Tài liệu Báo cáo khoa học: Solution structure of the matrix attachment region-binding domain of chicken MeCP2 ppt

Solution structure of the matrix attachment region-binding domain
of chicken MeCP2
Bjo¨ rn Heitmann
1
, Till Maurer
1
, Joachim M. Weitzel
2
, Wolf H. Stra¨ tling
2
, Hans Robert Kalbitzer
1
and Eike Brunner
1
1
Institut fu
¨
r Biophysik und physikalische Biochemie, Universita
¨
t Regensburg, Germany;
2
Institut fu
¨
r Medizinische Biochemie und
Molekularbiologie, Universita
¨
tsklinikum Hamburg-Eppendorf, Germany
Methyl-CpG-binding protein 2 (MeCP2) is a multifunc-
tional protein involved in chromatin organization and
silencing of methylated DNA. MAR-BD, a 125-amino-
acid residue domain of chicken MeCP2 (cMeCP2, origin-

ally named ARBP), is the minimal protein fragment
required to recognize MAR elements and mouse satellite
DNA. Here we report the solution structure of MAR-BD
as determined by multidimensional heteronuclear NMR
spectroscopy. The global fold of this domain is very simi-
lar to that of rat MeCP2 MBD and MBD1 MBD (the
methyl-CpG-binding domains of rat MeCP2 and methyl-
CpG-binding domain protein 1, respectively), exhibiting a
three-stranded antiparallel b-sheet and an a-helix a
1
.
We show that the C-terminal portion of MAR-BD also
contains an amphipathic helical coil, a
2
/a
3
. The hydrophilic
residues of this coil form a surface opposite the DNA
interface, available for interactions with other domains of
MeCP2 or other proteins. Spectroscopic studies of the
complex formed by MAR-BD and a 15-bp fragment of a
high-affinity binding site from mouse satellite DNA indi-
cates that the coil is also involved in proteinÆDNA inter-
actions. These studies provide a basis for discussion of the
consequences of six missense mutations within the helical
coil found in Rett syndrome cases.
Keywords: chicken methyl-CpG-binding protein 2
(cMeCP2); MAR-binding protein (ARBP); NMR spectro-
scopy; proteinÆDNA interaction.
Methylation of the DNA at cytosines in the dinucleotide

sequence CpG plays an important role in the regulation of
gene expression and imprinting as well as during develop-
ment. The information laid down in the methylation pat-
tern is read by a family of methyl-CpG-binding proteins:
MeCP2, MBD1, MBD2, MBD3, and MBD4 [1]. The
founding member of this family is MeCP2, methyl-CpG-
binding protein 2. Rat MeCP2 was identified through its
ability to recognize methylated DNA [2], and the chicken
homolog (originally named ARBP) was identified through
its ability to bind MAR elements, the putative bases of
chromatin loop domains [3]. MeCP2 acts as a transcrip-
tional repressor [4] and exerts this function through
interaction with the corepressor mSin3A and targeting of
histone deacetylases to methylated DNA [5]. An additional
histone deacetylase-independent mode of repression may
operate for a distinct set of promoters [6,7]. Targeting of
histone deacetylases is also involved in transcriptional
repression by MBD1 [8]. MBD3 is a component of a
multisubunit remodeling complex, NuRD, containing his-
tone deacetylase activities [9]. MBD2 interacts with the
NuRD complex and directs it to methylated DNA.
MeCP2 is expressed in all tissues of the human body and,
at particularly high levels, in neurons of the postnatal brain
[10,11]. This observation is in line with the fact that
mutations in the MECP2 gene cause Rett syndrome, an
X-linked, dominant neurological disorder that is one of the
most common causes of mental retardation in females [12].
At 6–18 months of age, affected girls gradually lose any
acquired speech and purposeful hand use. They also suffer
from microcephaly, severe mental retardation, autistic

