Vol. 174, No. 19

JOURNAL OF BACTERIOLOGY, OCt. 1992, p. 6207-6214

0021-9193/92/196207-08$02.00/0 Copyright © 1992, American Society for Microbiology

Structural Characterization and Corepressor Binding of the Escherichia coli Purine Repressort KANG YELL CHOI AND HOWARD ZALKIN* Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153 Received 27 April 1992/Accepted 27 July 1992

The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine. We have characterized the purified repressor. Chemical cross-linking indicates that PurR is dimeric. Each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal 289-residue domain for corepressor binding. Each domain was isolated after cleavage by trypsin. Sites for dimer formation are present within the corepressor binding domain. The corepressors hypoxanthine and guanine bind cooperatively to distinct sites in each subunit. Competition experiments indicate that binding of one purine abolishes cooperativity and decreases the affinity and the binding of the second corepressor. Binding of each corepressor results in a conformation change in the corepressor binding domain that was detected by intrinsic fluorescence of three tryptophan residues. These experiments characterize PurR as a complex allosteric regulatory protein.

Escherichia coli purR encodes a 38-kDa repressor protein (37) that regulates the expression of 10 pur regulon genes, which are required for de novo synthesis of IMP (11, 27), and guaBA, which are required for conversion of IMP to GMP (27). The purine repressor, PurR, also contributes to the coregulation of at least five additional genes: pyrC and pyrD, which are involved in synthesis of pyrimidine nucleotides (7, 49); glyA, which is required for generating one-carbon units for biosynthesis (42); codA, which encodes cytosine deaminase, a pyrimidine salvage enzyme (2, 17); and autoregulation of purR (27, 39). Hypoxanthine and guanine serve as corepressors in vivo (15, 28) and in vitro for binding of PurR to a 16-bp conserved operator sequence in the promoter (11, 27, 37) or coding (12, 39) region of the coregulated genes. A procedure for overproduction and purification of PurR has been reported (38). A 32-kDa PurR corepressor-binding domain has been crystallized, and a structure determination is in progress (41). Here we report a biochemical characterization of PurR which includes determination of quaternary structure, separation and isolation of functional domains, and analysis of corepressor binding and resultant changes in conformation.

with a 30 to 200 mM potassium phosphate (pH 7.4) gradient. The pooled fraction of repressor from the DEAE-Sepharose column was dialyzed against 30 mM KPO4 (pH 7.4) prior to chromatography on heparin-agarose. The repressor eluted from heparin-agarose at approximately 160 mM potassium phosphate and was concentrated to 10 mg/ml with an Amicon Centriprep concentrator (30-kDa cutoff). A concentration of 160 mM potassium phosphate is required to maintain solubility at this protein concentration. Corepressor-binding and DNA-binding domains were prepared by digestion of purified PurR with trypsin. PurR in 160 mM potassium phosphate (pH 7.4) was treated with 0.5% tosyl phenylalanine -chloromethylketone-treated trypsin for 10 min at room temperature. The proteolytic reaction was stopped with 1 mM phenylmethylsulfonyl fluoride, and proteolysis was checked by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. Domains were separated by gel filtration on Sephacryl-200 equilibrated with 160 mM potassium phosphate (pH 7.4). The corepressor-binding and DNA-binding domains were concentrated with Amicon Centricon membranes having 30-kDa and 3-kDa cutoffs. The separated domains were usually stored at -20°C in 160 mM potassium phosphate (pH 7.4), although high ionic strength was not required to maintain solubility of the concentrated corepressor-binding domain. The solubility properties of the DNA-binding domain were not investigated. Chemical cross-linking. Protein cross-linking with imidoesters was carried out as described by Davis and Stark (8). Reaction mixtures containing 2 mg of cross-linker (dimethyl adipimidate, dimethyl pimelimidate, or dimethyl suberimidate) per ml, 2 mg of PurR per ml, and 0.2 M triethanolamine (pH 8.0) were incubated at room temperature for various times. Reactions were stopped by addition of an equal volume of a solution containing 0.125 Tris-Cl (pH 6.8), 4% SDS, 10% mercaptoethanol, 20% glycerol, and 0.1% bromophenol blue, and the mixes were stored at -20°C until analysis by SDS-polyacrylamide gel electrophoresis. Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and rabbit muscle aldolase, cross-linked by the same procedure, served as reference proteins. In some experiments, crosslinked PurR was recovered from the reaction mixture. Ap-

