From the
The human serum serine protease inhibitor (serpin)
The human serum serine protease inhibitor (serpin)
In
the present work we employ mutagenesis and chemical modification to
identify lysine residues within two short regions of rACT that are
important for DNA binding, relating our results to other DNA-binding
proteins, and show that the DNA binding activity of rACT can be
completely decoupled from its serine protease inhibitory activity.
The rACT-K391T/K396T
double variant was constructed using polymerase chain reaction-mediated
mutagenesis. The N-terminal primer
(5`-ATGGCTAGCAACAGCCCACTT-3`) contained an NheI site
(italics). The C-terminal primer
(5`-TGGCTAGCCTAGGCTTGCGTGGGATTGGTGACTGTGCTCATGAAGAA-3`)
contained an NheI site (italics), a stop codon (underlined),
and Lys to Thr mutations (bold). The polymerase chain reaction product,
representing the entire coding region, was cut with NheI, gel
purified, and inserted in the correct reading orientation in pZMs, a
vector that does not have the N-terminal Cys, to create the variant C
terminus rACT, K391T/K396T-rACT. The N terminus of this variant is
MASNSPL-. The variant construction was confirmed by DNA sequencing.
As shown by PAGE
analysis (Fig. 1) rACT and the three variants (KTK, TTK, and
K391T/K396T) displaying some retention on DNA-cellulose were purified
to near homogeneity. The TTT and EEE variants were only approximately
50% pure following Mono Q chromatography, as estimated by titration
inhibition of a standard chymotrypsin solution.
Gel shift assays of rACT and rACT variant bound to double-stranded
and single-stranded DNA were conducted following either Garner and
Revzin (1981) or Maniatis et al.(1982). In the former case,
rACT (0-400 ng) was incubated with DNA (100-200 ng) in TE
buffer for 20 min at room temperature in a final volume of 20 µl,
then electrophoresed on 2% agarose gels. Detection was by ethidium
bromide (Sigma) staining. In the latter case, DNA was end-labeled with
Filter
binding assays were performed using Schleicher and Schuell
nitrocellulose filters (0.45 µm) and a 96-well suction manifold
(V& Scientific Inc., San Diego). Standard assays were performed in
DNA binding buffer containing the appropriate concentration of KCl.
Prior to use, filters were presoaked with this buffer, minus the bovine
serum albumin and Me
As shown in , rACT was most
strongly retained on the DNA-cellulose column, requiring the highest
KCl concentration for elution, while retention of the 210-212
variants fell in the order KTK>TTK>TTT
Identification of the amino acid residue(s) protected in the
DNA
rACT contains a total of 12 Met residues.
Cleavage after each by reaction with CNBr is expected to generate a
total of 11 peptide fragments of approximate molecular weights 12,000,
12,000, 6800, 4400, 2300, 1540, 1430, 1210, 660, 440, and 330 (there
are two Met-Met sequences). Tricine/SDS-PAGE analysis showed that none
of the largest 4 peptides, identified on the basis of their apparent
molecular weights, was differentially labeled by
[
The stoichiometry of
[
The involvement of the C terminus of rACT in DNA binding was
confirmed by mutagenesis. Expression of a mutant in which residues
390-398 were deleted yielded insoluble protein. Expression of a
double mutant, K391T/K396T-rACT, generated a soluble protein that,
while fully active as an inhibitor of chymotrypsin, required only 20
mM KCl for elution from the DNA-cellulose column (), and showed no observable binding to DNA by gel shift
assay (Fig. 2, lane 6).
Our results provide strong evidence that two elements within
rACT, a stretch of lysines (residues 210-212) and the C-terminal
peptide 390-398, are involved in the DNA binding interaction.
