THE JOURNAL OF BIOLOGICAL CHIZMISTRY Q 1989 by The American Society for Biochemista and Molecular Biologv, Inc

Vol. 264, No. 27, Issue of September 25, pp. 16067-16071,1989 Printed in U.S.A.

Evidence from Extended X-ray Absorption Fine Structure and Site-specific Mutagenesis for Zinc Fingers in UvrA Protein of Escherichia cloZi*

(Received for publication, April 27,1989)

Suppiah NavaratnamS, Gary M. Myless, Richard W. Strangell, and Aziz Sancaren From the $Research Diuision, North East Wales Institute, Clwyd CH5 4BR, United Kingdom, IISERC Daresbury hboratory, Warrington WA4 4AD. United Kingdom, and the §Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill, North CarolAa 27599

The UvrA protein is the damage recognition subunit of the Escherichia coli repair enzyme ABC excision nuclease. Sequence ;analysis of this 940-amino acid protein revealed two regions of sequence homology to the zinc finger motif found in many DNA binding pro- teins. Physical and chemical analyses indicate about 2 zinc atoms/molecule. We have used extended x-ray ab- sorption fine structure analysis to demonstrate that each of these zinc atoms is coordinat%d with 4 cysteine residues at a distance of 2.32 f 0.2 A. Substitution of one of the cysteines by a histidine, a serine, or an alanine in one of the potential finger sites resulted in a respective decrease in complementing activity. We thus conclude that the two zinc fingers identified by sequence analysis do indeed have zinc finger structure in UvrA protein.

Nucleotide excision repair in Escherichia coli is initiated by an ATP-dependent enzyme complex, the ABC excision nu- clease. The complex has three subunits, UvrA (Mr = 103,874), UvrB ( M , = 76,118), alnd UvrC (Mr = 66,038). The enzyme removes a wide variety of carcinogen adducts from DNA by incising on both sides of the damaged nucleotides, 7 bases away on the 5’ side and 3-4 bases away on the 3‘ side, thus producing a 12-mer whkh is released from the DNA (Sancar and Rupp, 1983). The enzyme has been characterized in considerable detail (see Sancar and Sancar, 1988, and Weiss and Grossman, 1987, for reviews). The UvrA subunit is an ATPase (Kacinski et al., 1981; Seeberg and Steinum, 1982) and a DNA binding protein (Sancar et al., 1981) with higher affinity for UV-irradiated DNA (Seeberg and Steinum, 1982), and it appears to be the damage recognition subunit of the enzyme as the other two subunits have no significant affinity for DNA in the absenc:e of UvrA. The uurA gene has been cloned and sequenced (Husain et al., 1986). Computer analysis of the sequence reveakd, in addition to two ATP binding sites, the amino acid sequence Cys-X-X-Cys-Xls-20-Cys-X-X- Cys in two places at residues 253-280 and 740-766 of the 940- amino acid protein (Doolittle et al., 1986) reminiscent of zinc

* This work was supported by National Institutes of Health Grant GM32833 and in part by grants from the Burroughs Wellcome Travel Fund and the Science and Engineering Research Council, United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this, fact.

7 To whom corespondence should be addressed Dept. of Biochem- istry, CB No. 7260, University of North Carolina School of Medicine, Chapel Hill, NC 27599. Tel.: 919-962-0115.

finger structure (Miller et al., 1985). Preliminary results in- dicated that UvrA contained intrinsic zinc atom(s); however, the environment of zinc in UvrA was not determined in the previous study and therefore physical evidence was lacking for coordination of zinc with amino acids to form zinc fingers in UvrA. In this study, we have used both physical and genetic methods to obtain strong evidence for the presence of two zinc fingers in UvrA.

