A Novel Zinc Snap Motif Conveys Structural Stability to 3-Methyladenine DNA Glycosylase I*

Keehwan Kwon, Chunyang Cao and James T. Stivers {ddagger}

From the Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185

Received for publication, January 28, 2003 , and in revised form, March 19, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The Escherichia coli 3-methyladenine DNA glycosylase I (TAG) is a DNA repair enzyme that excises 3-methyladenine in DNA and is the smallest member of the helix-hairpin-helix (HhH) superfamily of DNA glycosylases. Despite many studies over the last 25 years, there has been no suggestion that TAG was a metalloprotein. However, here we establish by heteronuclear NMR and other spectroscopic methods that TAG binds 1 eq of Zn2+ extremely tightly. A family of refined NMR structures shows that 4 conserved residues contributed from the amino- and carboxyl-terminal regions of TAG (Cys4, His17, His175, and Cys179) form a Zn2+ binding site. The Zn2+ ion serves to tether the otherwise unstructured amino- and carboxyl-terminal regions of TAG. We propose that this unexpected "zinc snap" motif in the TAG family (CX12–17HX~150HX3C) serves to stabilize the HhH domain thereby mimicking the functional role of protein-protein interactions in larger HhH superfamily members.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Excision repair of damaged bases in DNA is one pathway that cells use to protect the genome from the damaging effects of ionizing radiation, reactive oxygen species, and alkylating agents (1). Lesion repair begins by enzymatic hydrolysis of the glycosidic bond, the critical initiating step of the DNA base excision repair pathway. There are many different types of DNA glycosylases in cells. In general, each DNA glycosylase exhibits specificity for a cognate damaged base while ignoring undamaged bases in DNA. One alkylated base, 3-methyladenine, is highly toxic, because DNA replication is blocked at this lesion site (2). In Escherichia coli, the glycosidic bond of 3-methyladenine is hydrolyzed by two enzymes, 3-methyladenine DNA glycosylase I (TAG)1 and II (AlkA) (3).

It has been recently discovered that TAG is a member of the helix-hairpin-helix (HhH) superfamily of DNA glycosylases (4). The HhH motif is a sequence-nonspecific DNA binding module found in DNA polymerases, NAD+-dependent DNA ligases, and some DNA glycosylases (5). This motif consists of two {alpha}-helices connected by a consensus hairpin loop that interacts nonspecifically with the DNA backbone. Unlike other HhH family members, TAG does not possess the consensus hairpin sequence, (L/F)PG(V/I)G, nor does it contain the conserved aspartate group that was previously believed to be required for catalysis as a water activating group or, alternatively, for stabilization of a transition state with glycosyl cation character. Thus, TAG appears to be unique with respect to structure and mechanism within this superfamily (4).

Although it has been concluded previously that TAG does not require a metal ion for activity (6), the possibility of a metal binding site was suggested by our recent NMR structure and a coincident bioinformatics study (4, 7). According to the solution structure, two conserved sulfur and histidine ligands at opposing ends of the linear sequence are closely positioned in three-dimensional space (Cys4, His17, His175, and Cys179) (4, 7), but given the limitations of the NMR method, the presence of a metal ion was not established. As shown in Fig. 1, the sequence alignment of the TAG family shows that these potential metal ligands are also completely conserved across all species. Taken together, these observations clearly suggested the presence of a metal ion binding site. Here we establish this hypothesis and present the structure of a long overlooked Zn2+ binding site in TAG. The metal site stabilizes the structure of TAG by "snap-ping" together the largely unstructured N- and C-terminal regions. This "zinc snap" motif (CX12–17HX~150HX3C) is distinct from that of zinc finger proteins or the zinc binding sites in other DNA repair proteins such as formamidopyrimidine DNA glycosylase, poly(ADP-ribose) polymerase, and UvrA (8, 9, 10, 11). We propose that the minimal HhH domain is intrinsically unstable and that the zinc snap provides the stabilizing forces that are assumed by intramolecular protein-protein contacts in other larger HhH family members.



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FIG. 1.
Conservation of Cys2His2 cluster in the N- and C-terminal regions of the TAG family. The sequences of the TAG family were obtained by running a BLAST search using the E. coli TAG sequences. The alignment of the partial sequences was generated using T-COFFEE, version 1.41 (25). In the consensus sequences, an asterisk indicates identical amino acid residues and a colon indicates highly conserved residues. The metal-binding Cys2His2 cluster, CX12–17HXnHX3C, is highlighted in black. The NCBI gene identification (GI) numbers of the various TAG sequences are shown next to the names of the species.

