From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307
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ABSTRACT |
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Z Many protein domains that recognize DNA in both sequence- and
conformation-specific manners have been characterized (for a review,
see Ref. 1). These studies have resulted in an understanding of the
variety of ways in which protein-DNA interactions can result in
function. Identification of a peptide motif, Z Although many properties of Z The ADAR family of enzymes converts adenosine to inosine within
double-stranded regions of RNA (11). In mRNA, inosine is read as
guanosine by the translation apparatus, resulting in codon changes
within the synthesized protein. A-to-I editing has been shown to occur
in vivo in a number of mRNAs from higher animals (12-18). The best characterized of these, the editing of pre-mRNAs for subunits of the glutamate-gated cation channels in the brain, results in channels with dramatically altered functional properties (19). Double-stranded RNA structures required for ADAR activity are
formed by base pairing of an exonic sequence around the editing site
with a complementary sequence in the downstream intron; therefore, editing must take place in the nucleus before splicing removes the
respective intron(s). It has been proposed that Z To better understand the role of Z ADAR1: DNA Constructs and Protein Purification--
Different
portions of the cloned cDNA coding for human ADAR1
(GenBankTM accession number U10439) were polymerase chain
reaction-amplified and inserted into the expression vector pET28a
(Novagen), resulting in N-terminal His6-tagged fusion
proteins. In detail, Za131 (residues 96-226), Za77 (133-209), and
Zab236 (133-368) were amplified using complementary primers flanked
with restriction sites at their termini. Polymerase chain reaction
products were analyzed on an agarose gel; bands of the correct size
were extracted and subcloned into the
NdeI-HindIII sites (Za131 and Za77) or the
NheI-HindIII sites (Zab236) of the multiple
cloning site of pET28a, resulting in the vectors pZa131, pZa77, and
pZa236, respectively. Another construct, Zab Limited Proteolysis--
Protease digestion was performed by
treating 50 µg of protein (0.5 µg/µl) with trypsin, chymotrypsin,
thermolysin, or Staphylococcus aureus endoproteinase Glu-C
in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM DTT at a protein/protease mass ratio in the range of
50:1 to 1000:1 for various times at 24 °C. Reactions were stopped by
heat denaturation at 100 °C for 5 min. To examine the effect of
various DNA conformers on Zab digestion, the reaction was performed in
10 mM HEPES (pH 7.5), 20 mM NaCl, 5 mM DTT, and 10 mM MgCl2. DNA was
used in a base pair/protein molar ratio of 5:1.
Poly[d(5-MeC-G)] was used as substrate DNA, and
poly[d(A-G)]·poly[d(C-T)] as unspecific DNA. The digests were
separated by SDS-PAGE on 18% gels, followed by staining with Coomassie
Brilliant Blue G-250. In the case of protein digested for the
experiment shown in Fig. 6 (lanes 10-13), the
reactions were stopped by adding phenylmethylsulfonyl fluoride (1 mM) instead of heat inactivation to ensure nondenatured protein.
Protein Sequence Analysis--
The proteolytic fragments were
analyzed by mass spectrometry on a Voyager DE Workstation (PerSeptive)
using matrix-assisted laser desorption ionization-time of flight
technology. As a matrix, sinapinic acid (10 µg/µl) in
acetonitrile/H2O/trifluoroacetic acid (70:29.9:0.1) was
used. Alternatively, for fragments smaller than 10-kDa, the matrix was
prepared with DNA Binding Assay--
DNA binding was assayed by native PAGE
(23). The assay was carried out using
d(5-BrC-G)20 as the substrate, which is stable
in the left-handed Z-DNA conformation under the applied conditions
(24). The substrate was end-labeled with 32P and purified
by native PAGE prior to the experiment. A reaction mixture of 10 µl
containing the ADAR1 fragment (4-500 nM) with <1
pM substrate in 10 mM Tris-HCl (pH 7.8), 20 mM NaCl, 5 mM DTT, 5% glycerol, 100 µg/ml
bovine serum albumin, and 50 µg/ml poly[d(A-G)]·poly[d(C-T)] (Amersham Pharmacia Biotech) as an unspecific competitor was incubated for 30 min at 24 °C. The mixture was analyzed on a 6% native
polyacrylamide gel using 0.5× Tris borate (22.5 mM) as the
running buffer. After electrophoresis (10 V/cm, 90 min), the gel was
dried and autoradiographed at -70 °C on Kodak X-Omat Blue film with
intensifying screens.
CD Measurements--
CD spectra were recorded at 24 °C on an
Aviv Model 62DS spectrometer. Conformational change in DNA oligomers
was monitored between 235 and 305 nm. DNA samples used were annealed
prior to the experiment. For this purpose, a concentrated solution of
the self-complementary sequence d(C-G)6 or an equimolar
amount of d(C-A)7 and d(T-G)7 was heated to
85 °C for 10 min and then slowly cooled to <20 °C over 1 h.