Extended x-ray absorption fine structure (EXAFS)’ has been successfully used to obtain detailed structural informa- tion on metal centers in a variety of metalloproteins (Hasnain et al., 1984; Abrahams et al., 1986; Diakun et al., 1986; Hasnain et al., 1987; Fredman et al., 1988; Navaratnam et al., 1988). EXAFS provides interatomic distances, the number and type of atoms, and the statistical and/or thermal disorder of the shells of atoms around a metal in a metalloprotein (Teo, 1981). In this study we have used EXAFS to show that the zinc atoms in UvrA are ligate$ to 4 cysteine residues at a mean distance of 2.32 k 0.02 A. When one of the cysteines thought to be coordinated with zinc was replaced with His, Ser, or Ala, a protein with reduced activity was obtained. Thus, the physical and genetic data taken together support the prediction made from sequence analysis that UvrA con- tains two DNA binding zinc fingers.

MATERIALS AND METHODS

Purification of UvrA Protein-Approximately 1 g of UvrA protein was purified from an overproducing strain by a simplified form of previously published procedures (Sancar and Rupp, 1983; Thomas et al., 1985). Briefly, cells were lysed by sonication, and the cell debris was removed by centrifugation at 100,000 X g for 1 h. The clear supernatant (in 0.1 M KCl) was applied to a DNA-cellulose (Sigma) column (50-ml column bed for 50 g of cells as starting material). The column was washed with 5 column volumes of loading buffer (50 mM Tris-HC1, pH 7.5, 100 mM KCl, 1 mM EDTA, 20% glycerol, 5 mM mercaptoethanol), and UvrA was eluted with 1 M KC1 in the same buffer. The yield was approximately 5 mg of UvrA/g of cells a t about 95% purity.

Quantitation of Zinc in UurA-The zinc content of UvrA was determined by two methods, inductively coupled plasma analysis and the colorimetric method. Inductively coupled plasma analysis (con- ducted by Dr. John Olesik, Department of Chemistry, University of North Carolina) gave (5.7 2 2.0) X M and (16 f 4) X M zinc per 5 X M UvrA in two independent measurements. The colori- metric assay was conducted as follows (Giedroc et al., 1986, 1987). A 600-p1 sample of 10 p M UvrA in 10 mM Tris-HC1, pH 8.0, 200 mM NaCl, 5% glycerol (v/v) was titrated with 3.5 mM of the sulfhydryl agent P-chloromercuriphenylsulfonate (Sigma) to release cysteine- bound metal. The protein precipitated during titration, the supernate was removed and the pellet was washed with buffer, and both the

The abbreviation used is: EXAFS, extended x-ray absorption fine structure.

16067

16068 Zinc Fingers in UvrA Protein supernate and the wash were used for the colorimetric assay which was conducted by adding 10 mM 4-(2-pyridylazo)resorcinol (Sigma) to the solution and reading absorption at 500 nm. From the absorption of the supernate and the wash, the concentration of zinc was calcu- lated using a standard curve generated with ZnSOa. We obtained about 1.6 mol of Zn2+/mo1 of UvrA by this method. In both inductively coupled plasma analysis and the colorimetric assay, the zinc contents of buffers used as blank were below detection limits. Taking into account both the inductively coupled plasma and colorimetric assay results it appears that 2 zinc atoms/UvrA molecule is the best estimate. The concentration of UvrA preparations used in these determinations was measured spectrophotometrically based on the calculated molar extinction coefficient of the protein, eZw = 46,500 M" cm" (Husain et al., 1986).

