI-digested pET6H to generate pAFB105.
This plasmid therefore encodes residues 77-164 of MeCP2 with the
heterologous N-terminal sequence MHHHHHHAM. The portion of MeCP2
encoded is that previously defined as the minimum required for
recognition of methyl-CpG (2) and is identical to that used for
determination of the NMR structure of the MBD (19) except that it lacks
the heterologous, unstructured C-terminal residues GSGC. To generate
site-directed mutants of the MBD, the QuikChange kit (Stratagene) was
employed with 20-25-mer primers and pAFB105 as a template, according
to the manufacturer's instructions.
Protein Expression--
An overnight culture of a fresh
transformant of Escherichia coli BL21 ( Electrophoretic Mobility Shift Assay--
A double-stranded
27-mer oligonucleotide pair, symmetrically methylated (mC) at a single
site on each strand (5'-TCAGATTCGCGCmCGGCTGCGATAAGCT-3' and its reverse
complement; Ref. 1) was end-labeled with digoxygenin-dideoxy-UTP using
an oligonucleotide end-labeling kit (Roche Molecular
Biochemicals) according to the manufacturer's instructions. An
unmethylated version of the same oligonucleotide pair was labeled
similarly. Purified proteins were incubated with 2 nM
labeled duplex oligonucleotide plus 50 ng/µl poly[d(A-T)]
(Roche Molecular Biochemicals) as a nonspecific competitor in 20 mM HEPES, pH 7.6, 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM
dithiothreitol, 0.2% Tween® 20, 30 mM KCl in a final
volume of 30 µl at room temperature for 15 min. Loading buffer (60%
0.25× Tris-borate EDTA (TBE), 40% glycerol; 7.5 µl) was
added, and the samples were electrophoresed on pre-run 0.25× TBE, 6%
polyacrylamide (37.5:1 acrylamide-bisacrylamide) gels at 250 V for
3.5 h in 0.25× TBE buffer at 4 °C. Gels were blotted onto
Hybond-N+ (Amersham Pharmacia Biotech) in 0.25× TBE buffer
overnight, and the membranes were fixed by UV cross-linking followed by
baking at 80 °C for 10 min. Digoxygenin-labeled probes were detected using a nonradioactive DNA detection kit (Roche Molecular Biochemicals) according to the manufacturer's instructions, followed by exposure to
Hyperfilm ECL (Amersham Pharmacia Biotech). Scanned images were
quantified using ImageQuant software (Molecular Dynamics).
NMR Spectroscopy--
Spectra were collected on a 1 mM sample in 90% H2O, 10% D2O at
18 °C in a 50 mM sodium phosphate buffer adjusted to pH
6.0. Two-dimensional 15N-HSQC and three-dimensional
15N-edited nuclear Overhauser effect spectroscopy (NOESY)
spectra were recorded on a Varian INOVA 600 MHz spectrometers
using 5-mm probes. All NMR data were processed with the AZARA
package (see the CCPN website at the University of Cambridge)
with the application of an 80° sinebell-squared window function.
ANSIG (22, 23) was used to display spectra and maintain cross-peak
lists for resonance assignment. The resonance assignment for wild-type
MBD of MeCP2 was described previously (19). By overlaying
two-dimensional 15N-HSQC spectra collected on mutant MeCP2
MBD proteins, it was possible to assign the majority of peaks. Backbone
1HN and 15N atoms of the G114P
mutant were sequence-specifically assigned from three-dimensional
15N-edited nuclear Overhauser effect spectroscopy (NOESY)
experiments (75-ms mixing time) on the mutant. The chemical shift
perturbations for assigned peaks were quantitatively measured using
weighted averaging EMSA Analysis of DNA Binding by MeCP2 MBD Mutants--
We
generated a set of point mutations within the MBD that included
solvent-exposed charged and hydrophobic residues, core residues, and
side chains predicted to be important for the conformation of the
peptide backbone within the domain (Table
I). These mutant proteins were assayed
for binding to a methylated 27-mer duplex oligonucleotide
containing a single, symmetrically methylated CpG dinucleotide (see
"Experimental Procedures") at fixed protein and DNA concentrations
of 30 and 2 nM, respectively. At this protein concentration, the wild-type MBD is >80% bound. As quantified in
Table I, the mutant proteins exhibited a range of binding activities
from severely compromised to wild-type affinity; none of the mutants
nor the wild type showed any affinity for the unmethylated oligonucleotide under these conditions (data not shown). Of the mutations causing a significant reduction in DNA binding, the G114P and
D121A or D121E mutants were of particular interest. Gly-114 is
located at the tip of the disordered B-C loop, which otherwise seems
fairly amenable to substitution (see K112M and S113A, Table I). Residue
Asp-121, which we had previously predicted to be involved in bridging
interactions with arginine side chains on the DNA binding face (19),
was shown to be critical for binding, as its mutation to alanine or
even glutamate severely affected the DNA interaction. The mutation
S134A was also seen to cause a moderate reduction in binding affinity.