behavior, seizures, gait apraxia, and breathing abnormali-
ties. Studies on transgenic mice that mimic the Rett
phenotype indicate that MeCP2 is required for the main-
tenance of neuronal physiology rather than brain develop-
ment [13,14].
MeCP2 is an abundant component of the pericentromeric
heterochromatin of mouse chromosomes [2]. In methylated
murine major satellite DNA, MeCP2 recognizes in vitro two
sites (I and II) with high affinity: K
d
¼ (2.2–5.7) · 10
)10
M
[15]. In nonmethylated satellite DNA, MeCP2 binds
to these sites with slightly reduced affinity [K
d
¼
(6.2–13.2) · 10
)10
M
]. The DNA-binding region of MeCP2
is the most highly conserved portion of the protein. The
minimal sequence necessary to recognize methylated DNA
(named methyl-CpG-binding domain, MBD) comprises
Correspondence to E. Brunner, Institut fu
¨
r Biophysik und
physikalische Biochemie, Universita
¨
t Regensburg,

D-93040 Regensburg, Germany.
Fax: + 49 941943 2479, Tel.: + 49 941943 2492,
E-mail:
Abbreviations: MeCP2, methyl-CpG-binding protein 2; cMeCP2,
chicken MeCP2; rMeCP2, rat MeCP2; MBD, methyl-CpG-binding
domain; MBD1, 2, 3, and 4, methyl-CpG-binding domain protein 1, 2,
3, and 4; MAR-BD, matrix attachment region-binding domain;
ARBP, attachment region-binding protein; mSin3A, a mammalian
corepressor protein interacting with MeCP2; NuRD, a multisubunit
complex including MBD3; HSQC, heteronuclear single-quantum
coherence; HBHA(CO)NH and CC(CO)NH, names of 3D
heteronuclear correlation NMR experiments.
(Received 1 April 2003, revised 4 June 2003,
accepted 10 June 2003)
Eur. J. Biochem. 270, 3263–3270 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03714.x
amino-acid residues 78–162 [16], while the minimal frag-
ment required to bind to a chicken MAR element (here
called MAR-binding domain, MAR-BD) encompasses
residues 71–195 of human MeCP2 [corresponding to
residues 72–196 in chicken (c)MeCP2] [15].
The solution structure of the MBD of rat (r)MeCP2 has
recently been determined [17]. The MBD adopts a wedge-
shaped structure, composed of an antiparallel b-sheet on
one face of the wedge and a three-turn a-helix with an
antiparallel one-turn helix on the other face. It is thought
that the two inner strands of the b-sheet lie within the major
groove of the DNA and that a hydrophobic pocket formed
by the side chains of Y123 and I125 contacts the methyl
groups of methylated CpG. The DNA interface further-
more contains several arginine and lysine side chains

forming hydrogen bonds with the bases and contacting
the DNA backbone. The solution structure of the MBD of
MBD1 shows high similarity to that of MeCP2 except for
the C-terminus [18]. At the C-terminus of the MBD, MeCP2
exhibits a one-turn helix, while MBD1 is folded into a
hairpin loop. The MBD of MBD1 contacts the methyl
groups of a methylated CpG through a hydrophobic patch
formed by the side chains of five residues, V20, R22, Y34,
R44, and S45 [19].
Although the solution structure of the core region of the
MBD of MeCP2 has been determined and some essentials
of its interaction with DNA are grossly understood, many
questions remain to be solved. In particular, the structure
and function of the sequences flanking the core region are
not known. The importance of these flanking sequences is
highlighted by several missense mutations causing Rett
syndrome. Interestingly, six mutations cluster in the region
C-terminal to the a-helix. Here we describe the solution
structure of MAR-BD and show that its C-terminal portion
contains an amphipathic helical coil, a
2
/a
3
. This helical coil
mainly contributes to the surface opposite to the DNA
interface, providing a platform for interactions with other
domains of MeCP2 or other proteins [19]. The consequences
of six missense mutations within the coil found in Rett
syndrome cases are discussed.
Experimental procedures