Replacing one or two of lysines 210-212 by threonine results in
partial loss of binding affinity, whereas replacing all three results
in complete loss of binding. A more detailed study would be required to
determine which of these three lysines is (are) most critical for ACT
binding to DNA. Acetylation of Lys
Lys
Two additional
observations made in our laboratories (data not shown) provide further
evidence that both the tri-lysine and C-terminal elements are necessary
for DNA binding. Neither mouse contrapsin, which has a high degree of
sequence identity (58%) with ACT, especially at the C terminus
(MAKVNNPK for contrapsin (Hill et al., 1984; Suzuki et
al., 1990) versus MSKVTNPKQA for rACT (Rubin et
al., 1990)), but which lacks the lysine 210-212 loop, nor
carbonic anhydrase, which contains a stretch of three lysines (residues
111-113) but is not homologous to rACT at the C terminus, show
demonstrable binding to DNA, as measured by DNA-cellulose
chromatography (contrapsin) and gel shift assays (contrapsin, carbonic
anhydrase). We predict that two other serpins, horse leucocyte elastase
inhibitor (Potempa et al., 1991; Dubin et al., 1992)
and elastase inhibitor I (Remold-O'Donnell et al.,
1992), both of which possess the three lysine stretch but lack a C
terminus homologous to that of ACT, will display little or no binding
to DNA.
Examination of the crystal structure of both cleaved ACT
(Baumann et al., 1991) and an intact ACT variant (Wei et
al., 1994) reveals that the C terminus and lysines 210-212
occur in relatively close proximity (the distances between the
Another open question is whether the combination of
DNA binding elements we identify in this paper constitute a new DNA
binding motif (Harrison, 1991) or are unique to ACT. As summarized in , up to four known DNA-binding proteins, Bin3, the cell
cycle growth regulator p53, the accessory gene regulator protein, and
the 17-kDa protein in the DNAX 5` region (p17), have the same two DNA
binding elements as does ACT, supporting the notion of a motif. On the
other hand, it is unclear whether these elements function in the same
way as they appear to in rACT. Bin3 contains a lysine within a limited
C-terminal region known to be important for binding to DNA, but the C
terminus forms part of the common helix-loop-helix DNA-binding motif
(Rowland and Dyke, 1989). p53 has two lysines within the last 10
residues, but the DNA binding region has only been localized to within
a 47-residue region from the C terminus (Foord et al., 1991)
and no direct information is available identifying DNA binding regions
within proteins accessory gene regulator and p17.
ACT is an
acute-phase protein; its serum concentration increases as much as
4-fold (Katsunuma et al., 1980; Lindmark and Eriksson, 1985)
in response to injury or infection, and it is a provocative speculation
that its binding to DNA might provide a mechanism for the
self-regulation of ACT expression (Abraham, 1992). Our choice of DNA
40-mer as the test sequence for ACT binding was based on its being a
logical candidate as a specific site of ACT binding. However, the
rather weak affinity we measure for ACT binding to DNA 40-mer () belies this notion. In fact, by several measures ACT
failed to demonstrate sequence specificity in binding to DNA. Thus, ACT
formed complexes with all
Our work clearly demonstrates that the DNA
binding activity of rACT is completely independent of its protease
inhibitory activity. Thus mutations or chemical modifications that
decrease or abolish DNA binding have little or no effect on protease
inhibitory activity, and, reciprocally, destruction of the active site
loop necessary for protease inhibitory activity does not affect DNA
binding. Furthermore, complex formation with chymotrypsin has no effect
on DNA binding and complex formation with DNA binding does not affect
inhibitory activity of rACT. The KKK sequence is known to be part of a
nuclear localization signal (Dang and Lee, 1989; Addison et
al., 1990). It is not unlikely that the 210-212 ACT variants
lacking DNA binding activity, and perhaps ACT variants at Lys
WT and variant
ACTs were applied to a DNA-cellulose column equilibrated with 50 mM potassium P
Ionic strength in the
absence of added KCl was 0.03.
rACT
(10.5 µM), in the presence or absence of DNA 40-mer (12.5
µM), was acetylated with
[
A search (GCG Fasta and Findpatterns) of the Swissprot
database for sequences containing a tri-lysine element yielded a total
of 250 proteins, of which the 15 shown below, in addition to ACT, are
known to bind to DNA.