MATERIALS AND METHODS Purification of aporepressor and separated domains. PurR was overproduced in E. coli BL21(pPR1010) as described previously (38). The repressor was purified to homogeneity by two chromatographic steps with DEAE-Sepharose and heparin-agarose (38), with the following modifications. Cells were suspended in buffer solution containing 30 mM potassium phosphate (pH 7.4), 0.2 mM dithiothreitol, 0.1 mM EDTA, and 5% glycerol for preparation of the extract. Immediately prior to disruption in the French press, 1 mM phenylmethylsulfonyl fluoride was added. Both chromatographic columns were equilibrated with 30 mM potassium phosphate (pH 7.4). Elution from both columns was done *

Corresponding author.

t Journal paper number 13489 from the Purdue University Agri-

cultural Experiment Station.

6207

6208

CHOI AND ZALKIN

proximately 1.0 mg of PurR was incubated with dimethyl suberimidate for 3 h as described above. Insoluble material was removed by centrifugation for 1 min in a microcentrifuge, and the protein was isolated by centrifugal gel filtration with Sephadex G-50 (22). DNA-binding assay. Corepressor-dependent binding of PurR to purF operator DNA was routinely assayed by gel retardation with modified buffer system II (38). Glycerol was omitted from the previous formulation of buffer system II, and the dimethyl sulfoxide concentration was reduced to 2%. In some experiments, buffer system I (38), which permits repressor binding to purF operator in the absence of corepressor, was used. Equilibrium dialysis. Each 300-,u chamber contained 200 VI of buffer system II. One chamber contained 1.8 or 10 puM aporepressor- or corepressor-binding domain, and the other contained 1.2 to 18 ,uM '4C-labeled guanine or 10 to 100 puM '4C-labeled hypoxanthine. In some experiments, an unlabeled competitor purine was included. Radioactive purines were purchased from Moravek Biochemicals, Inc. Spectra/ Por membranes (12 to 14 kDa, cutoff) were soaked in 0.5 M EDTA for several days and washed thoroughly with distilled water prior to use. Dialysis was done for 16 h at 4°C in a rotating apparatus. Samples (160 pI) were counted for radioactivity. Molecular weights of 38,000 and 32,000 were used to calculate the subunit concentrations of the aporepressorand corepressor-binding domains, respectively. Binding data were analyzed in three ways: (i) fractional saturation versus free purine, (ii) Scatchard plot (40), and Hill plot (14). Data for the saturation plot, in which there was positive cooperativity, were fit to the equation Y = nl[1+(KIL)H], by using Sigma Plot software, where Y is the fraction saturation, n is the number of ligand-binding sites per monomer, K is the value for half-saturation, L is the concentration of free ligand, and H is the Hill coefficient. Data were fit to the Scatchard and Hill equations with the Macintosh Cricket Graph program. The number of binding sites, n, and the Kd were determined by Scatchard plot. The Hill coefficient was determined from the Hill plot. Intrinsic tryptophan fluorescence. Fluorescence spectra were recorded with a 1-cm quartz cuvette at 25°C in a Hitachi F-2000 fluorescence spectrophotometer equipped with a xenon lamp and thermostated cell holder. The excitation wavelength for emission spectra was 275 nm. Incubation mixtures (1.0 ml) contained modified buffer system II and 1.0 FM PurR or 1.25 ,uM corepressor-binding domain. In some experiments, 2 to 18 ,uM guanine or 1 to 30 ,uM hypoxanthine was added. All samples were incubated at room temperature for 2 h prior to measurement of fluorescence. Hypoxanthine and guanine, when excited at 275 nm, did not fluoresce but quenched protein fluorescence. To correct for nonspecific quenching by hypoxanthine and guanine, the fluorescence of three proteins (egg albumin, human gamma globulin, and egg white lysozyme) was analyzed. Standard curves for nonspecific quenching were determined for each of the reference proteins by titration with guanine and hypoxanthine, and average quenching factors were determined. The quenching correction was added to the fluorescence change of the repressor to obtain the final percent fluorescence changes that are plotted in Fig. 9. Maximal corrections of 10.6% at 16 p,M guanine and 8.4% at 30 ,uM hypoxanthine were applied to the fluorescence titrations of purine repressor for the experiments in Fig. 9. Protein analyses. PurR protein concentration was determined by using a value of A280 (1%) of 12.9 (38). The method of Lowry (19) was used to determine the concentrations of