Ser-134 was found to be mutated to cysteine in cases of Rett syndrome
(14, 16), and the reduction in binding affinity caused by mutating this residue may thus contribute to the pathology.
Constraining the B-C Loop by a G114P Mutation Severely Compromises
Binding--
As noted above, a mutation of residue Gly-114 within the
disordered B-C loop region to proline had a major effect on DNA binding (Table I). This is in contrast to the mild effects of other mutations within this loop examined, including the adjacent S113A. The
replacement of the flexible residue glycine with a structurally
constrained proline is predicted to preclude certain conformations of
the B-C loop when the MBD domain binds to DNA. To quantify the
magnitude of the effect of this change on binding, we carried out a
quantitative EMSA analysis with the G114P mutant (Fig.
1A). Minimal binding (10%)
was detectable at the highest protein concentration used (128 nM), indicating that binding was Structural Analysis of the D121A Mutant--
Because the
mutagenesis screen suggested that residue Asp-121 was particularly
critical for DNA binding and it had previously been suggested that this
side chain might form an important bridging interaction with the
Arg-111 or Arg-133 side chains (19), the D121A mutant was compared with
the wild-type domain by two-dimensional 15N-HSQC NMR
spectroscopy. An analysis of the backbone amide chemical shift
differences between the wild-type and mutant proteins for residues
throughout the domain (Fig.
2A) showed that this mutation causes significant changes in the environment of the B-C loop region
and toward the C terminus of the domain, which folds on top of strand
C, of which Asp-121 is a part. Moreover, the Arg-111 backbone amide
could not be assigned in the mutant by two-dimensional 15N-HSQC, whereas the Arg-133 chemical shift was not
greatly affected. Likewise, the Arg-111 side chain H Crucial Role of the Arg-111 Side Chain in DNA Binding--
We
mutated the Arg-111 residue of the MeCP2 MBD to glycine (Table
II) and examined the effect of the mutation on DNA
binding. The mutant protein exhibited no
affinity for methylated DNA at the concentrations tested (Fig.
3A), although the mutated
domain remained structured with only local perturbations in the
vicinity of Arg-111 and the B-C loop (Fig. 3B). We also
observed that the mutant protein, unlike all other MBD derivatives
tested, bound very poorly to Fractogel
SO32 Effects of Mutations in the "Hydrophobic Patches" Tyr-123 and
Ile-125--
It is interesting to note that the only significant
change in a backbone amide chemical shift remote from the mutated
residue in the R133C mutant was manifested in the strand C residue
Tyr-123 (indicated by an asterisk in Fig. 3C). In
the HSQC spectrum of R133C, the cross-peak due to the Tyr-123 amide was
not detectable at or near its position in the spectrum of the wild
type. Therefore it had either shifted significantly or become broadened
due to a substantial change in local relaxation properties. In either case, it is apparent that changing Arg-133 to Cys significantly perturbed residue 123. This (with Ile-125) is one of two semi-conserved hydrophobic side chains within the putative DNA binding face of MBD
proteins that we proposed earlier to form an interaction site for the
methyl groups of a methylated CpG dinucleotide (19). Mutation of
Tyr-34, the equivalent of Tyr-123 in MBD1, to alanine reduces
methyl-DNA binding (20), although Ile-125 is replaced by a glutamine in
the latter protein. The chemical shift of Tyr-123 may be affected by
the R133C mutation due to the spatial proximity of the two side chains.
In MeCP2 only, the Ala-131 side chain also contributes to the second
hydrophobic patch (19), and we observed that mutation of this side
chain to glutamate reduced the DNA binding affinity (Table I). We
therefore mutated Tyr-123 and Ile-125 of MeCP2 to alanine (Table II)
and analyzed the binding of the mutant proteins to methylated DNA (Fig.