We gratefully acknowledge the superb technical
assistance of Nora Zuo and Pirjo Tuominen in several aspects of this
work and Joseph Ippolito for generating Fig. 6.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1-antichymotrypsin (ACT) appears to be unique among serpins in its
ability to bind to double-stranded DNA. Using site-directed mutagenesis
and chemical modification, a tri-lysine sequence (residues
210-212) falling within a solvent exposed loop and the C-terminal
peptide containing two lysines (residues 391 and 396) were shown to be
important for DNA binding. Mutation of residues 210-212 from
lysines to either glutamates or threonines abolished DNA binding. The
Lys
-Thr
-Lys
and
Thr
-Th4
-Lys
variants
displayed reduced affinity for DNA, especially at higher ionic
strength. Limited acetylation of rACT with acetic anhydride led to loss
of DNA binding and, conversely, DNA protected rACT from acetylation. A
combination of CNBr digestion, peptide separation, and peptide
sequencing identified Lys
, two residues from the C
terminus, as the most reactive lysine in rACT. Acetylation of
Lys
is strongly decreased in the presence of DNA. The
double mutant K391T/K396T-rACT had very little affinity for DNA. The
-amines of lysines 210-212 are 8-15 Å across a
cleft from the
-amines in Lys
and Lys
,
and together these two elements may form an unusual DNA binding domain.
Attempts to isolate a DNA sequence to which ACT binds specifically have
been unsuccessful to date, raising the possibility that nonspecific
binding of ACT to DNA suffices to account for the ACT found in certain
cell nuclei. ACT variants not binding to double-stranded DNA retain ACT
protease inhibitory activity, a potentially important result for the
use of ACT variants as therapeutic agents.
1-antichymotrypsin (ACT)
(
)(Rubin et
al., 1990; Cooperman et al., 1993) appears to be unique
among serpins in its ability to bind to DNA (Katsunuma et al.,
1980), a property retained by the nonglycosylated recombinant form,
rACT, expressed in Escherichia coli (Rubin et al.,
1990). ACT, derived from serum, is found in carcinoma cell nuclei
(Takada et al., 1982, 1986), as well as in the nuclei of
non-malignant cells (Tahara et al., 1984),
(
)including those from human neural tissue (Abraham et al., 1988). ACT inhibits DNA synthesis in permeabilized
human carcinoma cells (Tsuda et al., 1987), possibly as a
result of its inhibition of DNA polymerase
(Tsuda et
al., 1986), and/or of DNA primase (Takada et al., 1988).
Furthermore, ACT inhibits natural killer cells, which are responsible
for tumor cell lysis, and it has been suggested that ACT present in the
nucleus may act as a protective agent for tumor cells (Travis and
Salvesen, 1983). ACT could also modulate the level of chymotrypsin-like
enzyme activity found in chromatin (Travis and Salvesen, 1983).
Materials
Buffers
DNA binding buffer contained 10 mM Tris-Cl (pH 7.5), 5 mM EDTA, 2 mM CaCl, 5% Me
SO, 0.1 mg/ml bovine serum
albumin (Promega); TAU: 200 mM NH
Cl, 5 M
urea, 20 mM Tris-HCl (pH 7.9); TE: 50 mM Tris-Cl (pH
7.9), 1 mM EDTA; TBE: 50 mM Tris borate (pH 8.3), 1
mM EDTA; TK: 50 mM Tris-Cl (pH 7.9), 50 mM KCl.
Cloning and Expression of rACT
Variants
Site-directed mutagenesis was carried out using the
Amersham M13 in vitro mutagenesis kit and the synthetic DNA
primers shown below. Lys to Thr or Glu mutations are denoted in bold:
GCACCATTACCCACTTTGTCTTGCTCAAGTAGAACC for KTK-rACT,
GCACCATTACCCACTCTTCCTCGCTCAAGTAGAACC for EEE-rACT;
GCACCATTACCCACGTTGTCTTGCTCAAGTAGAACC for TTK-rACT;
GCACCATTACCCACGTTGTCGTGCTCAAGTAGAACC for TTT-rACT. The altered
genes were excised from double-stranded M13 with EcoRI, the
overhanging ends made blunt by treatment with T4 polymerase, and
inserted into the expression vector pZM described earlier (Rubin et
al., 1990, 1994) to yield the 210-212 rACT mutants. The
variant construction was confirmed by DNA sequencing. All the
210-212 variants and rACT have a Cys derived from exon I of the
ACT gene at the N terminus: MGRDLCHPNSPL-.