J. BACTERIOL. 1

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FIG. 1. Chemical cross-linking of PurR. Protein samples were electrophoresed on an SDS-polyacrylamide gel and stained with Coomassie blue. Lane 1, glyceraldehyde-3-phosphate dehydrogenase; lane 2, PurR prior to cross-linking; lanes 3 to 7, cross-linked PurR after 15 min, 30 min, 1 h, 2 h, and 3 h, respectively; lane 8, cross-linked aldolase. The protein standards in lanes 1 and 8 were cross-linked for 1 h. Short arrows (left and right) mark cross-linked species of the reference proteins. Long arrows (right) mark monomeric (arrow 1) and dimeric (arrow 2) PurR.

the corepressor-binding and DNA-binding domains and purified reference proteins and for purification of PurR. Purified corepressor-binding and DNA-binding domains were prepared for N-terminal sequencing by dialysis against 30% formic acid. Approximately 1 nmol of each protein was used for six cycles of Edman degradation in an Applied Biosystems model 470 sequenator. The purity of proteins was assessed by SDS-polyacrylamide gel electrophoresis

(48). RESULTS

Oligomeric state. The oligomeric structure of PurR was determined by chemical cross-linking. Of a series of imidoesters (dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate), maximal cross-linking was obtained with dimethyl suberimidate. Representative cross-linking by dimethyl suberimidate, visualized by SDS-polyacrylamide gel electrophoresis, is shown in Fig. 1. Multimeric crosslinked PurR species were identified by comparison with two tetrameric proteins, glyceraldehyde-3-phosphate dehydrogenase (lane 1), and aldolase (lane 8). The multimer patterns for the reference proteins are similar to those reported previously (8). Figure 1, lane 2, shows the 38-kDa PurR monomer prior to cross-linking. As the period of crosslinking with dimethyl suberimidate was increased from 15 min to 3 h (Fig. 1, lanes 3 to 7), the monomer was consumed and the major species that was formed corresponded to a dimer. At all reaction times, there were two PurR side reactions. (i) Small amounts of intramolecularly cross-linked monomer were visible below the band corresponding to native monomer. (ii) More highly cross-linked species, initially corresponding to trimer, tetramer, and higher multimer forms, were visible after 15 min (Fig. 1, lane 3), and levels were maximal at 30 min (Fig. 1, lane 4). By 3 h (Fig. 1, lane 7), most of this material was further cross-linked into insoluble aggregates, which were removed by centrifugation prior to electrophoresis. Guanine and hypoxanthine interact with PurR and are required for specific binding of the repressor to pur gene operator sites (38). Guanine and hypoxanthine had no effect on the rate of cross-linking of PurR dimer and did not influence the pattern of side products (data not shown).

E. COLI PURINE REPRESSOR

VOL. 174,- 1992

6209

100 80-

0 1-

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Repressor, uM FIG. 2. DNA-binding curve of native (0) and cross-linked (@) PurR. Binding of PurR to 30 fmol of purF operator DNA was determined by gel retardation and was dependent upon guanine corepressor. The band containing free DNA was counted for radioactivity. The horizontal line marks the position for 50% binding.

Purine corepressors are therefore unlikely to convert the aporepressor monomer to the holorepressor dimer. To test function, a preparation of PurR that was more than 90% cross-linked dimer was isolated by gel filtration and assayed by gel retardation for DNA binding. Under standard conditions in which binding to purF operator DNA is dependent upon the addition of purine corepressor, approximately 0.4 ,uM PurR was required for half-maximal binding, compared with 0.2 ,uM for native, untreated repressor (Fig. 2). The decreased DNA-binding effectiveness of cross-linked PurR dimer may be due to structural perturbations resulting from intra- or intersubunit cross-links. Overall, these experiments support the conclusion that apo-PurR is dimeric and that, upon interaction with guanine or hypoxanthine, the resultant holorepressor can bind to pur gene operator DNA. Domain structure. PurR is subject to a single proteolytic cleavage by an endogenous E. coli protease (data not shown). This specific cleavage, which takes place during purification or in aged preparations, was inhibited by phenylmethylsulfonyl fluoride. In order to determine the site of cleavage by serine protease, purified repressor was treated with limiting amounts of trypsin. The data in Fig. 3 show 1