3A). Both mutant proteins exhibited reduced binding
affinity, although in each case this was only ~10-fold lower than the
wild-type affinity, and the majority of the probe was bound at the
higher concentrations used (200 nM and 2 µM).
Neither mutant had as severe an effect on binding as either of the
arginine mutants R111G or R133C. Mutating Tyr-123 to a negatively
charged residue, aspartate, instead of the hydrophobic alanine caused a
more severe effect on binding, but interaction with methylated DNA was
still observed (Table II). The Y123A mutation had no significant
structural effects on the domain according to two-dimensional
15N-HSQC NMR (data not shown), consistent with its
solvent-exposed location and relatively high binding affinity. Thus, it
seems reasonable to conclude that, although the Tyr-123, Ile-125, and Ala-131 side chains contribute to the interaction with methylated DNA,
there is redundancy in this component of the recognition event. The
role of basic side chains such as Arg-111 and, to a lesser extent,
Arg-133, together with the flexibility of the B-C loop is more
important in contributing binding energy to the reaction. How exactly
the methylated cytosines interact with the residues in the DNA binding
face and determine the specificity for methylated DNA is a question to
be addressed by the structure of the MBD·DNA complex.
Missense Mutations Found in Rett Syndrome Cases Affect the
Structure of the MBD--
As noted above, the R133C mutation, which
has a significant effect on DNA binding affinity, is found in cases of
Rett syndrome. We examined three other missense mutations within the
MBD identified in the initial study of MeCP2 mutations in Rett syndrome
(Ref. 10; Table II) to see if they also affected DNA binding. Amino acids Arg-106 and Phe-155 have side chains that both contribute to the
hydrophobic core of the MBD domain (19) and are mutated to tryptophan
and serine, respectively, in specific Rett syndrome cases. Further
occurrences of the R106W mutation have since been identified (13, 16,
18), and R106Q and F155I mutations have also recently been found in
Rett syndrome patients (14, 18). The R106W protein was found to bind
methylated DNA very poorly, whereas the F155S mutant, although forming
a complex at fairly low protein concentrations, did not shift the probe
to completion (Fig. 4). The latter result
suggested that a proportion of the F155S protein added to the reaction
may have been unfolded, and indeed two-dimensional 15N-HSQC
NMR spectroscopy of both of these mutant proteins showed the domain
structure to be unfolded, preventing re-assignment of the backbone
amides. It is likely that the F155S mutant in particular may be
stabilized in the EMSA assay by the presence of its target DNA and of
glycerol, by the low temperature at which the electrophoresis is
carried out and by the caging effect of the polyacrylamide gel. Indeed,
a recent study of the F155S mutation in the context of the intact
Xenopus MeCP2 protein in which mobility shift reactions were
incubated at 37 °C failed to detect any binding activity of the
mutant protein, presumably because the MBD became unfolded (25). The
presumptive instability of the R106W and F155S proteins in
vivo at 37 °C is likely to make them nonfunctional in DNA
binding, accounting for the observed phenotype. In contrast, a third
mutation, T158M, which has been found repeatedly in Rett syndrome cases
(10, 13, 14, 16, 18), had near-wild-type affinity for the methylated
oligonucleotide (Fig. 4; Table II). This has also been shown in the
context of Xenopus MeCP2 (25) and is perhaps unsurprising as
the Thr-158 side chain is on the opposite side of the domain from the
DNA binding face and has no obvious role in either structure or
function. Instead, Thr-158 may be involved in interactions between the
MBD and other domains of MeCP2 or other proteins, which could affect
the function of the intact protein.
In this study we have investigated those amino acid side chains
within the previously suggested DNA binding face of the MeCP2 MBD that
could make an important contribution to the recognition of methylated
DNA. It is apparent from our EMSA assays that the arginine residue 111, which is absolutely conserved in the MBD family, plays a critical role
in DNA binding, as its mutation results in an MBD which, although still
structured, has no detectable affinity for DNA. Arg-111 appears to be
involved in an interaction with the conserved aspartate side chain
Asp-121, which is also very sensitive to mutation, and as was
previously speculated (19), this interaction may orientate the arginine
functional group so that it is in the correct position to bind DNA.