Measurements were carried out in 10 mM sodium phosphate (pH
7.0), 10 mM NaF, 1 mM EDTA, and 2 mM DTT using a DNA concentration of 30.0 µM
base pairs and an optical path length of 5 mm. Spectra were recorded in
10-nm steps and averaged over 4 s. Protein was added to the sample
from a concentrated stock solution, in aliquots never exceeding 5% of
the total volume, and the mixture was equilibrated for 5 min before
each measurement. The spectra were corrected for buffer base line and
smoothed using software provided by Aviv. Protein spectra were recorded
between 190 and 250 nm. Za77 was measured at a concentration of 10.0 µM, and Zab
For comparisons of the spectra of Zab between 190 and 250 nm in the
presence and absence of substrate, poly[d(5-MeC-G)] was
used as substrate. A 2:1 base pair/protein molar ratio was used.
Defining the Boundaries of the Minimal Z-DNA-binding Domain of
Human ADAR1--
Protein domains are usually well structured regions
of 50-200 amino acids (25, 26). Larger proteins are built from
multiple, mostly independently folded domains. The regions connecting
those domains are often flexible and solvent-exposed. Limited
proteolysis is a classical approach to define domain organization
(27-30). It takes advantage of the fact that site-specific proteases
will cleave proteins preferentially in solvent-exposed unstructured regions, rather than within a folded domain.
Limited proteolysis was used to define a structured core containing
Z
An N- and C-terminally extended portion of the ADAR1 N terminus
comprising Gly96-Ser226 was overproduced as a
His6-tagged fusion protein in E. coli, and its
digestion with four different proteases (endoproteinase Glu-C,
chymotrypsin, thermolysin, and trypsin) was analyzed. Each of these
enzymes has a different sequence specificity; therefore, using them in
concert results in complementary information. The use of this
combination of proteases results in an even distribution of potential
cleavage sites throughout the studied protein, with gaps no longer than
4 residues between adjacent sites. A time course of cleavage with
endoproteinase Glu-C is shown in Fig. 1A. An 11-kDa band appeared
rapidly and increased in intensity over the observed time; the
full-length protein band gradually disappeared over the same period.
The intensity of the 11-kDa band was comparable to that of the
full-length band, indicating a stoichiometric conversion to a stable
product. The cleavage site was mapped to a preferential endoproteinase
Glu-C site, C-terminal to Asp132, using N-terminal
sequencing. Similar results were obtained using trypsin and
chymotrypsin to cleave this protein (data not shown).
To ensure that a minimum domain had been identified, the protein was
cleaved sequentially with two different proteases. Fig. 1B
shows the digestion with endoproteinase Glu-C followed by chymotrypsin. Before addition of the second protease, only the 11-kDa band was detectable. Chymotrypsin further truncated the fragment, producing the
stable product V8/Ch-8. The N and C termini of these fragments were
identified unambiguously using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The V8-11 fragment was
shown to contain residues 133-226. Chymotrypsin cut after Trp204; V8/Ch-8 consists of residues 133-204. A similar
digestion, carried out with endoproteinase Glu-C and thermolysin,
produced a stable product extending from amino acids 133 to 209 (data
not shown). Other combinations of enzymes produced consistent results
in all cases. From this, we conclude that there is a core domain
containing Z
These results were used to design a stable construct, Za, comprising
Leu133-Gly209. This protein was purified from
E. coli undegraded under nondenaturing conditions. Za showed
superior chromatographic behavior over previous Z The Two Motifs, Z
The results of the digestion of Zab with four different proteases are
shown in Fig. 2. Each enzyme cleaved in a
characteristic pattern and produced a small number of very stable
bands. Time points were selected to allow the identification of all
stable products, using mass spectrometry and N-terminal sequencing
where appropriate; minor products were identified wherever possible. In
each case, well resolved spectra were recorded. Table
I lists the peptides produced by each
enzyme, as determined from the molecular mass. For chymotrypsin,
trypsin, and endoproteinase Glu-C, the assignments are unambiguous and
in good agreement with SDS-PAGE analysis. Minor exceptions are
fragments Tr8 and Ch5, which were detected only by mass spectrometry,
as discussed below. In the case of thermolysin, it was not possible to
unambiguously assign the multiple transitory fragments; however, the
major fragments seen after 60 min of digestion could be identified. A
schematic diagram of the major transitory products and the stable
proteolytic fragments is shown in Fig.
3.
Endoproteinase Glu-C cleaved Zab rapidly at a single site,
Glu361 at the extreme C terminus (Fig. 2A). The
resulting peptide was very stable to further proteolysis, despite an
abundance of potential cleavage sites, including Asp132,
which is exquisitely sensitive in the shorter construct used to define
Za, as described above. After a long incubation with large amounts of
enzyme, additional cleavage occurred at Glu239,
Glu301, and Leu307. Glu239 lies
within the first 49-amino acid repeat; remarkably, the equivalent site
in the second repeat, Glu288, was uncut.