Purification of Wild-type rACT and Variants
rACT
was purified as described previously by successive chromatographic
steps: Fast Q followed by double-stranded DNA-cellulose (Rubin et
al., 1990; Kilpatrick et al., 1991). rACT and the
210-212 rACT variants exist in solution as a mixture of monomer
and dimer forms. Dimer formation results from disulfide bridge
formation between cysteine residues located in the N terminus. The
K391T/K396T variant, lacking an N-terminal cysteine, exists only as a
monomer. The variants KTK-rACT, TTK-rACT, and K391T/K396T-rACT retained
sufficient affinity for DNA that they could be purified to homogeneity
by double-stranded DNA-cellulose chromatography. These variants eluted
at considerably lower KCl concentrations than wild-type rACT (). The TTT-rACT and EEE-rACT variants that displayed no
binding to double-stranded DNA-cellulose were partially purified by
Mono Q FPLC chromatography. Following Fast Q chromatography (Rubin et al., 1990) these crude variant ACTs (10 mg of total
protein) were dialyzed against TK buffer, filtered by centrifugation
through a 0.2-µm filter unit (Rainin), and loaded onto a Mono Q HR
5/5 column (1 ml, Pharmacia) pre-equilibrated with TK buffer.
Chromatography was carried out with a 30-min linear gradient 50
mM KCl-350 mM KCl in 50 mM Tris-HCl (pH 7.9)
followed by a 20-min isocratic elution with the final buffer of the
linear gradient. The flow rate was maintained at 1 ml/min. Both variant
ACTs eluted between 250 and 350 mM KCl.
Figure 1:
SDS-PAGE
analysis of purified variant ACTs. Lane 1, standard molecular
weight markers; Lane 2, rACT; Lane 3, KTK-rACT; Lane 4, TTK-rACT; Lane 5, rACT-K391T/K396T; Lane
6, TTT-rACT; Lane 7, EEE-rACT.
Preparation of Cleaved rACT by Treatment with Human
Neutrophil Elastase (HNE)
Cleavage was carried out at an I/E
ratio of 20. HNE concentration was determined by its catalysis of N-methoxysuccinylAAPV-p-nitroanilide (10 mM)
hydrolysis (0.1 M HEPES, pH 7.5, 0.5 M NaCl, 25
°C), using the change in absorbance of the sample at 410 nm and a
specific activity of 0.0053 absorbance units/min/pmol/ml, a value based
on titration of HNE with standardized 1-protease inhibitor.
(
)HNE (0.22 µM) was added to ACT (4.4
µM) in 0.1 M Tris-HCl (pH 8.3) containing 0.025%
Triton X-100 and incubated at 25 °C for 1 h. The cleavage reaction
was stopped by the addition of phenylmethylsulfonyl fluoride to a final
concentration of 2 mM. Full cleavage was verified by SDS-PAGE
analysis (Schechter et al., 1993). rACT is cleaved by HNE in
the active loop region at positions P1-P1`, P3-P4, and P5-P6 (Rubin et al., 1994).
DNA Fragments Used in Binding Studies
X174
digested with HaeIII was from DuPont NEN. Double-stranded
fragments corresponding to the regulatory regions ATF/CREB, TFIIID,
NF1, MLTF, HIV-TATA, and E2-TATA, and varying in length from 22 to 38
base pairs (Naidoo, 1994), were the kind gifts of Dr. Roberto Weinmann
(Wistar Institute). The 40-residue double-stranded DNA fragment, DNA
40-mer, made up of residues corresponding to positions -56 to
-90 of the ACT gene plus a 5`-aggtt overhang used for radioactive
end labeling, aggtttgggaaatgccaggacaaccaagtgttctgttcta, as well as all
DNA primers, were synthesized by the DNA Service of the University of
Pennsylvania Cancer Center. This sequence lies upstream from the
putative TATA box (Chandra et al., 1983; Bao et al.,
1987) within a region usually associated with proximal upstream
regulatory sequences.
Methods
Assays
Determinations of second-order rate
constants of chymotrypsin inhibition by rACT and rACT variants were
carried out as described previously (Rubin et al., 1990).
-[
P]CTP (Amersham) using Klenow fragment
(NE Biolabs). Approximately 0.3 ng of
P-labeled DNA was
incubated with ACT (0-15 µg) in TE buffer in a final volume
of 15 µl for 20 min at room temperature. Samples were
electrophoresed on 10% polyacrylamide gels in TBE buffer and
DNA-containing bands were detected by autoradiography.
SO. Reaction mixtures containing DNA
binding buffer (70 µl), DNA (0.2 ng), and rACT or rACT variants
(0-7.5 µg) were incubated for 1 h at room temperature and
then filtered. The filters were washed with two 0.5-ml portions of DNA
binding buffer at 4 °C, dried, and counted in Scintiverse II fluid
(Fisher) using a Beckman LS 7500 scintillation counter. DNA 40-mer
concentration was calculated assuming a molecular weight of 26,400.