Sample preparation
15
N-labeled and
15
N/
13
C-labeled, His-tagged chicken
MeCP2
72-196
,namedMAR-BD,wasexpressedinEscheri-
chia coli BL21(DE3)pLysS from plasmid pET-cARBP-
Ex4.2 in isotope labeled Bio-Express media (Cambridge
Isotope Laboratories/Promochem, Wesel, Germany). The
labeling was nonspecific, i.e. no amino-acid-type selective
labeling was used. Purification of the protein on Ni
2+
/
agarose beads and on a Mono S FPLC column was
performed as described previously [20]. MAR-BD contains
the non-native sequence MGHHHHHH at its N-terminus.
NMR spectroscopy
NMR measurements on free MAR-BD, i.e. in the uncom-
plexed state, were performed at 298 K and pH 6.8 on
1–2 m
M
samplesinNSbuffer[10m
M
sodium phosphate
(46.3% Na
2

HPO
4
and 53.7% NaH
2
PO
4
), 0.5 m
M
NaN
3
,
with either 5% D
2
O or 100% D
2
O, and 0.1 m
M
2,2-
dimethyl-2-silapentanesulfonic acid] containing 154 m
M
NaCl. Complexes of MAR-BD with the unlabeled, non-
methylated, double-stranded oligonucleotide 5
0
-ATGACG
AAATCACTA-3
0
(MeCP2-binding site I in mouse satellite
DNA [15]) were generated by mixing the protein with the
oligonucleotide at molar ratios of 1 : 1.2 and 1 : 2.4 in NS
buffer containing 10 m

M
NaCl. NMR data were collected
on Bruker DRX-600 or DRX-800 NMR spectrometers
equipped with four channels, pulsed-field gradient triple-
resonance or quadruple-resonance probes with either z or
xyz gradients. The
1
H-NMR chemical shifts were referenced
using 2,2-dimethyl-2-silapentanesulfonic acid as an internal
standard. The chemical shifts of
15
N and
13
C were referenced
indirectly following the recommendations summarized in
[21]. In addition to the spectra recorded for sequential
assignment of the NMR signals of the backbone nuclei
described in [20], HCCH-TOCSY (9 ms mixing time),
HBHA(CO)NH, and CC(CO)NH spectra were measured
to further advance the extent of assignment of the side chain
nuclei. NOE distance restraints were obtained from 2D
NOESY and
13
C-edited and
15
N-edited 3D NOESY-HSQC
spectra measured in D
2
OandH
2

O, respectively, with a
mixing time of 150 ms, except for the
15
N-edited spectrum
(100 ms). These mixing times turned out to be optimal for
obtaining the maximum number of NOE contacts of
sufficient signal-to-noise ratio. NOESY spectra were
acquired with 1024 (complex) data points in direct dimen-
sion. For the 2D NOESY and 3D NOESY experiments,
1024 and 128 data points, respectively, were acquired in
the indirect
1
H dimension. In the indirect
13
Cand
15
N
dimension, 64 data points were acquired, and forward linear
prediction was used. Zero-filling was applied in the direct
and indirect spectral dimensions. ProteinÆDNA complexes
were analyzed by comparison of
1
H-
15
N HSQC spectra of
free MAR-BD with those of MAR-BD titrated with the 15-
mer oligonucleotide duplex at molar ratios of 1 : 1.2 and of
1 : 2.4.
Spectra analysis
NMR data were analysed and processed with the computer