2

3

4

5

6

38 kDa 32 kDa

6 kDa

FIG. 3. Specific proteolytic cleavage of PurR. Samples containing 1 ,ug of PurR per p.l were treated with 0.3% (wt/wt) trypsin as described in Materials and Methods. The samples were resolved by electrophoresis on a 10% polyacrylamide-SDS gel and stained with Coomassie blue. The digestion times were: lane 1, none; lane 2, 1 min; lane 3, 3 min; lane 4, 5 min; lane 5, 10 min; lane 6, 15 min. Arrows mark the positions of the 38-kDa PurR monomer, 32-kDa corepressor-binding domain, and 6-kDa DNA-binding domain.

FIG. 4. Separation of corepressor-binding and DNA-binding domains by gel filtration. The conditions for digestion with 0.5% trypsin are given in Materials and Methods. Domains were resolved on a Sephacryl-200 column (1.6 by 100 cm). Peak A contains pure corepressor-binding domain, and peak B contains pure DNA-binding domain.

nearly quantitative conversion of the 38-kDa monomner to proteins of approximately 32 and 6 kDa. No other products were detected. Preparative isolation of the 32- and 6-kDa subunits was achieved by gel filtration (Fig. 4), and the proteins were subjected to Edman degradation. By this analysis, the small fragment starts with Ala-2, the native PurR N terminus (38), and the large fragment starts with Ser-53. This result suggests that trypsin cleaves an N-terminal DNA-binding domain, residues Ala-2 to Arg-52, from a larger corepressor binding domain, residues Ser-53 to Arg341. By the criterion of chemical cross-linking, the corepressor-binding domain retained sites for subunit interaction. The overall pattern of dimethyl suberimidate cross-linking was virtually identical to that for native repressor, with the major product corresponding to cross-linked dimer (data not shown). Neither the isolated DNA-binding domain nor the corepressor-binding domain interacted with purF operator DNA even in buffer conditions that bypass the corepressor requirement (38) for DNA binding (data not shown). These results establish that the corepressor-binding domain provides contacts for dimerization which appear to be necessary for DNA binding. This conclusion is supported by an X-ray diffraction analysis of the corepressor-binding domain in which crystals diffracting to a resolution of at least 2.2 A contain two monomers per asymmetric unit (41). Equilibrium-binding measurements of corepressors to aporepressor. Guanine or hypoxanthine is required for specific binding of PurR to pur gene operator sites in vitro (28, 38) and for repression in vivo (15, 28). We have investigated the interaction of guanine and hypoxanthine with aporepressor by equilibrium-binding and fluorescence assays. Equilibrium dialysis measurements were carried out to quantitate the binding of guanine and hypoxanthine to aporepressor and to investigate the relationship between binding sites for the two corepressors. Data are shown for guanine binding in Fig. 5 and for hypoxanthine binding in Fig. 6. The curvature in the saturation plots for binding given in Fig. 5A and 6A suggests cooperativity. Cooperativity for guanine and hypoxanthine is more clearly apparent by the curvature of the Scatchard plots shown in Fig. SB and 6B, respectively. Hill coefficients of 1.5 for guanine and for hypoxanthine were calculated from the data in Fig. SC and 6C, respectively. The number of guanine and hypoxanthine binding sites was determined from the Scatchard plots in Fig. 5B and 6B. Extrapolation yields maximal binding of 1.0 eq of guanine and 1.1 eq of hypoxanthine per subunit. Ligand-binding

6210

CHOI AND ZALKIN

J. BACTERIOL.

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FIG. 5. Equilibrium binding of guanine (Gua) to PurR. (A) Saturation curve. PurR (0.925 FM monomer) was incubated with various guanine concentrations (0.6 to 9 p,M), and binding was determined by equilibrium dialysis. Y, mol fraction bound. (B) Scatchard plot. (C) Hill plot. n is the extrapolated number of guanine sites per monomer.