Protein concentration was estimated following Bradford(1976).
Acetylation
Acetylation of rACT and of the
rACTDNA 40-mer complex was carried out by initially reacting
sample with limiting amounts of [
H]acetic
anhydride followed by further reaction with unlabeled acetic anhydride
under conditions producing more complete acetylation of available sites
on rACT. The first acetylation step was carried out by adding
0.4-2.0 µmol of [
H]acetic anhydride (50
mCi/mmol, Amersham) to 1 ml of 10.5 µM rACT in the absence
or presence of 12.5 µM DNA 40-mer in 50 mM
NaP
buffer (pH 8.15). For reactions carried out in the
presence of DNA 40-mer, rACT was preincubated with DNA 40-mer for 20
min prior to acetic anhydride addition. Under these conditions,
approximately 95% of total rACT in solution is present as rACT
DNA
40-mer complex (I). The reaction was allowed to proceed
for 5 min at room temperature, after which it was quenched by addition
of 1.5 µl of 3.8 M NH
OH. Following quenching,
the rACT
DNA 40-mer complex was dissociated by addition of an
equal volume of 2
TAU buffer, and heating at 40 °C for 20
min. DNA 40-mer was removed from the partly acetylated rACT by
application of the dissociated complex to a DE-52 column (2 ml
pre-equilibrated with 5 M urea, 20 mM Tris-HCl, pH
7.9) and elution with TAU buffer. rACT was eluted within 4 ml while DNA
40-mer was retained. The rACT sample that was acetylated in the absence
of DNA 40-mer was treated in exactly the same fashion. Both rACT
samples were dialyzed against 50 mM NaP
(pH 8.15)
and concentrated by vacuum centrifugation (Savant) to approximately 10
µM in a volume of 0.96 ml. Unlabeled acetic anhydride
(2.5-µmol portions) was then added every 5 min at room temperature
to a total of 20 µmol, with a concommitant decrease in pH to 7.0.
The samples were then dialyzed against 50 mM NaP
(pH 8.15), subject to another round of reaction with unlabeled
acetic anhydride, exactly as described above, and dialyzed extensively
(Pierce Microdialyzer) against 100 mM Tris-HCl (pH 7.9). In a
separate experiment employing [
H]acetic
anhydride, these acetylation conditions were shown to result in a
stoichiometry of 26 acetyl groups/rACT (Johnson, 1990). There are a
total of 26 Lys residues/rACT (Chandra et al., 1983; Rubin et al., 1990).
CNBr Cleavage of Acetylated rACT
ACT acetylated in
the absence or presence of DNA 40-mer was cleaved with CNBr according
to the method of Gross and Witkop(1962). In a typical reaction about
500 µg (0.011 µmol) of rACT dissolved in 100 mM
Tris-Cl (pH 7.9) was lyophilized, the resulting solid was dissolved in
70% formic acid (500 µl), CNBr (5 mg, 60 µmol) was added, and
incubation was continued for 16 h under N at room
temperature. The solution was then concentrated to dryness over NaOH
pellets in a vacuum desiccator. Tricine/SDS-PAGE analyses of
CNBr-generated peptides were performed according to Schagger and von
Jagow(1987). Peptides were also separated by RP-HPLC and by microbore
capillary columns. Quantitative estimation of eluted peptides was
obtained by A
absorbance, as described by
Buck et al.(1989). The product of peak area and flow rate was
determined for elution of a known amount of a standard peptide, N-acetyl-LTLDADF (kindly provided by Alison Fisher of this
laboratory; the concentration of a stock solution of this peptide is
determined, setting
= 178). This value was
then used to calculate the amount of eluted, specifically acetylated
peptide derived from rACT from the product of its peak area and flow
rate.
Peptide Sequencing
Purified differentially labeled
peptide was subjected to automated Edman sequencing, on an ABI Model
473A sequencer in the Dept. of Pathology at the University of
Pennsylvania. The radioactivity in aliquots taken following each Edman
cycle was determined in a Beckman LS 1801 counter.