EXAFS Analysis-For EXAFS studies the enzyme was concen- trated in a collodion bag SM13200 (Sartorius membrane filter, Got- tingen, Federal Republic of Germany) to an approximate concentra- tion of 3 mM. EXAFS measurements were carried out at the zinc k- edge (9.661 keV) as fluorescence excitation spectra by using the fluorescence detection system (Hasnain et al., 1984; Hasnain and Garner, 1987) on the Wiggler beam line (station 9.2) a t the Synchro- tron Radiation Source, Daresbury Laboratory. The Synchrotron Ra- diation Source was operating at an energy of 2.0 GeV with an average current of 150 mA. A Si-220 double crystal order sorting monochro- mator was used to minimize harmonic contamination in the mono- chromatic beam. Copper foils were placed in front of the NaI detector to reduce scattered radiation. EXAFS data was recorded from a frozen sample at 77 K using a nitrogen cryostat. A number of scans were recorded, and each spectrum was checked individually and averaged until no further improvement in the quality of data was observed. No radiation damage was observed as no changes in the edge of EXAFS were seen between spectra. After calibration of monochromator po- sition to energy, the EXAFS was normalized to a unit zinc atom and extracted from the background absorption. The EXAFS thus obtained was then converted to k-space and analyzed using the nonlinear least squares minimization program EXCURV 88 (Binstead et al., 1988), which calculates the theoretical EXAFS using fast curved wave theory (Gurman et al., 1984). Atomic phase shifts were calculated by ab initio method as described previously (Lee and Pendry, 1975; Perutz et al., 1982). Curve fitting was carried out on k3-weighted raw EXAFS data by varying coordination number and atom types and iteratively refining the shell radii and Debye-Waller-type factors to a minimum in the fit index.

Site-specific Mutagenesis-The Cys-253 of UvrA which is presumed to be one of the 4 cysteines coordinated with zinc to make the first zinc finger in the protein was changed to other residues by site- specific mutagenesis. Mutagenesis was carried out by the method of Zoller and Smith (1983) as modified by Kunkel (1985). Briefly, the uurA gene was first inserted into pIBI24N, an NcoI site containing derivative of pIBI24 (International Biotechnologies, Inc.) and intro- duced into E. coli CJ236 (dut- ung-). Uracil containing template of pIBI24N-uurA was obtained by superinfection with M13K07 (Vieirra and Messing, 1987). Synthetic oligonucleotides (18-mers) bearing the appropriate mismatches were prepared on an Applied Biosystems DNA synthesizer model 380 and used without further purification. Mutants were screened by dideoxy sequencing using Sequenase" obtained from United States Biochemical Corp. Plasmid DNAs from the mutant pIBI24N-UvrA derivatives were then purified, and the mutant uurA genes were inserted on a NcoI-PstI fragment into pKK2332 (Pharmacia LKB Biotechnology Inc.) expression vector.

In Vivo Characterization of Mutant Proteins-pKK2332-derived plasmids carrying either the wild-type or mutant uurA genes were inserted into UNC522 (NM522 uurA::TnlO). Cultures of the appro- priate strains were grown (without induction) overnight in Luria broth, dilutions were plated on Luria agar plates containing ampicillin (200 pg/ml), and the plates were irradiated with various doses of UV (254 nm) light from a germicidal lamp. The plates were then incubated at 37 "C for 24 h, and the colonies were counted to obtain the UV survivals.

RESULTS

EXAFS of UvrA Protein-Fig. 1 shows the near edge and EXAFS spectrum of UvrA. The strong oscillations observed in the high energy region is indicative of back scattering from a heavy atom such as sulfur. Fig. 2A shows a comparison between the experimental and that calculated from the pa-

L:

9600 9700 9800 9900 10000 101 00 Energy (eV)

FIG. 1. Normalized EXAFS spectrum of zinc in UvrA.