Given the severe effect of mutating Arg-111, it is likely that its DNA
interaction is a specific one involving a guanine in the methylated CpG
recognition sequence rather than a nonspecific one with the phosphate
backbone. Recognition of guanine bases via a "buttressed arginine"
of this type is an important component of DNA binding by the zinc
fingers of the Zif268 protein (26). In contrast, mutation of
Arg-133 to cysteine, found to occur in cases of Rett syndrome, produced an MBD with low but detectable affinity for methylated DNA, whereas a
glycine substitution at position 133 had only a mild effect on DNA binding.
We have also defined an important role for flexibility in the
disordered B-C loop region in DNA binding. Although several mutations
within the loop region do not affect binding, a G114P mutation, which
is predicted to restrict the conformational flexibility in this region
of the protein, reduces its affinity for DNA substantially. The
mutation is seen by NMR spectroscopy to affect the environment of the
loop as expected but to leave the overall structure of the MBD
unaltered. This is consistent with the idea that the B-C loop may move
to fit into the major groove of the DNA as the protein binds. One
residue that may be brought into position by this movement is the
critical side chain of Arg-111, although the restriction of movement in
the loop does not have such a severe effect as the removal of the
arginine, since the G114P mutant still has affinity for DNA at protein
concentrations of greater than 100 nM. It is more likely
that a general nonspecific interaction is made with DNA by positive
charges in the loop region such as Arg-115 and Lys-119, whereas Arg-111
has a more specific role to play.
The hydrophobic residues exposed on the DNA binding face, Tyr-123,
Ile-125, and Ala-131 in MeCP2, have been shown to contribute to the
affinity of binding to methylated DNA. However, in contrast to
predictions from the original MBD-DNA interaction model (19, 20), their
individual contributions appear to be fairly weak, and specificity for
methylated DNA is retained when they are removed. More severe effects
on binding can be obtained when negative charges, which will repel the
phosphate backbone, are introduced at these positions (the Y123D and
A131E mutations), although binding is still not completely abolished.
This suggests that specificity for the exposed methyl groups in the
major groove of the DNA may be a result of additive hydrophobic
interactions within the protein-DNA interface. Given this and the
observation that a charged residue, Arg-111, seems to be more critical
for DNA binding, it is also possible that the aliphatic portion of the
arginine side chain may contribute directly to recognition of the
methylated base.
Finally, we have considered the effects that Rett syndrome-associated
missense mutations within the MBD have on its DNA binding ability. In
general, these were found to lead to a reduction in binding affinity,
but in only one case was this a direct effect of mutating a residue
implicated in protein-DNA interactions. This mutation, R133C, is unique
among the Rett syndrome mutations tested (Figs. 7 and 8) in affecting
binding without any structural effect on the domain. A study of the
R133C mutation in the context of Xenopus MeCP2 has suggested
that it may cause an altered protein structure (25), but the circular
dichroism analysis used in that study does not permit a precise
determination of structural changes. In contrast, our NMR analysis of
the R133C mutant MBD shows very limited changes in the chemical shifts
of backbone amides throughout the domain, indicating that it is
structurally virtually identical to the wild-type protein. Unlike
R133C, the R106W and F155S mutations both affect the structure and
stability of the domain, causing it to unfold under NMR conditions, as
expected for side chains that contribute to the hydrophobic core.
However, F155S in particular retained DNA binding activity in the EMSA assay, indicating that the domain is not completely unfolded under these conditions. The S134A mutation, analogous to S134C found in a
Rett syndrome case, also caused a reduction in DNA binding affinity,
but a fifth mutation, T158M, which has been found in several Rett
patients, did not seem to affect DNA binding affinity significantly.
The most likely explanation for the Rett syndrome phenotype of this
mutation is that the Thr-158 side chain, which is on the opposite side
of the MBD domain from the DNA binding surface, is important for
interactions between this domain and the rest of the intact MeCP2 protein.