affinities, estimated from values for half-saturation, were 1.5 FM for guanine and 9.3 ,uM for hypoxanthine. These values are in close agreement with apparent affinities of 1.7 and 7.1 ,M for guanine and hypoxanthine, respectively, that were previously determined from measurement of corepressordependent repressor-operator binding (38). Overall, these results are consistent with cooperative binding of guanine and hypoxanthine to single sites on each subunit of the dimeric repressor, but the data do not distinguish whether the two purines bind to a common site or two unique sites on each subunit. To determine the relationship between the sites for guanine and hypoxanthine, we examined the effect of one purine on the binding of the other. The Scatchard plot in Fig. 7A shows the effect of four levels of guanine on the binding of radioactive hypoxanthine. Addition of guanine had three distinctive effects on hypoxanthine binding. (i) Increasing levels of guanine initially reduced and finally abolished cooperativity between sites for hypoxanthine. As summarized in Table 1, the Hill coefficient for hypoxanthine binding was reduced from 1.5 to 1.3 by 0.5 ,uM guanine. At higher

Log Free Hyp, uM

FIG. 6. Equilibrium binding of hypoxanthine (Hyp) to PurR. (A) Saturation curve. PurR (5.0 pLM monomer) was incubated with various hypoxanthine concentrations (5 to 50 FM), and binding was determined by equilibrium dialysis. (B) Scatchard plot. (C) Hill plot.

concentrations of guanine, the Hill coefficient for hypoxanthine binding became 1.0, indicating complete loss of cooperativity. (ii) Increasing levels of guanine decreased the extrapolated saturation by hypoxanthine from 1.1 to 0.6 eq. (iii) In the absence of cooperativity, the Kd for hypoxanthine binding was increased from 9.3 to 12 to 18 p,M (Table 1). Similar results were obtained for guanine binding. Added hypoxanthine abolished cooperativity between guanine sites and also reduced maximal binding (Fig. 7B). In this case, however, addition of 12.5 ,uM hypoxanthine caused anomolous binding that extrapolated to 4.4 eq of guanine per subunit. The interpretation of this result is not apparent. Higher levels of hypoxanthine, however, resulted in a stepwise decrease in guanine binding. Qualitatively similar results were obtained with 6-mercaptopurine, a hypoxanthine analog, as a competitor purine (data not shown). Upon elimination of cooperativity, the Kd for guanine was increased to 11 to 16 ,uM (Table 1). Overall, these results indicate that binding of one purine abolishes the cooperativity and inhibits the binding of the second purine. Once the cooperativity is abolished, inhibition of binding results from reduced availability of sites, without a change in affinity. These properties are expected for noncompetitive inhibition in a protein having separate sites for guanine and hypoxan-

E. COLI PURINE REPRESSOR

VOL. 174, 1992

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FIG. 7. Scatchard plot showing competition of one purine with the binding of the other. (A) Effect of guanine on the binding of hypoxanthine (Hyp). Hypoxanthine binding was determined as for Fig. 6 except for the addition of various concentrations of guanine competitor. Guanine concentrations: 0, none; *, 0.5 p,M; A, 3.75 10 FM. (B) Effect of hypoxanthine on the p,M; 0, 6.25 ,M; binding of guanine (Gua). Hypoxanthine concentrations: 0, none; *, 12.5 pM; A, 25 pM;0, 100 pM. 0,

thine. Furthermore, it is striking that although guanine has a significantly higher affinity for repressor than hypoxanthine does, as a mixture, the purines have similar Kd values. Equilibrium binding of corepressors to isolated corepressorbinding domain. The binding characteristics of the two purines to the isolated corepressor-binding domain were similar to those of native PurR. Binding of approximately 0.73 and 1.0 eq of guanine and hypoxanthine, respectively, was determined from Scatchard plots (Table 1). Binding was cooperative, consistent with the dimeric structure deter-