programs
XWINNMR
,
AURELIA
[22], and
AUREMOL
[23]
(Bruker, Karlsruhe, Germany). In the final part of the
assignment of the NOESY spectra, the number of identified
NOEs was increased by comparison of back-calculated
spectra with the experimental data [24,25]. Based on a
preliminary structure, the NOESY spectra were simulated
using the relaxation matrix approach. Through comparison
with the corresponding experimental spectra, new NOE
restraints were obtained, which were used in subsequent
structure calculations. These newly calculated structures
were then used for the next step in the iteration process. This
procedure was continued until the quality of the structure
could not be further improved.
Structure calculations and analysis
Structure calculations were performed using the computer
program
DYANA
[26]. Distance information from NOEs was
included in the structure calculations assuming an error of
30%; 240 structures were calculated. The / and w angle
3264 B. Heitmann et al.(Eur. J. Biochem. 270) Ó FEBS 2003
restraints were obtained through a database search for
backbone chemical shifts and sequence homology using the
computer program

TALOS
[27]. Secondary structure ele-
ments and root mean square deviations (rmsd) were
determined using the program
MOLMOL
[28]. Rmsd values
were calculated for the best 10 structures with respect to the
value of the target function. Analysis of the / and w angles
of the nonglycine and nonproline residues was carried out
with the computer program
PROCHECK
-
NMR
[29]. The
secondary structure was calculated with the Kabsch-Sander
algorithm [30] implemented in the computer program
MOL-
MOL
. The atomic co-ordinates were deposited in the Protein
Data Bank under accession number PDB 1UB1.
Results and discussion
NMR signal assignment and secondary structure
Sequential NMR signal assignment of MAR-BD of
cMeCP2 has been described previously [20]. The relatively
high number of proline residues (>10%) and the fact that
only a core region of free MAR-BD appears to be well
folded made the assignment of the NMR signals difficult.
Some residues located in unfolded regions in the vicinity of
proline residues give rise to at least two sets of signals (with
one set having significantly higher intensities), indicating

slow exchange between different conformations on the
NMR time scale. Comparison of the
1
H
a
,
15
N,
13
C
a
,
13
C
b
,
and
13
C
0
chemical shifts with random coil values [31] and
the CSI plot [32], which is a consensus of the different
shifts, predicts a three-stranded b-sheet [strand b
1
, residues
104–110 (GWTRKLK); b
2
, residues 120–127 (KYDVY
LIN); b
3

, residues 131–135 (KAFRS)] immediately followed
by the three turns of an a-helix [a
1
, residues 136–145
(KVELIAYFEK); numbering refers to chicken MeCP2;
Fig. 1]. Interestingly, the
13
C
0
chemical shifts for the residues
in the vicinity of V160 show indications of an additional
helical structure. We therefore used the computer program
TALOS
[27] to further explore the secondary structure
elements. This program predicts / and w angle values on
the basis of a database search for chemical shifts of
backbone nuclei and sequence homology. The program
judges a prediction as ÔgoodÕ for (/,w) pairs, when nine or
10 matches occur with small dispersion of the angle values.
Besides the regions already shown to contain elements of
secondary structure by the CSI plot, two other regions are
predicted to exhibit secondary structure elements, namely
residues 96–101 and residues 152–163 (Fig. 2). Analysis of
the NOESY spectra reveals that residues 96–101 do not
show NOE contacts characteristic of b-sheet conformation
or other secondary structure elements [20]. In contrast,
residues 152–163 exhibit several NOE contacts indicative of
helical regions. This prediction will be further corroborated
below.
Tertiary structure

Structure calculations are based on a data set consisting of
891 different NMR-derived distance and torsion angle
restraints. Among the distance restraints, 447 intraresidual,
186 sequential, and 196 medium-range and long-range
restraints were found (Table 1). The number of NOE
contacts identified is plotted in Fig. 3A as a function of the
residue number. Obviously, the relatively high number of
NOE contacts (>10 per residue) commonly expected for
Fig. 1. Chemical shift analysis. Top, chemical shift index (CSI [32]) for
MAR-BD. Bottom, the
1
H,
15
N and
13
C chemical shift differences
(in 1 p.p.m.) relative to the random coil values [31] are given as a
function of the residue number.
Fig. 2. Database search for chemical shifts of backbone nuclei and
sequence homology. Residues judged as ÔgoodÕ by the computer program
TALOS
[27] and number of database matches for these residues indi-
cated by black bars. To keep the figure simple, the number of database
matches is not given for residues not judged as ÔgoodÕ by
TALOS
.
Ó FEBS 2003 Solution structure of chicken MeCP2 (Eur. J. Biochem. 270) 3265
structured regions is only observed for the central part of
cMeCP2 MAR-BD, i.e. for the region between residues 101
and 163. This indicates that the remaining N-terminal