Chymotrypsin cleaved the protein after Trp204 and
Trp253. These sites are at equivalent positions in the two
copies of the tandem repeat. The three generated fragments were stable
(Fig. 2B). The 5-kDa fragment was not visible on
SDS-polyacrylamide gel, although it generated a signal in mass
spectrometry of comparable intensity to Ch12 and Ch8. Coomassie Blue
staining depends largely on positive charges present in the peptide
(33). The 49-amino acid repeat contains only 1 positively charged
residue. Therefore, we speculated that although Ch5 was resolved on the
gel, it was not stained. Two other transitory fragments, Ch18 and Ch*,
were separated on the gel. Ch18 could be assigned to be the product of
a single cutting site. Ch* could not be unambiguously determined by
mass spectrometry.
Thermolysin produced similar stable products (Fig. 2C).
Again, symmetrical sites in the repeated linker, Gly209 and
Gly258, were cut, resulting in two stable products on an
SDS-polyacrylamide gel. Because thermolysin has low sequence
specificity, many transitory products were seen, especially at early
time points. Because of these products, it was not possible to
unambiguously identify the Tl5 fragment from among several candidates
seen by mass spectrometry.
Trypsin attacked the protein at two preferred sites, Arg232
in the first repeat and Lys302 near the N terminus of
Z One of the Two 49-Amino Acid Repeats Can Be Removed without
Destabilizing the Domain--
In all the ADAR1 genes
sequenced, there is a single copy of a 43-49-amino acid linker module
between Z Zab Consists of Two Ends with Regular Secondary Structure,
Connected by an Unconventionally Folded Linker--
Circular dichroic
measurements in the region between 190 and 250 nm are a useful tool to
assess the secondary structure of a protein. This method was used to
analyze Za, Zab, and Zab Zab Is Protected from Proteolysis When Bound to Its
Substrate--
The presence of substrate can affect the protease
sensitivity of a protein either because of a direct interference by the substrate molecule or by altering the conformation of the protein. To
test whether this is the case for Zab, protease digestions were carried
out in the presence of either B-DNA or Z-DNA. Although there were no
dramatic changes in the digestion profiles, B-DNA stabilized Zab
slightly against proteolysis, and Z-DNA had a very marked stabilizing
effect. Chymotrypsin, thermolysin, and trypsin all cut at their
established sites, but to an ~50-fold lower extent in the presence of
Z-DNA (data not shown).
Binding to Z-DNA offered striking protection against digestion with
endoproteinase Glu-C (Fig. 5). Although
there was no protection of the C-terminal site (Glu361),
the internal cleavage sites were strongly protected. Cleavage sites at
residues 301 and 307 were completely protected in the presence of
Z-DNA, resulting in the absence of the V20 and V19 bands. Cleavage at
residue 239 was reduced, with an ~50-fold increase in the stability
of the full-length Zab protein relative to the absence of DNA. In
contrast, B-DNA protected Zab against cleavage only ~5-fold and did
not alter the choice of sites.
These results suggest that the entire domain becomes more rigid and
less accessible in the presence of substrate. The protection of sites
within Z
When Zab in the presence and absence of Z-DNA was compared, there was
no change in the CD spectra between 190 and 250 nm (data not shown).
This indicates that there is no major change in the secondary structure
of the protein when substrate is bound.
The Intact Zab Domain Forms a Stable Complex with Z-DNA and
Binds with Sequence Preference--
The binding of Z
These results may indicate that binding of the
Z
As a second method of studying the binding of Z
Za, or any peptide containing Z Although the exact biological role of ADAR1 has not been
established, analysis of the amino acid sequence has allowed the assignment of functions to parts of the protein (38). These functions
contribute to the known in vitro activity of this enzyme, the conversion of A to I in regions of double-stranded RNA. The central
series of double-stranded RNA-binding motifs and the C-terminal catalytic domain are well characterized protein domains. The N-terminal region, on the other hand, contains a novel motif, Z The latter hypothesis was supported when we delineated the structural
domain Zab, containing both Z The overall structure of the Zab domain remains largely unchanged upon
binding to substrate DNA. Within the accuracy of the measurements, CD
spectra of Zab between 190 and 250 nm are identical in the presence or
absence of Z-DNA. Although there are many potential cleavage sites for
each protease throughout the protein, no new cuts are seen when the
protein-DNA complex is compared with protein alone. This result makes
it unlikely that major spatial reorientations take place within the
protein. The most striking effect of binding to Z-DNA is a marked
decrease in sensitivity to proteases. In particular, the endoproteinase
Glu-C sites within Z The hypothesis that Zab is a single domain with a single DNA-binding
site involving both Z is a peptide motif that binds to
Z-DNA with high affinity. This motif binds to alternating dC-dG
sequences stabilized in the Z-conformation by means of bromination or
supercoiling, but not to B-DNA. Z
is part of the
N-terminal region of double-stranded RNA adenosine deaminase
(ADAR1) , a candidate enzyme for nuclear pre-mRNA
editing in mammals. Z
is conserved in ADAR1 from many species; in each case, there is a second similar motif,
Z
, separated from Z
by a more divergent
linker. To investigate the structure-function relationship of
Z
, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Z
is
part of a domain, Zab, of 229 amino acids (residues 133-361 in human
ADAR1). This domain contains both Z
and Z
as well as a tandem repeat of a 49-amino acid linker module. Prolonged
proteolysis revealed a minimal core domain of 77 amino acids (positions
133-209), containing only Z
, which is sufficient to
bind left-handed Z-DNA; however, the substrate binding is strikingly
different from that of Zab. The second motif, Z
, retains
its structural integrity only in the context of Zab and does not bind
Z-DNA as a separate entity. These results suggest that Z
and Z
act as a single bipartite domain. In the presence
of substrate DNA, Zab becomes more resistant to proteases, suggesting
that it adopts a more rigid structure when bound to its substrate,
possibly with conformational changes in parts of the protein.