Since this study commenced, several other Rett syndrome-associated
mutations have been found within the MBD. These include changes at two
proline residues, 101 (to Thr, His, or Leu) and 152 (to Arg) (13, 15,
16), which probably have structural effects on the domain given their
locations between the A and B strands and at the terminus of the
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet on one face of the wedge and a C-terminal
-helix and small turn on the other face. At the thin end of the wedge, a large, flexible loop region (the B-C loop) between the second
and third
-strands appears, together with the exposed faces of the
second, third, and fourth
-strands (strands B, C, and D), to form a
binding surface for methylated DNA. Based on this information and the
surface of the DNA binding region mapped by NMR studies, a model was
proposed in which a conserved solvent-accessible hydrophobic patch
composed of the side chains of Tyr-123, Ile-125, and possibly Ala-131
might recognize the methyl groups of the methylated cytosine residues
in the major groove (19). A second NMR study on the MBD of the related
repressor MBD1 (20) revealed a highly similar fold, suggesting that all
the MBD-containing proteins are likely to recognize DNA in an
equivalent manner. These authors also proposed a similar model for DNA
recognition and confirmed that mutations in the conserved tyrosine and
charged side chains within the binding face affected methyl-DNA
recognition in mobility shift assays.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DE3)/pLysS was
diluted 1:100 into 1 liter of LB medium and grown at 37 °C to an
A600 of 0.5, whereupon the cells were induced
with 0.5 mM isopropyl-
-D-thiogalactoside and
grown for 3 h more. Cells were harvested, resuspended in 50 mM HEPES, 0.1 M NaCl, harvested again, and
resuspended in binding buffer (5 mM imidazole, 20 mM Tris-HCl, pH 8.0, 0.25 M NaCl, 10%
glycerol, 0.1% Triton X-100, 10 mM
-mercaptoethanol).
Cells were lysed by sonication, and debris was removed by
centrifugation at 16,000 rpm for 20 min. Clarified supernatants were
loaded onto 10 ml nickel-nitrilotriacetic acid Superflow-agarose
columns (Qiagen) equilibrated with binding buffer, washed with 2 × 10 ml binding buffer plus 30 mM imidazole, and eluted
with 5 ml of binding buffer plus 0.5 M imidazole. Elution
fractions were loaded onto 10 ml Fractogel EMD
SO32
650(M) columns (Merck),
washed with 2 × 10 ml binding buffer, and step-eluted with 5 ml
of binding buffer plus 0.25 M NaCl followed by 5 ml of
binding buffer plus 0.5 M NaCl; the majority of the protein
eluted in the first of these fractions. The pooled eluates were
dialyzed against 2 liters of 100 mM NaCl, 10% glycerol,
0.1% Triton X-100, and the resulting material was >95% pure as
judged by SDS-polyacrylamide gel electrophoresis. Proteins were
quantified by the Bradford assay kit (Bio-Rad). For the production of
labeled protein for NMR spectroscopy, cells were grown instead in M9
medium supplemented with 0.4% (w/v) glucose and 1.5 g/liter
[15N](NH4)2SO4 and
induced as above. Protein from the harvested cells was purified as
above, except that binding buffer lacking both Triton X-100 and
imidazole was used. The eluates from the Fractogel column were
buffer-exchanged into 50 mM sodium phosphate, pH 6.0, 50 mM NaCl and concentrated to a final concentration of
1
mM.
Av = ([
HN2 + (
N/5)2]1/2), where
Av is the
weighted average shift difference, and
HN and
N are
the differences in ppm between wild-type and mutant chemical shifts.
The ratio of 15N heteronuclear nuclear Overhauser effects
obtained from experiments with a 3.01-ms saturation pulse and without a
saturation pulse was used to probe the backbone dynamics of the G114P
MBD mutant (24).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Mutations in the MBD of MeCP2 and DNA binding abilities of the mutants
25-fold weaker than
that of the wild-type MBD, which showed 10% binding at
5
nM protein under these conditions. Thus it appears that
flexibility in the B-C loop region is a critical component of DNA
recognition by the MBD, possibly reflecting the ability of this region
to fit into the major groove of DNA and make multiple contacts with the phosphate backbone (19). Two-dimensional 15N-HSQC NMR
analysis of the structure of the G114P mutant indicated major changes
in the loop region from residues 110-121, consistent with the
structural constraint of this loop as a result of the mutation (Fig.
1B). In contrast, the structure of the rest of the MBD
remained unaffected, indicating that the overall fold remained intact
as predicted. However, analysis of the backbone dynamics of the loop
region in the G114P mutant revealed that it is still relatively
disordered (Fig. 1C), suggesting that the proline
substitution precludes specific conformations rather than imposing a
general reduction in flexibility. It is also possible that the proline
substitution introduces a steric clash with the DNA in the mutant.