and C-terminal regions of the domain are unstructured, as
already predicted by the chemical shift and
TALOS
analyses.
However, we note that the region between residues 152 and
163 is obviously structured, confirming our conclusions
drawn from the
TALOS
analysis (Fig. 2). A superimposition
of the best 10 structures with respect to the target function is
shown in Fig. 3B together with a ribbon plot of one selected
structure. As can be seen, the described secondary-structure
elements are well defined, as also indicated by the rmsd
values given in Table 1. Analysis of the Ramachandran plot
shows that the dihedral angles / and w for the secondary
structure elements are all found in the most favored or the
additionally allowed region. A selected example of the 10
structures shown in Fig. 3B is compared in Fig. 4 (middle)
with the structure of rMeCP2 MBD (left) and MBD1 MBD
(right) [17,18]. The global fold of these three domains turns
out to be identical, which is not surprising considering the
high degree of sequence similarity. The core of MAR-BD
consists of the above described three-stranded antiparallel
b-sheet followed by a-helix a
1
. For these secondary
structure elements, the Kabsch-Sander algorithm imple-
mented in the computer program
MOLMOL
always identifies

the following: b
1
, residues 106–110 (TRKLK); b
2
, residues
122–126 (DVYLI); b
3
, residues 132–133 (AF); and a
1
,
residues 136–144 (KVELIAYFE). Strands b
1
and b
2
are
separated by a flexible loop. The core of free MAR-BD is
hydrophobic, consisting mainly of residues T106, K108,
V123, L125, F133, L139, F143, F158, and T161. The
C-terminus of helix a
1
isfollowedbyanextendedloop
ending in a one-turn helix [a
2
, residues 153–155 (PND)].
After a short interruption by three residues, a third short
helix [a
3
, residues 159–163 (TVTGR)] could be identified.
This Ôhelical coilÕ, a
2

/a
3
, is arranged antiparallel to a
1
.The
N-terminal (72–100) and C-terminal residues (164–196)
exhibit a significantly reduced number of NOE contacts
(compare with Fig. 3A). This behavior is characteristic of
unfolded regions and agrees with the results of the chemical-
shift analysis (see above). Additional efforts were made to
confirm the helical structure of residues 153–155, as these
residues were not always shown to exhibit a helical structure
by the Kabsch-Sander algorithm implemented in the
computer program
MOLMOL
[28,30]. The / and w angles
of these residues, however, are clearly found in the region
characteristic of residues located in a-helices. This observa-
tion is made for all the other calculated structures, strongly
supporting the observation that residues 153–155 form a
short helix.
DNA-binding site
In murine metaphase chromosomes, MeCP2 preferentially
localizes to the pericentromeric regions containing highly
Table 1. Structural statistics and rmsd values.
Type of restraint Number
Total 891
Intraresidual NOEs 447
Sequential (i, i + 1) NOEs 186
Medium-range (i, i + j;1<j < 5) NOEs 65

Long-range (i, i + j;4<j) NOEs 131
Angle restraints
a
62
Atoms used for the calculation of root mean
square deviations rmsd/nm
Backbone atoms (N, C
a
, and C
0
) for residues 95–170 0.268
Heavy atoms for residues 95–170 0.329
Backbone (secondary structure elements, see text) 0.037
Heavy atoms (secondary structure elements, see text) 0.102
Ramachandran plot
analysis
% residues found
in this region
b
Most favored region 89.7 (58.3)
Additionally allowed region 10.3 (28.1)
Generously allowed region – (8.3)
Disallowed region – (5.2)
a
Using the computer program
TALOS
[27].
b
These data were
determined using the computer program