Identification of a Portion of the DNA Binding Domain
by Inspection and Site-specific Mutagenesis
Comparison of the
primary structure of ACT with those of well-studied serpins such as
antithrombin III, 1-proteinase inhibitor, or C1-inhibitor (Huber
and Carrell, 1989) showed it to contain a stretch of three consecutive
lysines (residues 210-212) not present in the others. The x-ray
crystal structures of cleaved ACT (Baumann et al., 1991) and
an intact rACT variant (Wei et al., 1994) show these three
lysines to fall in a solvent-exposed loop. To test the possibility that
this tri-lysine stretch was directly involved in rACT binding to DNA,
four 210-212 variants, EEE-rACT, KTK-rACT, TTK-rACT, and
TTT-rACT, were prepared and tested for DNA binding and chymotrypsin
inhibitory activity.
EEE. In accord with
the DNA-cellulose results, rACT (Fig. 2, lane 1) and
both KTK-rACT (lane 2) and TTK-rACT (lane 3) showed
at least some binding to
X174-HaeIII fragments by gel
shift assay (Fig. 2), but TTT-rACT (lane 4) and EEE-rACT (lane 5) did not. In similar experiments using DNA 40-mer,
cleaved ACT was found to bind DNA as strongly as intact rACT,
demonstrating that binding does not depend on an intact active-site
loop of rACT (residues 345-365, Huber and Carrell, 1989), which
is critical for the binding to and inhibition of chymotrypsin. No
binding of rACT was observed to either + or - strands,
demonstrating that binding is specific for double-stranded DNA.
Figure 2:
Gel retardation assay of rACT and rACT
variant binding to X174-HaeIII fragments. ACT or ACT
variants (300 ng) were incubated with
X174-HaeIII DNA
(200 ng) following Garner and Revzin (1981) as described under
``Methods.'' All lanes contained
X174-HaeIII
DNA. Lane 1, plus rACT; Lane 2, plus KTK-rACT; Lane 3, plus TTK-rACT; Lane 4, plus TTT-rACT; Lane 5, plus EEE-rACT; Lane 6, plus K391T/K396T-rACT; Lane 7, no added protein.
Measured by filter binding assay at low ionic strength, rACT binds
to DNA 40-mer approximately 1 order of magnitude more tightly than
either KTK-rACT or TTK-rACT, and neither TTT-rACT nor EEE-rACT show any
binding (Fig. 3). The different plateau values may reflect
varying efficiencies with which rACTDNA complexes are retained on
the filter (Yarus and Berg, 1970; Riggs et al., 1970; Hinkle
and Chamberlain, 1972; Strauss et al., 1980). rACT and
rACT-chymotrypsin complex were found to bind to DNA with similar
dissociation constants. The effects of added KCl on K
values for rACT, KTK-rACT, and TTK-rACT
binding to DNA 40-mer are summarized in .
Figure 3:
Filter assays of rACT and variant ACT
binding to DNA 40-mer at 10 mM KCl, as described (see
``Methods''). Curves are drawn assuming 1:1 complex formation
between the indicated ACT and DNA 40-mer, using the K values
shown in Table II and an efficiency of complex retention on the filter
that differed for different ACTs (90% for rACT, 60% for KTK-rACT, 40%
for TTK-rACT). ACT concentration was calculated as a monomer. In all
cases, monomer was the dominant species in solution. The assumption was
made that each ACT within a dimer bound independently to
DNA.
In contrast
to the dramatic effects observed on DNA affinity, each of the
210-212 variants described above had a second-order rate constant
for inhibition of chymotrypsin similar to that for rACT.
Identification of a Portion of the DNA Binding Domain by
Acetylation and Site-specific Mutagenesis
Acetylation of rACT
with [H]acetic anhydride led to loss of DNA
binding activity at a stoichiometry of
4 acetyl groups/rACT (gel
shift assay, data not shown). Strong evidence that this effect was due
to a specific loss of DNA binding activity rather than some more
generalized conformational change came from the observation that
acetylation up to a level of 20 acetyl groups/rACT has little effect on
chymotrypsin inhibitory activity (Johnson, 1990). Formation of a
complex with DNA reduced the stoichiometry of rACT acetylation, under
conditions of limited acylation, by 40-50% (I).
rACT complex was accomplished by first labeling rACT with
limiting amounts of [
H]acetic anhydride in the
presence and absence of DNA and then, following dissociation of the
complex in the sample containing DNA, subjecting both samples to
vigorous acetylation with repeated additions of unlabeled acetic
anhydride. This procedure is designed to leave both rACT samples
chemically similar, differing only in that the residues labeled
preferentially by [
H]acetic anhydride have higher
specific radioactivity.
