©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Lysines within 1-Antichymotrypsin Important for DNA Binding
AN UNUSUAL COMBINATION OF DNA-BINDING ELEMENTS (*)

Nirinjini Naidoo (1), Barry S. Cooperman (1)(§), Zhi-mei Wang (2), Xu-zhou Liu (2), Harvey Rubin (2)(§)

From the (1)Departments of Chemistry and (2)Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human serum serine protease inhibitor (serpin) 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.


INTRODUCTION

The human serum serine protease inhibitor (serpin) 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).

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.


EXPERIMENTAL PROCEDURES

Materials

Buffers

DNA binding buffer contained 10 mM Tris-Cl (pH 7.5), 5 mM EDTA, 2 mM CaCl, 5% MeSO, 0.1 mg/ml bovine serum albumin (Promega); TAU: 200 mM NHCl, 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-.

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.

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.

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.


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).

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 -[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.

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 MeSO. 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 rACTDNA 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 NHOH. Following quenching, the rACTDNA 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.


RESULTS

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.

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>TTTEEE. 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).

Identification of the amino acid residue(s) protected in the DNArACT 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.

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 [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.

The stoichiometry of [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.

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).


DISCUSSION

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 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.

Lys 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.

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 -NHs in Lys and Lys and the -NHs 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.

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 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.

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 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

WT and variant ACTs were applied to a DNA-cellulose column equilibrated with 50 mM potassium P, 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

Ionic strength in the absence of added KCl was 0.03.


  
Table: Effect of DNA on acetylation of rACT

rACT (10.5 µM), in the presence or absence of DNA 40-mer (12.5 µM), was acetylated with [H]AcO 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

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.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant AG-10599 and the Lexin Corp. Drs. Cooperman, Wang, and Rubin have equity in Lexin. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Authors to whom correspondence should be addressed. Cooperman: Tel.: 215-898-6330; Fax: 215-898-2037. Rubin: Tel.: 215-662-6475; Fax: 215-662-7842.

The abbreviations used are: ACT, 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.

N. Naidoo, B. S. Cooperman, Z-M. Wang, X-Z. Liu, and H. Rubin, unpublished data.

N. Schechter, personal communication.


ACKNOWLEDGEMENTS

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.


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