Protein

Native PurR

mined for this domain. Hill coefficients of 1.4 were calculated for both guanine and hypoxanthine (Table 1). Values for half-saturation of 2.1 and 7.6 ,M were determined from the plots for guanine and hypoxanthine, respectively. These data indicate that cooperative binding of guanine and hypoxanthine is retained in the corepressor-binding domain. Conformational perturbation accompanying corepressor binding. We have used intrinsic tryptophan fluorescence as a probe of conformation to investigate how binding of guanine and hypoxanthine increases holorepressor-pur gene operator affinity. PurR contains four tryptophan residues, at positions 37, 98, 147, and 215 (37). Trp-37 is in the DNA-binding domain, whereas the three other tryptophan residues are in the corepressor-binding domain. The peak for PurR fluorescence excitation was at 275 nm (not shown) and for PurR fluorescence emission was at 340 nm (Fig. 8). The fluorescence emission spectra in Fig. 8A and B show two perturbations upon binding of guanine and hypoxanthine, respectively: (i) a long-wavelength shift in the peak of maximal fluorescence from 340 to 342 nm, and (ii) increased fluorescence intensity. The increased fluorescence intensity, corrected for quenching as described in Materials and Methods, exhibited saturation. Saturation curves are given in Fig. 9. The apparent affinity, estimated from half-saturation, was 2.0 and 5.0 ,uM for guanine and hypoxanthine, respectively. The correspondence of these estimates of apparent affinity with the values obtained from equilibrium-binding measurements indicates that perturbations of fluorescence reflect changes in conformation resulting from binding of guanine and hypoxanthine to PurR corepressor sites. As an initial approach to localizing the conformational perturbations, the fluorescence titrations were repeated with the isolated corepressor-binding domain. Excitation and emission spectra for the corepressor-binding domain were similar to those of the aporepressor, with fluorescence maxima at 275 and 337 nm, respectively. The maximal fluorescence emission for an equivalent concentration of the corepressor-binding domain was 86% of that of apo-PurR. Titration with guanine and with hypoxanthine each gave a wavelength shift from 337 to 341 nm and increased fluorescence intensity. The increased fluorescence intensity is shown as a function of purine concentration in Fig. 9. These data show that the interaction of guanine and hypoxanthine with the corepressor-binding domain resulted in a larger structural perturbation than was obtained with native PurR. Values for apparent affinity of 4.0 and 8.0 ,uM for binding of guanine and hypoxanthine, respectively, were calculated

TABLE 1. Summary of corepressor binding data Affinity' (>M) Competitor (>M) Ligand

Hypoxanthine

No. of sites

Hill coefficiente

Hypoxanthine (12.5) Hypoxanthine (25.0) Hypoxanthine (100)

0.75 12 13 12 18 1.5 ± 0.42 16 11 16

1.1 1.0 1.0 0.78 0.60 1.0 4.4 1.9 1.1

1.5 ± 0.04 1.3 1.0 1.0 1.0 1.5 + 0.09 1.0 1.0 0.9

None None

7.6 ± 0.64 2.1 ± 0.85

1.0 0.73

1.4 ± 0.03 1.4 + 0.06

None Guanine (0.50) Guanine (3.75)

Guanine (6.25) Guanine (10.0) Guanine

Corepressor-binding domain

Hypoxanthine Guanine

6211

None

9.3

+

a Determined from half-saturation (mean of two or three experiments + standard deviation) or Kd from a single linear Scatchard plot. b Values are means for two or three experiments ± standard deviation or are from a single Hill plot.

6212

CHOI AND ZALKIN

200.0

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350 400 Wavelength (nm) FIG. 8. Effect of corepressor on intrinsic tryptophan fluorescence emission. Each sample contained 1 ,uM PurR or 1.25 RM corepressor-binding domain. Emission fluorescence spectra were measured with different concentrations of (A) guanine (0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 ,uM) and (B) hypoxanthine (0, 3, 6, 9, 12, 15, 18, 21, 27, and 30 ,uM). In each case, the lowest curve is the emission spectrum without corepressor. Maximum fluorescence intensity was obtained with 10 ,uM guanine and 27 ,uM hypoxanthine. Higher concentrations of guanine and hypoxanthine lowered the emission spectra due to nonspecific fluorescence quenching.

from these data. These results thus indicate that the conformational change resulting from the binding of purine, as monitored by intrinsic tryptophan fluorescence, resides in the corepressor-binding domain. The environment of tryptophan residues is in some way restrained by the DNAbinding domain, so that larger perturbations can result from corepressor binding when the DNA-binding domain is excised. DISCUSSION The interaction of the E. coli purine repressor with guanine and hypoxanthine results in the formation of a holorepressor which binds to a conserved 16-bp operator sequence and regulates the expression of a series of genes involved in the synthesis of purine and pyrimidine nucleotides (2, 7, 11, 17, 27, 42, 49). The overall picture that emerges from the biochemical analyses in this study is that of a complex allosteric regulatory protein. PurR is a homodimeric protein in which each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal domain of 289 residues for corepressor binding. The amino acid side chain interactions required for dimerization are found within the corepressor-binding domain. Guanine and hypoxanthine bind cooperatively to distinct sites on each subunit; however, binding of one purine abolishes cooperativity and decreases the affinity and binding of the second corepressor. Binding of each purine corepressor is accompanied by a conformational change in the corepressor-binding domain. Here we provide a further analysis of these properties and