rameters given in Table I; the corresponding Fourier trans- forms are given in Fig. 2B. Data analysis was limited to 10.5 8," because of glitches in the high energy region. The overall profile of the experimental EXAFS is typical of back scatter- ing fromesulfur atoms and could be fitted with 4 sulfur atoms at 2.32 A with a value for Debye-Waller-type factor, which Gescribes the effects of static and thermal disorder, of 0.008 A2. Simulations which included low zinc atoms instead of sulfur gave calculated EXAFS oscillations which were about 180" out of phase with experimental EXAFS. Negative values for the Debye-Waller factor were obtained when simulations were carried out with 2 sulfur and 2 or more low zinc atoms, indicating that zinc in UvrA is coordinated to 3-4 sulfur atoms. There are a number of examples where zinc is coordi- !ated to 3-4 sulfur atoms (see Hasnain, 1988) at about 2.32 A. We compared the EXAFS data of UvrA with that of zinc- 7-metallothionein from rabbit (Hasnain et al., 1987). Both the EXAFS and the Fourier transform of metallothionein fitted very well with those of UvrA as seen in Fig. 3, A and B. Hasnain et al. (1987) have proposed that zinc in metallothi- onein is coordinated to 4 sulfur atoms at 2.31 8, with Debye- Waller factor of 0.005 8,' which i t in good agreement with our data for UvrA. The value of 2.32 A for the zinc-sulfur distance in UvrA is also in very good agreement with that observed in glucocorticoid receptor DNA binding domain (Freeman et al., 1988) and the prototype "zinc finger" protein, the Xenopus transcription factor TFIIIA (Diakun et al., 1986). However, the absence of any significant outer shell peaks in the Fourier transform of UvrA together with the good matching with zinc- metallothionein spectrum would suggest that the environment of zinc in UvrA is unlike that in TFIIIA where the zinc is coordinated to 2 cysteine and 2 histidine residues (Diakun et al., 1986), but that the coordination sphere of zinc consists of 4 cysteine residues.

Evidence from Site-specific Mutagenesis for Zinc Finger- Although the EXAFS data and amino acid sequence of UvrA point very strongly toward the regions predicted from the sequence to be indeed zinc fingers, we sought additional evidence for this structure by site-specific mutagenesis. Be- cause the two putative fingers apparently arose by gene du- plication (Doolittie et al., 1986), we concentrated our efforts on the first finger in the sequence, assuming that any results obtained with this finger would be equally applicable to the second finger. We replaced one of the 4 cysteines presumed

Zinc Fingers in UvrA Protein 16069

1.5

1.0

0. 5

Y

x 0. 0 (I) LL

5 -0.5 W

-1.0

-1.5

- 2 . 0 -1 V

3 4 5 6 7 8 9 10

Energy (A" )

I. e

I . 6

1 . 4

m I . 2 U J

1.0 -J

Q 0 . 0

c- LL 0. 6

1 2 3 4 5 6

R ( A ) FIG. 2. Zink k-edge lr3-weighted, EXAFS ( A ) and its phase

corrected Fourier transform of UvrA protein ( B ) . Solid lines, represent experimental data; dashed lines represent theoretical single scattering simulations with parameters given in Table I.

TABLE I Parameters used for simulations of the EXAFS g iven in Fig. 2.

Atom type No. R 2' '[distance to zinc) (Debye-Waller factor)

A A' Sulfur 4 2.32 0.008

to coordinate with zinc with histidine, serine, or alanine. The resulting proteins were stable in E. coli as evidenced by overproduction of mutant proteins to levels comparable to that of the wild-type proteins (Fig. 4), suggesting that any functional differences ,would be solely due to the local effect of amino acid replacement. The effect of mutagenesis of protein function was tested by measuring the UV survival of cells containing the mutant proteins. Note that survival meas- urements were not conducted under inducing conditions and therefore all proteins were produced at constitutive levels which presumably are the same for wild-type and mutant proteins. The results are presented in Fig. 5. Two conclusions emerge from this daba. First, replacement of Cys with His,

1.5

1.0

Y

X

0.5

m 0. 0 L < X

-0 .5

- I . 0

-1.5

4 5 6 7 8 9 IO Energy ( A ' )

1

1.5

3 3 u J : . o - d

Q E < 7

L- 0.5

0. 0

1 2 3 4 5 4 7 8 9 1 0

R ( A ) FIG. 3. EXAFS spectra at zinc k-edge of UvrA (solid lines)

and of zinc-7-metallothionein from rabbit (dashed lines) ( A ) and their respective s phase-corrected Fourier transforms ( B ) .