H]acetic anhydride in the presence or absence of
DNA. The CNBr-generated peptide fragments were also analyzed by
RP-HPLC. Typical results are presented in Fig. 4. This analysis
consistently showed that two peaks, a and b, as well
as a broad region of the chromatogram (elution time 60-80 min),
were
H-labeled to considerable extents, and that such
labeling was markedly reduced (2-4-fold) in the presence of DNA.
Figure 4:
RP-HPLC
analysis of CNBr digest of H-acetylated rACT. rACT (10.5
µM) was reacted with [
H]acetic
anhydride (0.9 µmol) as described (see ``Methods'') in
the presence or absence of DNA 40-mer (12.5 µM).
Separation was carried out on a C-18 RP-HPLC column (Synchrom, Inc.).
Column conditions: solvent A, ACN; solvent B, water; solvent C, 1%
trifluoroacetic acid. All elution solvents contained 10% solvent C.
Gradient employed: 10-min isocratic at 1% A; 30-min linear gradient,
1-20% A; 100-min linear gradient, 20-45% A; 30-min linear
gradient 45-65% A; 20-min linear gradient, 65-90% A, flow rate, 0.7 ml/min; 1-min fractions were collected and counted. Panel A, A
trace for CNBr-treated rACT
labeled in the absence of DNA 40-mer. Peaks a and b and subregions c (66-68 min) and d (69-71 min) are indicated. Protein labeled in the presence
of DNA 40-mer gave an essentially identical trace. Panel B,
radioactivity profile for rACT acetylated in the absence of DNA 40-mer. Panel C, radioactivity profile for rACT acetylated in the
presence of DNA 40-mer. Panel D, difference radioactivity
profile, Panels B-C.
The peaks in panels A and B and two subregions (C, 66-68 min, and D, 69-71 min)
corresponding to the most highly labeled portions of the broad
radioactive region, were collected separately, and each was further
resolved by microbore capillary HPLC (Fig. 5). Although the
peptide compositions of a-d were different, each gave a single
radioactive peptide. In each case the sequence of the labeled peptide () was SKVTNPKQA, corresponding to the C-terminal peptide
(residues 390-398) following the final Met in rACT.
Figure 5:
Capillary microbore analyses of peaks a and b and subregions c and d from
Fig. 4. Fractions corresponding to a-d were
separately pooled, lyophilized, dissolved in 0.1% trifluoroacetic acid
and applied onto a microbore capillary C-18 column (Applied
Biosystems). Solvent A, 0.1% trifluoroacetic acid in water; solvent B,
0.1% trifluoroacetic acid in ACN. Gradient, 0-70% B in 30 min,
flow rate, 0.3 ml/min. Radioactivity levels in the collected fractions
are shown in the histograms.
The
labeled peptides derived from b, c, and d are identical, and differ from the labeled peptide derived from a in that Lys is acetylated in b-d but not in a. This accounts for the separation of a and b on RP-HPLC analysis, and also demonstrates that the
acetylation procedure with unlabeled acetic anhydride is insufficient
to completely derivatize all lysines remaining underivatized after the
initial reaction with
H-labeled acetic anhydride. That c and d are resolved from b on RP-HPLC
analysis (Fig. 4) probably reflects formation of nonspecific
complexes between the diacetylated form of SKVTNPKQA and acetyl
derivatives of other peptides, generated by CNBr cleavage, that persist
during RP-HPLC analysis.
H]acetylation determined for each of the
resolved peaks in Fig. 5, isolated from rACT labeled to the
extent of 3.0 [
H]acetyl groups/protein, was 0.62
± 0.02. As is clear from determination of the radioactivity
released during each cycle of Edman degradation of the radioactive
peptide isolated from a (), only Lys
is acetylated by [
H]acetic anhydride.
is both protected
against by complex formation with DNA and accompanied by loss of ACT
binding to DNA which, along with the low DNA binding activity of the
rACT-K391T/K396 variant, provides strong evidence for the involvement
of Lys
in DNA binding. Expression and testing of the
single-site mutants K391T and K396T will be required to resolve the
remaining ambiguity concerning the involvement of Lys
in
DNA binding.
is the most reactive lysine in rACT,
acetylated to a stoichiometry of 0.62 for an ACT sample that has
incorporated only 3.0 acetyls/protein. Such high reactivity may reflect
not only accessibility to solvent, but also an abnormally low
pK
, since it is the basic form of the
-amino group that is required for reaction with acetic anhydride
and the normal
-amino pK
is
10.5. Lysines 210-212 fall within a 21-amino acid peptide
(residues 195-215) expected to result from CNBr cleavage of rACT.