INTRODUCTION
Top
Abstract
Introduction
References
, which
binds specifically to Z-DNA, opens up a new vista and invites the
investigation of the similarities and differences between domains that
bind right- and left-handed DNAs. The conformation specificity of
Z
binding has been characterized in many ways. Peptides
including this motif bind to alternating dC-dG that has been stabilized in the Z-conformation using bromination or supercoiling, as shown by
band shift assays, competition experiments, and BIAcore measurements (2). When linked to the nuclease domain from FokI, the resulting chimeric nuclease cuts supercoiled plasmid DNA to bracket a
d(C-G)13 in the Z-conformation (3). The protein also binds
to short oligonucleotides of suitable sequence and converts them from
the B- to the Z-conformation, as detected by CD and Raman spectroscopy (4, 5). The binding of Z-DNA by Z
occurs even in the
presence of a 105-fold excess of B-DNA (6). Z
binds poly(dC-dG), stabilized in the Z-conformation by bromination,
with an equilibrium dissociation constant (Kd) in
the lower nanomolar range, as shown by BIAcore measurements (2).
have been studied, its
biological function in the context of ADAR1 remains unknown. The Z-DNA binding activity of Z
was first identified in proteolytic
fragments of double-stranded RNA adenosine deaminase (ADAR1) (6) and then in the full-length enzyme (7). Z
has been shown to
be a conserved feature of human, rat, bovine, chicken, and
Xenopus ADAR1 (2). A second related motif, Z
,
has been identified in all the ADAR1 enzymes whose sequences are known.
These two motifs are separated by a linker region of conserved size; an exception is the human enzyme, in which the linker is twice as long and
consists of two nearly identical copies of a module (8). The presence
of a conserved N-terminal region containing these motifs distinguishes
ADAR1 from other members of the ADAR family (9, 10), and the N terminus
has been shown to be differentially expressed (8). Therefore, we
conclude that this region is of importance for the biological function
of ADAR1.
serves to target ADAR1 to its preferred substrates by binding to Z-DNA formed
close to actively transcribing genes (20).
, we have characterized
the N-terminal region of ADAR1 functionally and structurally. Using human ADAR1 as a model, the classical approach of limited proteolysis was employed to define the boundaries of this domain. Both motifs, Z
and Z
, together are shown to form a
single functional domain, Zab; Zab is stable and protected from
proteolysis. Za, containing Z
, but not Z
,
can be regarded as a stable subdomain; this subdomain contributes the
main binding activity. There is no equivalent subdomain containing
Z
: this region is poorly structured and unstable when
isolated. The intervening linker region is unexpectedly well
structured, In humans, the second copy of the linker module appears to
have a structure similar to the first. Removing one copy of the linker
modules reduces the DNA binding affinity, indicating the importance of
the distance between the Z
and Z
motifs. Za binds Z-DNA in a conformationally specific, but not sequence-specific manner. The binding is modified by the presence of the entire Zab
domain to confer preference for d(C-G)n over
d(C-A)n·d(T-G)n.
EXPERIMENTAL PROCEDURES
l, missing one of the
two 49-amino acid linker modules separating the Z
and
Z
motifs, was created from pZab236 as follows. The
1.1-kilobase SphI-HindIII restriction fragment was digested with the restriction enzyme DrdI, resulting in
two cleavage sites at identical locations at nucleotides 789 and 936 (numbers according to GenBankTM accession number U10439).
The resulting DNA fragments were deproteinized and precipitated (21).