Ser, or Ala only partially reduces the activity of the protein suggesting that if this Cys is involved in zinc finger formation, zinc coordinated with 3 sulfur atoms must be able to maintain the finger structure. Second, the His mutant is more active than the Ser mutant which is more active than the Ala mutant. The His residue is known to coordinate metals and may do so in the mutant. It is also conceivable that the hydroxyl group of Ser may coordinate zinc; however, Ala, which is isosteric with Cys, has no metal binding capacity. Thus, the fact that His, which is structurally quite dissimilar from Cys, confers near wild-type activity upon the mutant, whereas replacement by the two isosteric amino acids Ser and Ala results in less active protein, and the fact that Ser, which may have a partial chelating activity, is somewhat better substitution than Ala lend strong support to the prediction based on amino acid sequence that UvrA has two zinc fingers and that the 4 sulfur-zinc ligations detected by EXAFS are the cysteines shown in Fig. 4.

16070 Zinc Fingers in UvrA Protein

kD - 116 - 914 - 66

- 45

- 29

FIG. 4. Overproduction of mutant proteins. Cells carrying a plasmid with either the wild-type (W/T) UurA gene or with a uurA gene with the indicated mutations were grown in Luria broth to Am = 0.6 and induced with 1 mM isopropyl 0-D-thiogalactoside for 12 h. Cells from 200 liters of induced culture were collected by centrifuga- tion, and total cellular protein content was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacryl- amide) followed by staining with Coomassie Blue. Purified UvrA protein and a commercial protein mixture containing proteins of indicated molecular weights ( M W STDS) were used as standards. The amino acid substitution at Cys253 (C253) by His (H), Ser (SI, and Ala ( A ) are as indicated. AUurA, strain with a deletion of uurA gene.

NH, C '

1 I

253 I

'C c' ' C COOH I I I I

280 740 766 940

WILD-TYPE C253H C253S C253A AUvrA

UV SURVIVAL

5.4 x 10" 4.1 x 10" 1.1 x 10" 8.0 x 10-2 5.8 X 10-7

FIG. 5. Location of zinc fingers in UvrA and effect of mu- tations in the first finger on cell survival. The cell survivals at 300 ergs/mm* are averages from two experiments with stationary phase cells. The absolute values were variable depending on growth phase of cells; however, the relative order of UV sensitivity was under all conditions tested.

DISCUSSION

Since the description of the zinc finger as a structural motif for binding DNA in the Xenopus transcription factor TFIIIA, similar motifs have been found in over 50 actual or potential DNA binding proteins (Klug and Rhodes, 1987; Evans and Hellenberg, 1988). Zinc fingers are proposed to be made up of a 12-20-amino acid-long loop stabilized at the base with zinc

tetrahedrally coordinated to 4 Cys, 3 Cys and 1 His, or 2 Cys and 2 His. In contrast to the abundance of sequence infor- mation regarding this interesting motif, of perhaps more versatility than the helix-turn-helix DNA binding motif, ge- netic and/or structural studies on the proposed structure have been few. In E. coli, T4 phage gene 32 protein zinc was replaced by cobalt and from the spectral properties of the cobalt-containing protein (a set of absorption bands in the near UV-visible characteristic of tetrahedral cobalt (11) com- plexes), it was concluded that, in native protein, zinc was coordinated with 3 Cys and 1 His residues (Giedroc et al., 1986). However, it has since been shown that the zinc finger in gene 32 protein is involved in protein-protein interaction

