THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 by The American Society for Bioc - PDF document

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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 265, No. 22, Issue of August 5. PP. 1282%X335,1990 Printed in U.S. A. Reaction of LexA Repressor with Diisopropyl Fluorophosphate A TEST OF THE SERINE PROTEASE MODEL* (Received for publication, March 5, 1990) Kenneth L. Roland+ and John W. LittleSQll From the Departments of $Biochemistry and §Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721 The LexA repressor of Escherichia coli modulates the expression of the SOS regulon. In the presence of DNA damaging agents in vivo, the 202-amino acid LexA repressor is In vitro, LexA cleavage requires activated RecA at neu- tral pH, and proceeds spontaneously at high pH in an intramolecular reaction termed autodigestion. A model has been proposed for the mechanism of autodigestion in which serine 119 serves as the reactive nucleophile that attacks the Ala-84/Gly-85 peptide bond in a man- ner analogous to a serine protease, while uncharged lysine 156 activates the serine 119 hydroxyl group. In this work, we have tested this model by examining the effect of the serine protease inhibitor diisopropyl fluo- rophosphate (DFP) on autodigestion. We found that DFP inhibited autodigestion and that serine 119 was the only serine residue to react with DFP. We also examined [3H]DFP incorporation by a number of The LexA protein of Escherichia coli regulates the expres- sion of a set of genes, collectively termed the SOS regulon (1, which are involved in the repair of DNA damage. When cells are treated by a DNA damaging agent, such UV irradiation, the LexA repressor is inactivated by a proteolytic cleavage event at the Ala-84/Gly-85 peptide bond in vivo requires functional RecA protein (4),

which becomes activated by an inducing s
which becomes activated by an inducing signal, probably single-stranded DNA (5). Activated RecA is also required for the cleavage of an SOS protein, UmuD (6-8), as well as a number of bacte- riophage repressors, including Xc1 (5, LexA cleavage is also observed in vitro at physiological pH in the presence of RecA protein and two types of cofactors, single-stranded DNA and nucleoside triphosphate such * This work was supported by Grant GM24178 (to J. W. L.) and Grant GM12390-03 (to K. L. R.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ?l To whom correspondence should be addressed: Dept. of Biochem- istry, Rm. 347, Biological Sciences West, University of Arizona, Tucson, AZ 85721. dATP (10). RecA and its cofactors form a ternary complex (11-13) which is the activated form of RecA (1). RecA- independent cleavage is also observed in vitro at alkaline pH, yielding fragments identical to those observed for the RecA- mediated reaction (14). The RecA-independent cleavage re- action is termed “autodigestion” (14), and its existence sug- gests that the effect of RecA is indirect. In this view, the catalytic residues important in the cleavage reaction are lo- cated in the LexA protein, and RecA serves to in vivo repressor activity and normal in vitro thermostability but are completely deficient in auto- digestion and RecA-mediated cleavage (15), indicating that these two residues are absolutely required for both types of cleavage reactions. Additional support for this model comes from a recent study in which 20 independently DIP, diisopropyl phosphoryl; CAPS, 3-(c&lohe

xylamino)-i-pro- panesulfonic acid; Pipe
xylamino)-i-pro- panesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; HPLC, high performance liquid chromatography. 12828 DFP Inhibits .LaA Autodigestion 12829 N-Terminal Domain - Ala -C N-Terminal + C-Terminal Fragments FIG. 1. Model for LexA autodigestion. Taken from Ref. 15. proteolytic cleavage. Preliminary experiments in our labora- tory indicated that concentrations of DFP up to 1 mM did not inhibit LexA autodigestion, as judged by visual examination of Coomassie Blue-stained gels (15). This negative result was reconciled with the model by either or both of two interpre- tations. First, since autodigestion is an intramolecular reac- tion, the active site serine may not be accessible to solvent, and therefore to the DFP. Second, since the rate of autodiges- tion is rather slow as compared with the catalytic rates of most serine proteases, the active site serine in LexA may not be as highly activated as the respective serines found in trypsin or chymotrypsin, and is therefore less reactive to DFP. In MT118’ was constructed by site-directed mutagenesis as described (15). The oli- godeoxyribonucleotide used to introduce the single nucleotide substi- tution was 5’TCAGCGGGACGTCGATGAA3’ (the nucleotide - change is underlined). Purification of Proteins-LexA repressor (14) and the mutant derivatives of LexA protein; GD80, GV80, VM82, AT84, VF115, GE117, MT118, SA119, KR156, and KA156, (19) were purified as described. The carboxyl-terminal cleavage fragment was purified as described (23). Purified proteins were stored in buffer D (IO mM Pipes-NaOH, pH 7.0, 0.1 mM EDTA, 200 mM NaCI, and 10% glyc- erol). The protein concentration of LexA’ was determined spectro- photometrically using .&,, M-’ . cm-’ (24). Due to a slightly