After incubation with T4 DNA ligase (25 °C, 4 h), the reaction
mixture was analyzed on an agarose gel. The 930-base pair ligation
product was isolated and subcloned in the 5-kilobase
SphI-HindIII restriction fragment of pET28a,
resulting in the vector pZab
l. To ensure that the plasmids were
correct, they were analyzed by restriction digestion, and the coding
regions were sequenced using Sequenase Version 2.0 (U. S. Biochemical
Corp.). according to the manufacturer's instructions. The proteins
were overproduced in Escherichia coli strain Novablue(DE3)
(Novagen). Bacteria were grown at 37 °C in Luria-Bertani medium and
induced with 1 mM
isopropyl-
-D-thiogalactopyranoside at 0.7-0.9
A600 nm units. Cells were harvested after a further 3 h of growth at 37 °C. All subsequent steps were done at 4 °C. The proteins were purified essentially to homogeneity under
nondenaturing conditions as follows. A cell pellet obtained from a
1-liter culture was resuspended in 15 ml of buffer A (50 mM
Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM
imidazole, 5 mM
-mercaptoethanol, 20 µg/ml RNase A,
and 100 µM phenylmethylsulfonyl fluoride), and the cells
were lysed using a French press. The lysate was then centrifuged for 30 min at 25,000 × g, and the clear supernatant was
separated and incubated with 2 ml of Ni2+-nitrilotriacetic
acid metal affinity resin (QIAGEN Inc.) for 1 h. The resin was
washed three times with 20 ml of buffer A in a batch and then washed
with 40 ml of buffer B (50 mM Tris-HCl (pH 8.0), 1 M NaCl, 10 mM imidazole, and 5 mM
-mercaptoethanol) in a column. Overproduced His6-tagged
fusion protein was eluted with an imidazole step gradient in buffer C
(50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 5 mM
-mercaptoethanol). Steps were 30, 50, and 200 mM imidazole, respectively. Fractions were analyzed by
denaturing SDS-polyacrylamide gel electrophoresis
(PAGE)1 on 15 or 18% gels.
Fractions containing protein were pooled and dialyzed against buffer D
(20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM dithiothreitol (DTT)). After 1 h of dialysis, 15 units of thrombin (Calbiochem) were added to cleave the N-terminal
His6 tag. 12 h later, the cleaved protein was dialyzed
against buffer E (20 mM HEPES (pH 7.5), 20 mM
NaCl, and 2 mM DTT) and finally purified by cation-exchange
chromatography on a Mono S HR5/5 column (Amersham Pharmacia Biotech).
Proteins were eluted with a 30-ml linear gradient of NaCl (0.05-0.3
M) in 20 mM HEPES (pH 7.5) and 1 mM
DTT at a flow rate of 0.7 ml/min, resulting in sharp peak profiles.
Za77 eluted at 220 mM NaCl, Zab
l at 200 mM,
and Zab236 at 180 mM. The yield of electrophoretically
homogeneous protein was determined using extinction coefficients of
14,000 M
1 cm
1 (Za77 and Za131),
22,400 M
1 cm
1 (Zab
l), and
28,020 M
1 cm
1 (Zab236) at the
absorbance maximum at 278 nm (calculated as described in Ref. 22).
8-12 mg of protein were obtained per liter of bacterial culture.
-cyanocinnamic acid (10 µg/µl) instead of
sinapinic acid. Various fragments were further analyzed by
amino-terminal sequencing on an Applied Biosystems Model 475/477A
protein sequencer.
l and Zab were measured at 5.0 µM and an optical path length of 1 mm. Spectra were
measured in 1-nm steps and averaged over 10 s.
RESULTS
, the Z-DNA-binding motif present in the N-terminal region of ADAR1. Z
has been defined to comprise residues
121-197 of human ADAR1 using functional assays (2). However, a variety of results from nondenaturing electrophoresis, chromatographic elution,
and NMR studies have suggested that the recombinantly produced peptide
is not stably
folded.2,3
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Fig. 1.
Limited proteolysis reveals a stable
Z core domain. A, a
Z
-containing peptide, comprising
Gly96-Ser226 of human ADAR1, was digested with
endoproteinase Glu-C (V8) at a protease/protein mass ratio
of 1:250 at room temperature. Reactions were stopped by heat
denaturation after the indicated incubation times. Samples were
resolved by SDS-PAGE (18% gels) and visualized by staining with
Coomassie Brilliant Blue. The V8-11 fragment resulted from a single
cleavage and comprises residues 133-226. B, the same
construct was digested consecutively with two site-specific proteases.
After preincubation with endoproteinase Glu-C (1:100 protease/protein)
for 1 h, chymotrypsin (1:250 protease/protein) was added, and the
samples were analyzed after the indicated reaction times. V8/Ch-8 is a
protease-insensitive core fragment containing Z
, which spans
residues 133-204 of human ADAR1. Lane M, molecular mass
markers.
. Trp204 is a potential target
for cleavage by both chymotrypsin and thermolysin. Chymotrypsin cut
well, but thermolysin cut only marginally at Trp204.
Therefore, we define the core domain as comprising
Leu133-Gly209. This core was in no case
significantly degraded, whereas the regions on either end were rapidly
degraded to pieces too small to detect.
constructs, purifying from a Mono S cation-exchange column homogeneously as a sharp peak; this indicated structural uniformity. Samples yielded a single band when analyzed by native
PAGE.4 When challenged with
exogenous proteases, only Za showed striking stability; other
Z
constructs were rapidly degraded (data not shown).
and Z
, Form a Single Structural
Entity--
Both Z
and Z
are present in
every species in which the sequence of ADAR1 is known. The motifs are
separated by one or two copies of a module, weakly conserved in
sequence, but consistently lacking positively charged residues and
43-49 amino acids in length. 12 residues from this module are an
essential part of Za, the stable Z
core domain. It seemed
possible that Z
, Z
, and the linker
module(s) together form a single structural and functional unit. To
investigate this possibility, we examined the structural organization
of a peptide spanning both DNA-binding motifs. This peptide, termed
Zab, comprising Leu133 (the previously defined N terminus
of Za) to Asn368 (C-terminal to Z
, from human
ADAR1), was soluble when overproduced in E. coli, and
full-length protein could be obtained with high yield. These results
indicate proper folding with no significant instability. Improper
folding often leads to the formation of inclusion bodies inside the
overproducing bacterial cell (31). Highly flexible proteins are
frequently degraded if expressed in a foreign host (32).