0 S

0-

10

20

30

Hypoxanthine, uM

FIG. 9. Effect of corepressor on fluorescence emission intensity for PurR (0) and corepressor-binding domain (-) for binding of (A) guanine and (B) hypoxanthine. The data obtained from the measurement of highest intensity for each corepressor concentration (from Fig. 8) were corrected for quenching as described in Materials and Methods.

the relationship of PurR to other bacterial repressor proteins. PurR is a member of the lac repressor family, which includes LacI (4, 9), GalR (46), RbsR (25), MalI (36), RafR (3), RbtR (50), CytR (44), EbgR (43), CcpA (13), and FruR (16, 45). Sequence identities suggest that these proteins are homologous (26, 30, 37, 45). Each is a DNA-binding protein having an N-terminal helix-turn-helix motif. Sequence analyses (26, 30, 47) and structure profiles (5) indicate that a C-terminal sugar-binding domain in the lac, gal, and mal repressors and the corepressor-binding domain in PurR are structurally similar to those of periplasmic sugar-binding proteins for arabinose (10), galactose (29), and ribose (47). These analyses thus predict a two-domain structure for the lac repressor family. Proteolytic separation of PurR DNA and corepressor-binding domains by cleavage after Arg-52 is comparable to the earlier-reported (35) separation of the corresponding domains in Lacd by cleavage after Lys-59 and provides direct support for a two-domain structure. PurR residue Arg-52 is likely in a short, exposed hinge region, since cleavage by trypsin was not detected after Lys-55 or Lys-60. Chemical cross-linking experiments have provided evidence that PurR is predominantly, if not entirely, dimeric. Although small amounts of monomeric or higher oligomeric states would escape detection by this procedure, we consider their existence unlikely. Since binding of corepressor did not influence chemical cross-linking and corepressor binding was cooperative, monomers should not contribute to the observed binding of guanine or hypoxanthine. We sus-

VOL. 174, 1992 pect that an earlier M, estimate of 56,000 by gel filtration (38)

resulted from the interaction of protein with the gel matrix rather than from a monomer-dimer equilibrium. On the other hand, if functional tetramers were possible, we would expect eventual cross-linking of dimer to tetramer. This conversion was not seen, and therefore the formation of functional tetramers is unlikely. PurR does not contain a C-terminal leucine zipper motif similar to that which is involved in LacI tetramer formation (1, 6). Tetramer formation in LacI is required for DNA loop formation resulting from binding of the protein to two operators (18, 32). PurR, on the other hand, resembles GalR, which is also dimeric (21). In the only case in which PurR interacts with dual operators, DNA looping was not detected (39). Since the isolated PurR corepressor-binding domain is dimeric and binds corepressors with positive cooperativity, similar to the native aporepressor, this domain contains the side chain interactions required for dimerization. In contrast to LacI (23, 24), the PurR corepressor-binding domain does not bind to specific operator sites. Rather, the corepressorbinding domain would appear to have two roles in modulating the affinity for DNA: (i) provide sites for dimer formation and (ii) utilize a conformation change that results from binding of corepressor to increase the affinity of the DNAbinding domain for specific pur gene operator DNA. The PurR DNA-binding domain, in contrast to the 59-residue lac repressor DNA-binding domain (33), does not exhibit highaffinity binding to operator DNA. The formation of purine holorepressor requires the binding of guanine, hypoxanthine, or both purines to the aporepressor. The utilization of guanine and hypoxanthine to sense availability of purine nucleotides for biosynthesis demonstrates the important relationship between nucleotide degradation and uptake as sources of purine bases (31) and the de novo and salvage pathways for purine nucleotide biosynthesis. Here we have focused on a biochemical characterization of the interaction of the purine corepressors with aporepressor. There are two key elements for this interaction: (i) cooperative binding of guanine or hypoxanthine to independent sites and (ii) loss of cooperativity and an increased Kd for a mixture of guanine and hypoxanthine. Cooperative binding of guanine and hypoxanthine amplifies the response to low concentrations of a single purine corepressor. As a result of cooperativity, the affinity of the repressor for a purine corepressor was increased. The presence of hypoxanthine abolished cooperativity and resulted in an approximately 10-fold decreased affinity for guanine (Table 1). The mechanism by which one purine abolishes cooperative binding of the second purine is not understood but likely involves a conformational alteration. Measurements of intrinsic tryptophan fluorescence have provided direct evidence for a conformation change in the corepressor-binding domain that results from binding of the purine. A simple one-site model, in which binding of a purine, guanine for example, to a single site on one subunit prevents cooperative binding of the second purine, hypoxanthine, to the corresponding site on the other subunit, appears to have been excluded by the data in Fig. 7A. These data show that guanine inhibits hypoxanthine binding without a change in Kd and conform to a model for noncompetitive inhibition. Noncompetitive inhibition for binding of one purine by a second purine leads to a model in which each subunit has distinct sites for guanine and hypoxanthine. In attempting to ascribe physiological significance to the complex data for corepressor-repressor binding, we are