lower level of protein purity in the pr
lower level of protein purity in the preparations of mutant proteins (approximately 90 to 95% purity of mutant proteins versus approxi- mately 99% purity of LexA’), the concentrations of the mutant proteins were estimated by visual examination of SDS-polyacryl- amide gels (25) stained with Coomassie Blue (26). The estimated concentrations may vary from actual levels by much as 20%. ‘The scheme for naming the LexA mutants makes use of the single-letter designation for the amino acids. The first letter denotes the amino acid residue in the wild-type protein, and the second letter designates the amino acid substitution. The number specifies the position in the LexA amino acid sequence where the mutation has occurred. Thus, in the mutant protein MT118, a threonine residue replaces the normally occurring methionine residue at amino acid 118 of the LexA sequence. Chemicals and Enzymes-CNBr and trypsin were obtained from Sigma. Trifluoroacetic acid, acetonitrile, and isopropyl alcohol were HPLC-grade and were obtained from Aldrich and was dissolved in isopropyl alcohol to a concentration of 1 M. [1,3-3H]DFP was obtained from Du Pont-New England Nuclear or Amersham Corp. and was provided dissolved in propylene glycol. The specific activity varied between lots from 3.0 to 5.8 Ci/mmol. We found no qualitative differences in our results between [1,3-3H]DFP obtained from either source. Because the [3H] DFP was provided in propylene glycol, we tested the effects of several concentrations of propylene glycol on [3H]DFP incorporation by LexA. We found that [3H]DFP incorporation was not inhibited by propylene glycol concentrations of up to 11% (data not shown). However, at a propylene glycol concentration of 20% (Figs. 8 9), there was a 20% decrease in the total number of

counts incorporated (data not shown). Es
counts incorporated (data not shown). Estimation of the Stability of DFP-[3H]DFP (6.6 pM, 3.0 Ci/ mmol) was incubated at 37 “C in the presence of either 50 mM CAPS, pH 10.5, or 400 mM CAPS, pH (time 0) and at subsequent 15-min intervals, and to 90- J reaction mixtures, such that the final concentration of reactants was 0.66 pM [3H]DFP (at time 0), 21 pM trypsin, 100 mM ammonium bicarbonate, pH 7.8, and mM CaC12. After incubation for 10 min at 37 “C, reactions were stopped by the addition of 1 ml of cold 5% trichloroacetic acid and the mixture kept on ice for at least 20 min. The samples were further treated and analyzed by electrophoresis as described for Fig. 2. Protein bands were cut out of the gels and incorporation of [3H]DFP into trypsin was determined by scintillation counting, as described (20). The half-life of [3H]DFP was estimated by plotting the log of counts incorporated into trypsin versus time. The half-life in 50 mM CAPS, pH 10.5, mM CAPS, pH 10.5, was 30 min, respectively. These values are shorter than those previously reported (approximately 1 h, Ref. 27), but those authors measured stability at 25 “C with a different buffer. The difference in half-lives at the two concentrations of CAPS may be to general base catalysis (28). In experiments at 20 mM DFP, it was necessary to use 400 mM CAPS: 50 mM CAPS did not adequately buffer the reactions (data not shown), since a proton is released upon hydrolysis of DFP. Purification and Analysis of PHIDIP Peptides-For preparative digests, LexA protein was reacted with DFP (final volume 1 ml) in a solution whose final composition was 90 pM LexA, 20 mM [3H]DFP (5 mCi/mmol), 400 mM CAPS, pH 10.5, 2% isopropyl alcohol, 10% propylene glycol, and 48% buffer D. After incubation at

37 “C for 30 min, 4 ml of 100 mM
37 “C for 30 min, 4 ml of 100 mM ammonium bicarbonate, pH 7.8 was added, and the sample was dialyzed at mM ammonium bicarbonate, pH 7.8. The LexA-DIP formed a white precipitate during dialysis, but this did not interfere with subsequent digestions. For analytical digests, the labeling reactions were the same, except the protein concentration was 9 /LM. For digestion with trypsin, 4 ml of dialyzed material (approximately 1.5 mg) was used. Twenty ~1 of trypsin (5 ng/Fl), dissolved in 100 mM ammonium bicarbonate, pH 7.8, was added. The sample was heated to 65 “C for 10 min. Another 20 ~1 of trypsin was added the sample was incubated for 3 at 37 “C. Twenty ~1 of trypsin was added after the first and second hours of incubation. The sample was dried under vacuum, washed twice with 1 ml of water, and suspended in 0.1% trifluoroacetic acid. The 3H-labeled peptide was purified on Varian (Vista Series) model 5000 HPLC with a Vydac C,s reverse phase column equilibrated with 0.1% trifluoroacetic acid (solution A). For each purification DFP Inhibits LexA Autodigestion ml cyanogen hromide (29). For preparative digests, the protein con- centratlon was 1 me/ml. For analvtical dieests of LexA-[‘HIDIP. A min+ 0 -,4,Orr~~OD~ MT118-[“HIDIP, and the purified tryptic peptide, the protein’con: centrations were 05, 0.5, and 0.025 mg/ml, respectively. The labeled cyanogen bromide peptide was purified hy HPLC on Vydac C,$ column and eluted as described for the isolation of labeled tryptic peptide, except that the gradient was started at 10% solution B and increased at a rate of 0.2%/min. Peak fractions were pooled and either run again using the same conditions for further purification or dried under vacuum to a small volume for amino acid analysis. Analyti