Our failure to detect major DNA-sensitive labeling of this peptide on
RP-HPLC analysis may reflect low reactivity of lysines 210-212
toward acetic anhydride under the reaction conditions employed. One
possibility is that the amino groups of these lysines retain high
basicity within rACT, which would diminish their reactivities toward
acetic anhydride at pH values
8.15, but would allow them to
participate in DNA binding by coulombic attraction.
-NH
s in Lys
and Lys
and
the
-NH
s in Lys
, Lys
, and
Lys
fall in the range 8-15 Å) and could form
a specific combination of elements for DNA binding (Fig. 6). The
crystal structures show further that lysines 210-212 occur in a
turn between two
sheets. This region displays very weak electron
density, implying that it is quite flexible. We speculate that DNA may
bind to ACT by docking within a shallow cleft created by the very
flexible lysine loop stretching out as a ``thumb,'' with the
C-terminal lysines lying at the base of the cleft.
Figure 6:
Positions of lysines 210, 211, 212, 391,
and 396 (in red) within the backbone structure of an intact rACT.
Depicted is the structure of the active-site loop variant rACT-P3P3`
(Wei et al., 1994). The active-site loop is at upper
right. Inset, a detail of the five-lysine region, with side chains
included.
It remains an
open question as to whether other elements within ACT are also
important for binding to DNA. Under conditions in which ACT alone is
modified to the extent of 3 acetyls/protein, the addition of DNA
decreases this amount by 1.4 acetyls (I), of which 0.6 are
incorporated into Lys. The remaining 0.7-0.8
acetyls that are incorporated in a DNA-sensitive manner are presumably
distributed in small amounts over several Lys residues that we could
not identify, and one or more of such residues could also be important
for DNA binding.
X174-HaeIII fragments (73 base
pairs to 1.6 kilobase pairs) as analyzed by gel shift assay (Fig. 2). Furthermore, no evidence of sequence specificity was
observed for rACT binding to double-stranded fragments known to
represent regulatory sequences, ATF/CREB, AP1, TFIID, NF1, MLTF, HIV
TATA, and E2 TATA. Of course, negative results are never definitive and
it remains possible that a specific genomic site for ACT binding exists
and remains to be identified. On the other hand, the presence of ACT in
nuclei could simply result from nonspecific binding of ACT to DNA
serving to increase the local concentration of ACT within the nucleus
of certain cell types.
and Lys
as well, will lack the ability of wild-type
ACT to incorporate into certain cell nuclei. Such variants could have
considerably altered physiological effects in vivo when
compared with wild-type ACT, an important consideration in our ongoing
efforts to develop rACT variants as therapeutic agents (Rubin et
al., 1994).
Table: DNA-cellulose chromatography
, 50 mM KCl (pH 6.9) and eluted
with a linear gradient of KCl 50-500 mM in 50 mM potassium P
(pH 6.9). A gradient of 10-70 mM KCl in 50 mM potassium P
(pH 6.9) was used
for the K391T/K396T variant.
Table: Dependence of rACT and 210-212 rACT
variant binding to DNA 40-MER on added KCl
Table: Effect of DNA on acetylation of rACT
H]Ac
O in a total volume of 1 ml (see
``Methods'') and stoichiometries of incorporation were
determined.
Table: Sequencing
data for microbore resolved peaks
Table: DNA binding proteins containing a trilysine
sequence
1- antichymotrypsin; HNE, human
neutrophil elastase; PAGE, polyacrylamide gel electrophoresis; p17, the
17-kDa protein in the DNAX 5` region; RP-HPLC, reverse phase-high
performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
-Antichymotrypsin from Human Serum and the Study of Its Interaction with the Superoxide Alteration of Stimulated Human Neutrophils. Masters thesis, University of Pennsylvania
-Antichymotrypsin. Ph. D. thesis, University of Pennsylvania
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.