PROCHECK
-
NMR
[29]
taking into account only residues located in secondary structure
elements. The values determined for the entire molecule are given in
parentheses.
Fig. 3. NOE statistics and structure of cMeCP2 MAR-BD. (A)
Number of NOE contacts as a function of residue number. Filled bars,
intraresidual NOEs; dark grey bars, sequential NOEs; light grey
bars, medium-range NOEs (sequential distance 2–4 residues); white
bars, long-range NOEs (sequential distance >4). (B) Superimposition
of the best 10 structures with respect to the target function (see text)
and ribbon plot of one of these 10 structures.
3266 B. Heitmann et al.(Eur. J. Biochem. 270) Ó FEBS 2003
methylated satellite DNA [2]. Biochemical studies showed
thatMeCP2recognizestwosites(IandII)inmethylated
satellite DNA with high affinity (see above) [15]. Binding
to these sites in nonmethylated satellite DNA occurs with
slightly reduced affinity. When cMeCP2 MAR-BD was
complexed with methylated DNA, only poorly resolved
spectra could be obtained (data not shown). In contrast,
spectra of satisfactory resolution were acquired if a
nonmethylated 15-mer oligonucleotide duplex encompas-
sing site I was used (Fig. 5B). Distinct chemical shift
changes were observed for the signals in the
1
H-
15
N HSQC

spectrum on complex formation (Fig. 5A). To evaluate the
shift of a signal in
1
Hand
15
N dimension, we introduce the
term Ôtotal induced chemical shiftÕ, D
T
, defined as the sum
of the absolute values of the
1
H
N
and
15
N chemical shift
changes measured in Hz. Obviously, the entire molecule is
affected by DNA binding, as most of the residues exhibit a
total induced chemical shift, D
T
, far beyond the experimen-
tal error of  10 Hz. Strongly affected residues are located
in the loop between strands a
1
and a
2
, in helix a
1
,andinthe
helical coil a

2
/a
3
.
The involvement of residues located in the unfolded parts
of unbound MAR-BD is hard to predict, because a
considerable fraction of the NMR signals of those regions
could not be assigned in the spectra of the proteinÆDNA
complex. For such residues, the total induced chemical shift
plotted in Fig. 5A is a minimum value. This minimum value
was estimated from the difference between the chemical shift
observed for the free protein and that of the next nearest
unassigned signal. For a better understanding of this
procedure, a section of the
1
H-
15
N HSQC spectrum of free
MAR-BD (solid grey lines) is overlayed in Fig. 5B by the
corresponding section of the spectrum of MAR-BD com-
plexed with DNA (dashed black lines).
Biological and medical implications
The MAR-BD of MeCP2 extends the methyl-CpG-
binding domain (MBD) by seven N-terminal and 33
C-terminal residues [15]. Consequently, we found that the
core of MAR-BD folds into the same structure as the
core of MBD, i.e. an antiparallel three-stranded a-sheet
followed by an a-helix, a
1
[17]. This fold is also shared by

the MBD of MBD1 [18]. Moreover, the structure of
MAR-BD described here and the reported structure of its
MBD [17] coincide in possessing the short helix a
2
(residues P153, N154, and D155 in chicken MAR-BD).
This short helix is located at the side of the domain
opposite to the DNA-binding face with solvent exposed
residues N154 and D155. In addition to helix a
2
,wehave
identified a third helix, a
3
, in MAR-BD at T159 to R163.
Helices a
2
and a
3
are connected by a stretch of three
residues (F156, D157, and F158), which are conserved at
comparable positions among all the other members of the
MBD protein family [18]. The helical coil a
2
/a
3
is
amphipathic. P153 and G162 are buried in the protein
core. Also, F156, F158, and V160 are tightly packed into
the hydrophobic core of the domain. On the other hand,
residues D152, N154, D155, D157, and R163 are solvent
exposed and cluster in a small patch located opposite to