but is not essential for DNA binding (Giedroc et al., 1987). The second (out of nine) finger of TFIIIA was synthesized in E. coli as a fusion protein and then separated by cyanogen bromide cleavage, and its folding and spectroscopic properties were investigated (Frankel et al., 1987). It was found that the peptide folds only in the presence of zinc or cobalt. However, the folded peptide had no DNA binding activity. The first physical evidence for the reality of zinc fingers as structural motifs came from the work of Diakun et al. (1986), who obtained evidence from EXAFS that there were 7-9 zinc atoms/TFIIIA molecule an! that these were coordinated to 2 Vitrogen atoms (His) at 2 A and 2 sulfur atoms (Cys) at 2.32 A distances. A similar study was conducted on the DNA binding region of the glucocorticoid receptor, and it was established that the two putative zinc fingers in this region did indeed contain two zinc atoms, each coordinated to 4- cysteine residues (Freedman et al., 1988). The EXAFS study on the glucocorticoid receptor was complemented by another study using site-specific mutagenesis (Severne et al., 1988) in which the amino acids with zinc-ligating potential were re- placed with Ala, Ser, or His. It was found that replacement of Cys with His at certain positions reduced the activity to 15-54%, whereas at others it completely abolished it. Simi- larly, replacement with Ala or Ser of any of the cysteines thought to be involved in ligating zinc completely abolished activity.

In this paper, we have presented both EXAFS and site- specific mutagenesis data to prove that the two zinc finger motifs identified by sequence analysis of UvrA protein are in fact zinc fingers. The EXAFS data only yields the atoms coordinated to heavy metals but it does not differentiate which atoms (amino acids) in the protein are ligated to the metal. Such information can only be obtained from crystal structure, or the amino acids may be identified by site-specific mutagen- esis. In fact because of this shortcoming of EXAFS, the amino acids ligated to zinc in the second finger of the glucocorticoid receptor were misidentified in the EXAFS study on that protein (Freedman et al., 1988). The correct assignment was made possible by the site-specific mutagenesis study (Severne et al., 1988). We believe our assignment of the amino acids coordinated to zinc in UvrA is correct because it is supported by genetic evidence. In addition, in UvrA the base of the finger does not contain any Cys or His residues other than the 4 Cys proposed to be ligated and therefore there is no alternative ligation patterns.

In light of these facts, the phenotypes of the cells carrying the mutant proteins were somewhat surprising. It has been reported that simultaneous replacement of 2 Cys by 2 His in the estrogen receptor inactivates the protein (Green and Chambon, 1987), Similarly in glucocorticoid receptor Cys + His substitutions resulted in 6-54% of wild-type receptor activities depending on the substituted cysteine (Severne et al., 1988). In this same protein substitution of any of the

Zinc Fingers in UvrA Protein 16071

cysteines by Ser or Ala resulted in near total loss of activity. However, both the study with the estrogen receptor and that with the glucocorticoid receptor were conducted in uiuo, and the amounts of mutant proteins produced were not measured and therefore it is conceivable that some of these results may be due to global change in protein conformation and/or deg- radation due to improper folding. In contrast, in this study we have shown that the mutant proteins are folded properly as evidenced by comparable levels overproduction. Therefore, we conclude that the observed phenotypes are of significance. Based on the phenotypes of mutants, we are inclined to think that in the Cys -P Ala mutant the zinc is coordinated only to 3 Cys residues and that this form must be reasonably stable to give near 20% activity. We also think that Cys + Ser may result in partial coordination of the hydroxyl group of Ser to give the reproducibly higher activity compared to the Cys -P

Ala substitution. Finally, in contrast to the glucocorticoid receptor, it appears that His can be a reasonable substitute for Cys in the first zinc: finger of UvrA protein. More impor- tantly, the activity pattern of these three mutants is entirely consistent with the predicted structure with the zinc fingers and identifies the Cys residues that are known to be ligated according to EXAFS.

Acknowledgments-We are grateful to Wen& Carlton for excellent technical help, Dr. J. Olesik for carrying out inductively coupled plasma analysis, and Dr. S’. S. Hasnain for making available to us the metallothionein data.

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