Either possibility is, however, inconsistent with "docking models"
that show a lack of close contact between the B-C loop and the target
DNA (20, 25). Instead, we suggest that an induced conformational change
in this region of the protein upon DNA interaction contributes to the
binding affinity.
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Fig. 1.
Effects of the G114P mutation on DNA binding
and structure of the MBD. A, binding of the wild type
(wt) MeCP2 MBD and its G114P mutant derivative to a
methylated 27-mer duplex oligonucleotide (see "Experimental
Procedures") measured by EMSA. Concentrations of the proteins used
were 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, and 128.0 nM. The EMSAs from which the binding curves were derived
are shown to the right of the graph. B, chemical shift
perturbation for the backbone amides of the G114P mutant compared with
wild type as measured by two-dimensional 15N-HSQC NMR
spectroscopy. Significant perturbations are those greater than 0.04 ppm, as indicated by the horizontal dotted line. The G114P
mutation is indicated by the vertical dashed line,
and a schematic of the regular secondary structural features of the MBD
is shown above the graph (arrow, -strand;
coil,
-helix). The backbone amide of Arg-115 could not be
unambiguously assigned in this spectrum. C, value of
1H,15N-NOE ratio as a function of residue
number for the G114P mutant. Significant disorder is indicated by
values below the horizontal line (0.6). A schematic of the regular
secondary structural features of the MBD is shown above the graph
(arrow,
-strand; coil,
-helix).
atoms were significantly perturbed by the D121A mutation and moved from
an abnormal chemical shift to a position in the spectrum occupied by
the majority of arginine side chain H
atoms (Fig.
2B). This suggests that the primary interaction of Asp-121
is with Arg-111 and that the effect of the D121A and D121E mutations on
DNA binding may largely be due to the abolition of this interaction
given the critical role of Arg-111 in DNA binding (see below).
It seems probable that the Asp-121-Arg-111 interaction positions the
basic arginine side chain so that it can interact correctly with the
DNA target.
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Fig. 2.
Structural effects of the D121A
mutation. A, chemical shift perturbation for the
backbone amides of the D121A mutant compared with wild type as measured
by two-dimensional 15N-HSQC NMR spectroscopy. Significant
perturbations are those greater than 0.04 ppm, as indicated by the
horizontal dotted line. Amides that could not be
unambiguously assigned in the spectrum of the mutant protein are
indicated by asterisks, and the D121A mutation is indicated
by the vertical dotted line. A schematic of the
regular secondary structural features of the MBD is shown above the
graph (arrow, -strand; coil,
-helix).
B, a portion of the two-dimensional 15N-HSQC NMR
spectrum for the D121A mutant overlaid with that of the wild-type MBD.
Backbone amides are indicated for the wild-type (blue) and
mutant (red), and arginine H
atoms are
indicated for the wild-type (magenta) and mutant
(green). The large chemical shift change for the Arg-111
H
atoms caused by the D121A mutation is indicated.
resin during purification (data
not shown). These are the most severe phenotypes seen for any single
MBD missense mutation and indicate that the positive charge provided by
the Arg-111 side chain is crucial for the interaction of the domain
with both DNA and the negatively charged resin. It also suggests that
the reduced binding affinities seen for the D121A and G114P mutants
(above) may in part be due to their disruptive effects on the
environment of this arginine side chain. A second arginine side chain,
that of Arg-133, also projects from the DNA binding face of the domain, is conserved in the MBD family, and undergoes a large chemical shift
change upon DNA binding (19). This latter residue is found mutated (to
cysteine) in a case of Rett syndrome (10). We made a mutant MBD
containing the R133C mutation (Table II) and compared its effect on
binding to that of R111G. Unlike the Arg-111 mutant, R133C showed weak
binding to methylated DNA at a concentration of 2 µM
(Fig. 3A) and had minimal effects on the overall structure of the domain (Fig. 3C). Also unlike the R111G mutation, the
R133C mutation did not affect the chemical shift of the Asp-121
backbone amide, confirming that Asp-121 interacts with Arg-111 rather
than Arg-133. We also observed that mutation of Arg-133 to glycine had
a less significant effect on binding than the cysteine substitution at
this position (Table I), suggesting that the positive charge of this
arginine side chain does not contribute substantially to the DNA
interaction. Thus, although the reduced binding affinity of the R133C
mutant probably accounts for its phenotype as manifested in Rett
syndrome, this side chain is not of comparable importance to Arg-111 in
binding DNA.