cal Seyuence Analysis-Amino acid analy
cal Seyuence Analysis-Amino acid analysis of purified peptides was carried out using an Applied Biosystems 420A Derivatizer/Analyzer Amino Acid Analyzer. Amino acid se- quencing of purified peptides was done Applied Biosystems 477A Protein Sequencer. RESULTS LexA Autodigestion Is Inhibited by DFP-Although pre- vious attempts to inhibit LexA autodigestion with DFP were unsuccessful (see Introduction), a more sensitive assay showed an interaction between LexA and DFP. When 15 ELM LexA protein was incubated with various concentrations of [ ‘H]DFP at pH 10.5, we found that a constant proportion of DFP, roughly 0.04%, reacted with LexA after a lo-min incu- bation (data not shown). Therefore, we reasoned mM DFP, about 50% of the LexA should react, resulting in an easily detectable fraction of the mole- cules resistant to autodigestion. When the DFP concentration was increased to 20 mM, we found that the extent of auto- digestion was significantly diminished (Fig. 2), although the initial rate of autodigestion did not appear greatly reduced (compare the l-, 3-, and 5-min time points between panels A and B). As a control for this experiment, DFP was inactivated by preincubation in 400 mM CAPS, pH 10.5, at 37 “C for 120 min before adding the LexA (the half-life of DFP under these conditions was 10 min; see “Materials and Methods”). This material had mM DFP, only about 3% of the LexA would have been resistant to autodigestion. This minor resistant fraction would not have been obvious by visual inspection of Coo- massie Blue-stained gels, since those experiments were de- signed to examine the effect of DFP on initial rates, and therefore LexA autodigestion was not followed to completion.” DFP Reacts Selectively with Serine mM [‘HIDFP

(5 mCi/mmol). Unreacted [‘H]DFP was
(5 mCi/mmol). Unreacted [‘H]DFP was removed by dialysis, and the labeled LexA protein was digested with trypsin. The LexA-[“HIDIP digest was fractionated on Cl8 reverse-phase HPLC column. A major peak, representing 63% of the total recovered radioac- tivity, was observed (Fig. 3, peah II). The peak was further purified. Amino acid analysis of the purified peak yielded the following composition: Ala, Gly, Lys, 2 Ser, and Met. This composition is consistent with that expected from the pre- dicted sequence of the tryptic peptide (7-mer) that contains Ser-119 (30, Val-Ser-Gly-Met-Ser-Met-Lys 115 ’ ,J. W. Little, unpublished results B NI-, -(IEponm FIG. 2. Kinetics of inhibition of LexA autodigestion by DFP. These SDS-polyacrylamide gels show LexA autodigestion in the presence (panel A) or absence (panel R) of 20 mM DFP. The reactlon volume was 400 ~1, and the final concentration of components was 15 @M LexA, 400 mM CAPS, pH 10.5, 20 mM DFP. 5% isopropyl alcohol, and 50% buffer D. All components except CAPS were com- bined and incubated at 37 “C for 3 min. An aliquot was taken (24 pl), and then prewarmed 1 M CAPS, pH 10.5, was added with rapid mixing to a final concentration of 400 mM. Incubation was continued at 37 “C. Aliquots (40 ~1) II 2,000 - 100 ** x! .- ** 1,500 - .' .- ** g r .* ,- z B ** 3 1,000 - -* .- -50 m a- .* .' .* .- 500 2. III 10 Fractton Number FIG. 3. Radioactivity The asterisk denotes the proposed nucleophile, Ser-119. This tryptic peptide is the only one in LexA with the composition indicated (30, The amino acid sequence of the peptide was then deter- mined by Edman degradation to be the sequence shown above. Only 25% of the radioactivity applied to the sequencer was recovered in the Edman degradation fractions, presum