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struck by the similarity in binding affinities for the two purine corepressors in a mixture. Thus, the level of repression should be relatively independent of the hypoxanthine/guanine concentration ratio. This is in contrast to situations in which only a single purine corepressor is present. In this case, guanine will half-saturate the repressor at sixfold-lower concentrations than hypoxanthine. To further understand PurR function, it will be necessary to identify the sites involved in binding guanine and hypoxanthine and to determine how the corepressor-dependent conformation change that is detected by tryptophan fluorescence modulates the affinity for operator DNA. The study of the X-ray structure of the corepressor-binding domain which is in progress (41) should contribute important information. ACKNOWLEDGMENTS We thank Henry Weiner for help with tryptophan fluorescence experiments and helpful comments on the manuscript. This work was supported by Public Health Service grant GM 24658 from the National Institutes of Health. Protein sequence analyses were performed by the Laboratory for Macromolecular Structure, supported by the Diabetes Research and Training Center (PHS grant P60DK20542). REFERENCES 1. Alberta, S., S. Oehler, B. von Wilcken-Bergmann, H. Kramer, and B. Muller-Hill. 1991. Dimer-to-tetramer assembly of lac repressor involves a leucine heptad repeat. New Biol. 3:57-62. 2. Andersen, L., M. Kilstrup, and J. Neuhard. 1989. Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K12. Involvement of theglnLG andpurR genes in the regulation of codA expression. Arch. Microbiol. 152:115118. 3. Aslanidis, C., and R. Schmitt. 1990. Regulatory elements of the raffinose operon: nucleotide sequences of operator and repressor genes. J. Bacteriol. 172:2178-2180. 4. Beyreuther, K. 1978. Revised sequence for the lac repressor. Nature (London) 274:767. 5. Bowie, J. V., R. Luthy, and D. Eisenberg. 1991. A method to identify protein sequences that fold into a known three-dimensional structure. Science 253:164-170. 6. Chakerian, A., V. M. Tesmer, S. P. Manly, J. K. Brackett, M. J. Lynch, J. T. Hoh, and K. S. Matthews. 1991. Evidence for leucine zipper motif in lactose repressor protein. J. Biol. Chem. 266:1371-1374. 7. Choi, K. Y., and H. Zalkin. 1990. Regulation of Eschenchia coli ,pyrC by the purine regulon repressor protein. J. Bacteriol. 172:3201-3207. 8. Davis, G. E., and G. R. Stark. 1970. Use of dimethyl subrimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc. Natl. Acad. Sci. USA 66:651-656. 9. Farabaugh, P. J. 1978. Sequence of the lacI gene. Nature (London) 274:765-769. 10. Gilliland, G. L., and F. A. Quliocho. 1981. Structure of the L-arabinose-binding protein from Escherichia coli at 2.4 A resolution. J. Mol. Biol. 146:341-362. 11. He, B., A. Shiau, K. Y. Choi, H. Zalkin, and J. M. Smith. 1990. Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction. J. Bacteriol. 172: 4555-4562. 12. He, B., J. M. Smith, and H. Zalkin. 1992. Eschenichia coli purB gene: cloning, nucleotide sequence, and regulation by PurR. J. Bacteriol. 174:130-136. 13. Henkin, T. M., F. J. Grundy, W. L. Nicholson, and G. H. Chambliss. 1991. Catabolite repression of alpha-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lacI and galR repressors. Mol. Microbiol. 5:575-584. 14. Hill, A. V. 1910. The possible effects of the aggregation of the molecules of hemoglobin on its dissociation curves. J. Physiol.

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