DNA binding by arginine, hydrophobic patch, and Rett
syndrome-associated mutants of the MeCP2 MBD
View larger version (47K):
[in a new window]
Fig. 3.
Conserved arginine and hydrophobic side
chains are important for DNA binding by the MBD. A,
binding of the R111G, Y123A, I125A, and R133C mutants to the methylated
27-mer, as measured by EMSA. Concentrations of the proteins in
nM are shown above the panel. B,
chemical shift perturbation for the backbone amides of the R111G mutant
compared with wild-type as measured by two-dimensional
15N-HSQC NMR spectroscopy. Significant perturbations are
those greater than 0.04 ppm, as indicated by the horizontal
dotted line. Amides that could not be unambiguously assigned in
the spectrum of the mutant protein are indicated by
asterisks, and the R111G mutation is indicated by the
vertical dotted line. A schematic of the regular
secondary structural features of the MBD is shown above the graph
(arrow, -strand; coil,
-helix). C, chemical
shift perturbation for the backbone amides of the R133C mutant compared
with wild type as measured by two-dimensional 15N-HSQC NMR
spectroscopy. Significant perturbations are those greater than 0.04 ppm, as indicated by the horizontal dotted line. Amides that
could not be unambiguously assigned in the spectrum of the mutant
protein are indicated by asterisks, and the R133C mutation
is indicated by the vertical dotted line. A
schematic of the regular secondary structural features of the MBD is
shown above the graph (arrow,
-strand; coil,
-helix).
View larger version (73K):
[in a new window]
Fig. 4.
Rett syndrome-associated mutations within the
MBD affect DNA binding. Binding of the R106W, F155S, and T158M
mutants to the methylated 27-mer as measured by EMSA compared with the
wild-type (wt). Concentrations of the proteins in
nM are shown above the panel. C,
complex; F, free DNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix, respectively. Subtle aspartate to glutamate mutations at
positions 97 (within strand A) and 156 (at the C terminus of the
domain) have also been found in Rett patients (13, 18); although the
latter residue is well conserved in the MBD family, neither mutation is
predicted to have a particularly severe effect on the properties of the domain itself. In contrast to previous statements (13), Asp-156 is not
implicated in any way in DNA binding. Instead, the interfaces between
the MBD and other domains of the protein (or other proteins) could be
affected by the D97E and D156E mutations. It seems that in general, the
missense mutations found in Rett syndrome may result in structural
changes in the domain rather than specifically targeting side chains
involved in DNA recognition, the exception being the R133C mutation.
However, it must be remembered that the mutation frequency at different
sites within the DNA encoding the MBD probably varies widely, with many
of the mutations identified so far being at highly mutable CpG sites.
It would be interesting to determine the phenotypes caused by some of
the DNA-binding site mutations constructed in this study, such as the
loop mutation G114P and the severe R111G change, in an in
vivo model system.
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ACKNOWLEDGEMENTS |
---|
We thank Dusan Uhrin for assistance with NMR spectroscopy, Vicky Clark and Helen Barr for DNA sequencing, Aileen Greig and Joan Davidson for technical assistance, and Megan Porter and Padraig Deighan for insightful comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by Wellcome Trust (United Kingdom) Advanced Training Fellowship 052284/Z/97/Z (to A. F.) and by the Edinburgh Center for Protein Technology, which is funded by the United Kingdom Biotechnology and Biological Sciences Research Council and Department of Trade and Industry.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.
To whom correspondence should be addressed: Institute of Cell and
Molecular Biology, University of Edinburgh, Swann Bldg., King's
Bldgs., Mayfield Rd., Edinburgh, EH9 3JR, UK. Tel.: 44-131-6507079; Fax: 44-131-6505379; E-mail: andrew.free@ed.ac.uk.
¶ This author holds a Medical Research Council (United Kingdom) Career Development Award.
This author was the recipient of a University Research
Fellowship from the Royal Society. Present address: Dept. of Chemistry, University of Edinburgh, King's Bldgs., Mayfield Rd., Edinburgh, EH9
3JJ, UK.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007224200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MBD, methyl-CpG binding domain; HSQC, heteronuclear spin quantum correlation; EMSA, electrophoretic mobility shift assay.
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