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Fig. 2.
Digestion of Zab with different
proteases. Zab (residues 133-368 from human ADAR1) was incubated
with endoproteinase Glu-C (V8) (A), chymotrypsin
(B), thermolysin (C), and trypsin (D)
at the indicated protease/protein mass ratios for the indicated times.
Fragments were resolved by SDS-PAGE (18% gels) and visualized by
staining with Coomassie Brilliant Blue. Arrows indicate
stable fragments, which where further analyzed by mass spectrometry.
The results of this analysis are shown in Table I. Lane M,
molecular mass markers.
Mass spectroscopic analysis of Zab236 fragments
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Fig. 3.
Structure and protease cleavage map of the
Z-DNA-binding domain of ADAR1. At the top is a schematic
representation of human ADAR1 (hADAR1). Below are the stable
fragments produced by limited proteolysis. Numbers above
ADAR1 are residue positions. The illustrations are proportional.
dsRNA, double-stranded RNA.
(Fig. 2D). (The site equivalent to Arg232 in the second repeat is Ser280, not a
substrate for trypsin.) Two sites near the C terminus, Lys366 and Arg367, resulted in heterogeneity of
the full-length protein and in the Tr15 fragment. Most of the expected
products were stable; however, the C-terminal region peptide, starting
at Ile303, was not detected by SDS-PAGE or mass
spectrometry. A similar result was seen after extensive endoproteinase
Glu-C digestion; again, the C-terminal fragment was not stable. It
appears that Z
, intrinsically more accessible than
Z
, is stable only in the context of the larger domain.
and Z
. In human ADAR1, this
module is repeated. To determine the effect of this repeat on the
structure of Zab, a protein lacking one module was constructed. This
protein, Zab
l, was produced in high yields as a soluble protein in
E. coli and could be purified to homogeneity. Protease mapping showed results similar to those for Zab (data not shown). Trypsin and endoproteinase Glu-C cleaved at identical residues. Chymotrypsin and thermolysin had only a single site each. Therefore, the overall structure of the domain was not altered by the presence of
the repeated module.
l. The spectra are shown in Fig.
4, along with a difference spectrum between Zab and Zab
l, which reflects the contribution of a single copy of the linker module. Results of the analysis of the curves using
the program K2d (34) are shown in Table
II. The Z
and Z
motifs contain significant amounts of
-helix and
-sheet structures. In contrast, to a large extent, the linker adopts
an alternate structure. This is consistent with the secondary structure
analysis of the primary sequence with computer programs, such as PHD
(35). Those analyses predict that no significant areas in the linker are structured as
-helices or
-sheets. It must be emphasized that
the proteolysis studies clearly indicate that the linker module is
structurally well defined, although in the majority, neither
-helical nor
-pleated.
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Fig. 4.
Protein CD spectra. Spectra were
recorded as described under "Experimental Procedures" and are
expressed in terms of mean residue ellipticity in units of
degrees·cm2·dmol 1. The curves show the
protein spectra of Zab (------), Zab
l ([- - -]), and Za
(-----). A difference spectrum, Zab minus Zab
l, is also shown
(····). The corresponding percentages of secondary structure
motifs were calculated using the program K2d (34) and are listed in
Table II.
Secondary structure analysis of recorded CD spectra (Fig. 4)
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Fig. 5.
Zab is protected from proteolysis in the
presence of Z-DNA. Zab was digested with endoproteinase Glu-C
without DNA (lanes 3-5), in presence of B-DNA (lanes
6-8), and in the presence of Z-DNA
(poly[d(5-MeC-G)]) (lanes 9-11). Digestion
and analysis were performed as described for Fig. 1, except that the
protein/protease ratio was 1:30. Lane M, molecular mass
markers.
from endoproteinase Glu-C cleavage may occur because these sites are involved in DNA interaction. On the other hand,
conformational changes occurring in the protein as a consequence of
binding to DNA could prevent endoproteinase Glu-C from cutting. It is
of note that the nearby trypsin site, Lys302, was protected
in the presence of Z-DNA, but not to the same extent (data not shown).