the DNA interface (Fig. 6). This patch is negatively
charged through three aspartic acids, but also contains
one positive charge through R163. It has been proposed
that the negative charges on the surface of the domain
Fig. 4. Comparison of cMeCP2 MAR-BD with rMeCP2 MBD and MBD1 MBD. Ribbon plots of rMeCP2 MBD [17] (left), the core region of
cMeCP2 MAR-BD (residues 95–170) (middle; this study), and MBD1 MBD [18] (right).
Ó FEBS 2003 Solution structure of chicken MeCP2 (Eur. J. Biochem. 270) 3267
opposite the DNA interface have a role in interactions
with another protein or with another domain within
MeCP2 [19,
1
33]. Notably, the C-terminal region of MAR-
BD differs significantly from that of MBD1 MBD. In
MBD1 MBD, helix a
1
is shortened by one turn, helix a
2
is lacking, and helix a
3
is replaced by a hairpin loop. First
of all, these differences and the corresponding amino-acid
changes generate a characteristic protein interaction site in
each of the domains. Secondly, they cause differences in
the mode of interaction with the periphery of the DNA
target site. For example, a lysine residue at the tip of the
hairpin loop that mediates a backbone contact with
DNA is unique in MBD1. In MAR-BD, the significant
chemical shift changes of the helical coil region found in
titration experiments with a mouse satellite DNA-derived
oligonucleotide duplex are noteworthy (Fig. 5A). They

probably indicate that the helical coil is also involved in
proteinÆDNA interactions. Chemical shift changes further-
more indicate that residues close to the N-terminus and
C-terminus also contribute to these interactions. In this
study, we used a nonmethylated high-affinity binding site
from mouse satellite DNA for complex formation with
MAR-BD [15]. As the chemical shifts obtained with this
DNA fragment closely resemble those obtained with a
methylated CpG sequence [17], our data corroborate pre-
vious findings that MeCP2 also recognizes nonmethylated
sequences [3,15,34].
Considerable interest in the structure of MeCP2 was
generated by the discovery that mutations in MECP2
cause Rett syndrome, an X-linked, dominant neurological
disorder primarily affecting young girls [12]. Intriguingly,
six missense mutations cluster in the helical coil a
2
/a
3
,
emphasizing the importance of this region: P153(152)R;
F156(155)I,S,C; D157(156)G,E; F158(157)I; T159(158)
M,A; and G162(161)R,W (here and in the discussion
below, human numbering is given in parentheses).
F156(155), D157(156), and F158(157) are conserved
Fig. 5. Effects of mouse satellite site I on chemical shifts of MAR-BD.
(A) Total induced chemical shift, D
T
, observed for complex formation
with a 15-mer oligonucleotide duplex from MeCP2 high-affinity

binding site I of mouse satellite DNA at a molar protein/DNA ratio of
1 : 2.4 vs. the residue number. The dotted line indicates D
T
¼ 100 Hz.
White bars denote residues where the signals could also be assigned
unambiguously for the proteinÆDNA complex. Black bars indicate
residues with ambiguous signal assignment for the proteinÆDNA
complex. In such cases, the minimum induced chemical shift, i.e. the
distance to the next nearest unassigned signal in the spectrum is shown.
Grey bars indicate proline residues. (B) Selected region from the
1
H-
15
N HSQC spectra of free MAR-BD (solid grey lines) and of the
proteinÆDNA complex (nonmethylated DNA, dashed black lines).
Fig. 6. Structural models depicting residues affected by DNA binding
and residues mutated in Rett syndrome cases. Ribbon plot of the
backbone of cMeCP2 MAR-BD for two orientations of the molecule
(left) and total induced chemical shift, D
T
, projected on the surface of
the MAR-BD (right). Blue, residues with D
T
<100 Hz.Red,residues
with D
T
> 100 Hz. Orange, residues with D
T
< 100 Hz but sequen-
tially neighbored to residues exhibiting D

T
> 100 Hz. Three residues
at the surface opposite the DNA interface (bottom) and mutated in
Rett syndrome cases (D98, D157, and T159) are marked by arrows
(left) and by white boxes (right).
3268 B. Heitmann et al.(Eur. J. Biochem. 270) Ó FEBS 2003
among all MBD-containing proteins, P153(152) and
T159(158) only in MeCP2 and MBD4, but G162(161)
solely occurs in MeCP2 [18]. The structure of the helical
coil a
2
/a
3
allows us to interpret the consequences of the
six mutations. As P153(152) and G162(161) are buried in
the protein core, replacement of each of these residues
with positively charged arginines [or the bulky tryptophan
in the case of G162(161)W] is predicted to generate gross
structural disturbance of the fold. Likewise, as the side
chains of F156(155) and F158(157) contribute to the
hydrophobic core of MAR-BD, their replacement with
isoleucine [or serine in the case of F156(155)S] probably
causes unfolding of the domain. In fact, the Rett mutation
F156(155)S has been previously shown to disrupt the
domain and to cause severe reduction of the binding
affinity to methylated DNA [33,35]. Residue F158(157) is
equivalent to F64 in MBD1; mutation of this residue,
F64A, has been shown to disrupt the tertiary structure of
the domain, resulting in total loss of binding to methy-
lated DNA [18].

Residue D157(156) is a Rett mutation site of considerable
interest, because its replacement with glutamic acid is only a
minimal change with conservation of the negative charge.
Continuing with the hypothesis that the negatively charged
surface opposite the DNA interface serves as a protein
interaction site, we have to infer that insertion of the small
methylene group by the D157(156)E mutation disrupts such
interactions. D98(97), which is located in close vicinity to
D157(156) (Fig. 6, bottom), is also mutated in Rett
syndrome cases. In one patient, D98(97) is replaced with
glutamic acid, reminiscent of the D157(156)E mutation
which causes the same minimal change. Thus, the negatively
charged surface critical for interactions with another
domain of MeCP2 or another protein probably includes
D98(97).
T159(158), another target residue at a Rett mutation
site, is located adjacent to D157(156) (Fig. 6, bottom). As
the putative interaction of D157(156) with another
protein or domain is disrupted by the insertion of a
methylene group, it follows that replacement of the
neighboring T159(158) with alanine or methionine may
compromise this interaction as well. Consistent with the
location of T159(158) at the surface opposite the DNA
interface, Rett mutation T159(158)M, the most common
mutation in MeCP2 [36], was previously shown to cause
little reduction in the affinity for methylated DNA
[33,35]. Collectively, these observations suggest that
D98(97), D157(156), and T159(158) form a region critical
for the interaction between MAR-BD and another
domain of MeCP2 or another protein (see Fig. 6,

bottom). Candidate interacting sequences within MeCP2
are evolutionarily conserved basic regions, such as
residues 249–271 (human numbering), which contains
the nuclear localization signal, and residues 284–309, the
terminal portion of the transcriptional repression domain
[6]. Intriguingly, several Rett missense mutations that
affect basic residues cluster in these two regions. Know-
ledge of the structure of a larger portion of MeCP2
including the transcriptional repression domain would
resolve these speculations and clarify the role of the
helical coil as well as of residues 164–195 in the DNA-
recognition process.
Acknowledgements
We thank Susanne Giehler for excellent technical assistance, and Ingrid
Cuno for carefully proofreading the manuscript. Financial support
from the Deutsche Forschungsgemeinschaft is gratefully acknowledged
(grants SFB 545-B2, Str145/12-3, and Br 1278/8).
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