to
Z-DNA has been previously characterized using electrophoretic mobility
shift assays (2, 6, 7). This assay was used to compare the binding of
Za with that of Zab. d(5-BrC-G)20 was used as a
substrate; this oligonucleotide is stabilized in the Z-form by the
presence of bromine in the 5-position of cytosine (24). Binding was
tested in the presence of a 104-fold excess of B-DNA. The
results are shown in Fig. 6. Four
different proteins were compared at four concentrations (500, 100, 20, and 4 nM protein). Zab bound well at 500 and 100 nM, producing a stable, high molecular mass protein-DNA
complex (Fig. 6A, lanes 2-5). At lower
concentrations, the complex appeared to break down during electrophoresis, resulting in a smear. This behavior suggests that the
most stable complex is formed when the sites on the probe are
saturated. Zab
l showed a similar behavior, although the stable complex was formed only at the highest concentration (Fig. 6A, lanes 14-17). In contrast, Za produced two complex bands, which appeared smeared at all concentrations (Fig. 6A, lanes
6-9). Compared with Zab, Za had a slightly higher affinity for
the substrate. The smearing is the result of the instability of the
complex under electrophoretic conditions and the longer migration path
of Za-DNA complexes as compared with Zab-DNA complexes.
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Fig. 6.
Binding of Z-DNA by Zab and subdomains.
A, electrophoretic mobility shift assays were performed with
32P-labeled d(5-BrC-G)20 as the
substrate, which is stable in the Z-conformation under the applied
conditions (24). Zab (a, lanes 2-5), Za
(b, lanes 6-9), Zab digested with chymotrypsin
to separate the Z and Z
motifs
(c, lanes 10-13), and Zab
l (d,
lanes 14-17) were each assayed in a 5-fold
dilution series (500, 100, 20, and 4 nM). Lanes
1 and 18 show the migration of free substrate. Reaction
conditions are described under "Experimental Procedures." The
spot at the top of lane 16 is an artifact, which
was not reproducible. B, 200 nmol of the protein preparation
used for the band shift assay in A were subjected to
SDS-PAGE on 18% gels. The bands were visualized by Coomassie Brilliant
Blue staining. Proteins are labeled as described for A. The
digestion of Zab with chymotrypsin (lane c) was complete,
leaving no full-length protein. The difference in the size of Za and
the small digestion fragment in lane c is due to 5 additional C-terminal residues present in the Za expression construct.
Lane M, molecular mass markers.
moiety of Zab is responsible for the difference in
binding behavior between Za and Zab. To test this hypothesis, Zab was
digested with chymotrypsin and then assayed in the band shift (Fig.
6A, lanes 10-13). Complete digestion, yielding the
Z
and Z
motifs as separate peptides, was
confirmed by SDS-PAGE (Fig. 6B). This mixture showed a band shift pattern very similar to that for Za. No additional bands were
observed; therefore, Z
alone does not bind to the
substrate. Since the molecular masses of the Z
- and
Z
-containing fragments differ substantially, it is
extremely unlikely that any complex formed by Z
and DNA
would comigrate with the observed Z
-DNA complexes (23).
That the isolated Z
is not capable of binding Z-DNA under
these conditions is remarkable considering the conservative substitution of functionally important residues (2).
to DNA,
circular dichroism was used to monitor the transition of the DNA conformation from the B- to the Z-form (2, 4, 5). The spectrum of Z-DNA
is inverted as compared with that of B-DNA in the near-UV region
between 240 and 300 nm (36, 37). Fig. 7 shows the spectra of two Z-DNA-forming oligomers of different sequence,
d(C-G)6 and d(C-A)7·d(T-G)7, in
the presence of either Za or Zab. The DNAs adopted the right-handed
B-DNA conformation in the absence of protein. Protein was added in
aliquots, resulting in protein/base pair molar ratios of 1:6, 1:4, 1:2,
and 1:1.5. When Za was added, the spectra of both d(C-G)6
and d(C-A)7·d(T-G)7 became inverted,
indicating the shift from the B- to the Z-DNA conformation, with
saturation at a 1:2 ratio (Fig. 7, C and D). Further addition of protein did not change the spectrum significantly above 250 nm, where the contribution of the protein to the spectrum was
negligible. Below 250 nm, the spectrum was dominated by the contribution of the protein. This result is in agreement with Herbert
et al. (5), which showed that a Z
motif (amino
acids 121-201) binds without sequence specificity to a variety of
Z-DNA-forming sequences. Zab converted d(C-G)6 to the
Z-form with a similar stoichiometry to Za (Fig. 7A). In
contrast, there was only a limited effect on
d(C-A)7·d(T-G)7 of the addition of Zab, even
at a ratio of 1:1.5 (Fig. 7B).
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Fig. 7.
CD studies of the conformational change of
Z-forming sequences in the presence of Za or Zab. Traces were
generated as described under "Experimental Procedures." The spectra
show the titration of d(C-G)6 (A and
C) and d(C-A)7·d(T-G)7
(B and D) with Zab (A and
B) and Za (C and D), respectively. The
curves represent the spectra of the DNA alone (····) and in
presence of protein at protein/base pair molar ratios of 1:6
(- · -), 1:4 (- - -), 1:2 (-----), and 1:1.5 (------),
respectively. Spectra are expressed in absolute values of ellipticity
in millidegrees (mdeg).
alone, was able to bind
to Z-DNA in a sequence-independent manner. However, when
Z
was in the context of the entire domain, Zab, a
sequence preference for d(C-G)n was observed. Band shift data
suggests that the mode of binding is different between Za and Zab,
probably reflecting a difference in the degree of cooperativity.
DISCUSSION
,
with a binding specificity for Z-DNA. Analysis of the primary structure of the N-terminal region has led to a number of hypotheses about the
structure and function of this region. In addition to Z
, a second similar sequence, Z
, was identified (2). This
similarity and the conservative substitution of functionally important
residues made it possible that this, too, is a Z-DNA-binding motif.
These two motifs are separated by a linker, which is conserved in
length but not in sequence, suggesting that correct spacing between
Z
and Z
may be important. Prediction of the
secondary structure and low resolution NMR analysis suggest that
Z
may bind to Z-DNA using a unique application of the
helix-turn-helix motif common to many B-DNA-binding proteins (2, 39).
Z
and Z
might form separate domains or make
up a single bipartite domain.
and Z
as well
as two 49-amino acid linker modules. Limited proteolysis demonstrated that the domain extends from Leu133 to Glu361.
A smaller core domain, Za, from Leu133 to
Gly209, contains Z
and a portion of the
linker. C-terminally further shortened Z
peptides are
functional in binding specifically to Z-DNA (2). However, they lack the
structural uniformity seen only for the proteolytically defined domain
Za. These earlier constructs also contain additional N-terminal
residues (positions 121-132). These residues have been reported to
modulate the results of band shift assays (5); however, CD experiments
are unaffected by their presence. Although Za is stable to proteolysis,
we conclude that Zab is the functional entity. Domain boundaries are
frequently protease-hypersensitive, with cleavage sites for different
proteases clustered in close proximity. This is observed both
N-terminal to Leu133 and C-terminal to Glu361.
In contrast, the cleavage sites in the linker region are not clustered,
but rather specifically selected from a number of alternatives for each
enzyme. In contrast to Za, the stable domain containing Z
, Z
is not organized independently into a
stable domain. Instead, peptides containing Z
require
almost all of one linker module to be stable. Finally, although
secondary structure analysis of the primary sequence predicts mostly
non-
-helix or
-sheet in the linker modules, a prediction
confirmed by CD spectra, we have shown that these modules are well
structured. They are not as susceptible to proteolytic cleavage as it
would be expected for unstructured regions. Most important in this
respect is the result of cleavage by endoproteinase Glu-C, with 16 potential cleavage sites in the linker region. In fact, only one of
these is attacked even at high protease concentrations. Other proteases
provide similar results. Both linker modules are cut at a single
repeated site by chymotrypsin and thermolysin. Trypsin cuts the first
copy of the linker module once; the equivalent site in the second copy is missing due to a nonconserved residue. In each case, there are a
large number of unattacked potential cleavage sites. A single linker
module is sufficient for proper folding; removal of one linker module
to form Zab
l does not appear to change the overall domain structure.
become completely protected in the
presence of substrate DNA. We conclude that the protein becomes less
flexible when bound to its substrate. It is also possible that DNA
contacts shield the protein surface from proteases.
and Z
is supported
not only by proteolytic studies, but also by functional assays. In
electrophoretic mobility shift experiments, Zab yields a distinct and
stable product when bound to a left-handed DNA substrate. Zab
l forms
an equivalently stable complex, although with an ~5-fold reduced
affinity. There is no evidence that the linker modules are directly
involved in DNA binding, and their high negative charge makes this
unlikely. From the significant differences observed between Zab and
Zab
l, it is clear that the relative orientation of Z
to Z
is important for DNA binding. In contrast, Za
produces a less stable product, although it appears to bind with a
somewhat increased affinity. Chymotrypsin-digested Zab, in which
the halves of the domain are separated, binds identically to Za;
therefore, it is very unlikely that Z
binds to DNA
independently of Z
. In addition, if Z
and Z
acted as physically linked independent motifs, Zab
would be expected to have a higher affinity for Z-DNA than Za.
Therefore, we conclude that Z
and Z
must
interact within the Zab domain to form a single binding site, involving
both motifs. The results obtained by CD measurements strongly support
this conclusion. Zab binds with sequence preference for alternating
d(C-G)n, whereas Za does not discriminate between Z-forming
sequences, but rather is conformation-specific. This is in agreement
with previous studies on Z
-(121-201) (5). The preference
of Zab for d(C-G)n needs further investigation. Identification of the optimal substrate in vitro may elucidate the role of
Zab as part of ADAR1 in vivo and lead to the identification
of the actual binding sites of ADAR1 on chromatin.
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ACKNOWLEDGEMENTS |
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We thank Marina Voulova for help in the proteolytic digestion experiments. We especially thank Dr. Stefan Maas for many helpful discussions and for carefully reading the manuscript.
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FOOTNOTES |
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* 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.
Supported by a predoctoral fellowship from the German Academic
Exchange Service.
§ To whom correspondence should be addressed.
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol.
2 K. Lowenhaupt and T. Schwartz, unpublished results.
3 M. Schade and I. Berger, personal communication.
4 T. Schwartz, unpublished results.
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REFERENCES |
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