 


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Enzyme Purification—Wild-type TAG was overexpressed from pET21a(TAG) in BL21(DE3) and was purified by two sequential chromatography steps as described previously by Drohat et al. (4). A His6-tagged version was overexpressed from pET28a(TAG) in BL21(DE3)pLysS and purified using a nickel-nitrilotriacetic acid-agarose column. The amino-terminal tag was cleaved and removed using biotinylated thrombin, streptavidin agarose resin, and a second nickel-nitrilotriacetic acid-agarose column. Final purification was achieved using a Poros 20-S HPLC column (Applied Biosystems, Foster City, CA) using a linear gradient of 0.1–1 M NaCl. The purity of TAG was >95% by both procedures.

Atomic Absorption Spectroscopy (AAS)—The presence of zinc in the holoenzyme was determined using a PerkinElmer model 370 atomic absorption spectrophotometer. The purified enzyme was dialyzed extensively against buffer A (50 mM Tris-HCl, 200 mM NaCl, pH 7.5) containing 4 ml of hydrated chelex-100 resin (Sigma)/500 ml of buffer. A 2-ml volume of TAG (2 or 5 µM) was injected into the atomic absorption spectrophotometer with monitoring at the absorption maximum for Zn2+ (213.5 nm). AAS on apoTAG was performed by the following procedures. Partial removal of Zn2+ from holoTAG was achieved using nondenaturing conditions by dialysis of the enzyme against low pH buffer B (20 mM MES, 200 mM NaCl, pH 5.5) containing 50 mM EDTA and chelating resin. Complete removal of Zn2+ was achieved by denaturing the enzyme in 0.1 M HCl and then separating the denatured apoenzyme from the released Zn2+ by gel filtration chromatography (PD-10, Amersham Biosciences). The TAG concentration was determined by UV absorbance using a calculated extinction coefficient of 30,800 M-1cm-1 in a buffer containing 20 mM sodium phosphate (pH 6.0) and 6 M guanidinium chloride.2 Standard solutions of ZnCl2 were used to calibrate the atomic absorption spectrophotometer.

UV-visible Absorption Spectroscopy of Co2+ Complex—A solution of TAG with 78% of the Zn2+ removed was prepared by dialysis against buffer B as described above. To remove EDTA and raise the pH, the enzyme (100 µl, 25 µM total protein) was exchanged into buffer C (20 mM Tris-HCl, 100 mM NaCl, pH 7.5) using two sequential Bio-Gel P-6 gel filtration columns (Bio-Rad). An absorption spectrum of the 78% apoTAG sample (as determined from AAS) was collected in the wave-length range of 320 to 800 nm using a Beckman DU 640 spectrophotometer. Then, 1 molar eq of CoCl2 was added quickly, and the spectrum was reacquired. A polynomial base-line subtraction was used to correct the spectrum for light scattering by apoTAG.

NMR Spectroscopy—The NMR sample, prepared as described in a previous report (4), contained ~0.5 mM TAG, 10 mM phosphate buffer (pH 6.6), 100 mM NaCl, 3 mM dithiothreitol, 0.34 mM NaN3, and 10% D2O in a total volume of 300 µl. The two-dimensional 1H-15N LR HSQC experiment was conducted at 20 °C on a Varian Unity Plus 600-MHz spectrometer equipped with four frequency channels and pulse-field gradients as described (12). This experiment was simply a conventional HSQC used for backbone amide correlations collected with an optimized two-bond 2JNH value of 22 Hz in order to observe signals from the weak two-bond couplings in the histidine rings and suppress the signals from the one-bond JNH amide couplings. The 15N dimension was collected with 256 complex points, a 120-ppm sweep width, 128 scans, and the 15N carrier set at 205 ppm. The 1H dimension was collected with 1024 complex points and a 13.3-ppm sweep width centered at 4.82 ppm. Decoupling of 15N was accomplished with the WALTZ16 sequence (13) using a 5.1-kHz field.

NMR Structural Calculations—Starting from the published NMR structure of TAG (Protein Data Bank code: 1LMZ [PDB] ), which was originally refined without any constraints to Zn2+, a set of 100 structures that included restraints from the enzyme ligands to Zn2+ were calculated using the program XPLOR-NIH, version 2.0.23 (14). These calculations employed the same NOE constraints, dihedral angle, and H-bond restraints as the original structures. Additionally, because of new assignments obtained from the 1H-15N LR-HSQC experiment, a total of 11 new NOEs and three new hydrogen bonds were used as restraints in the calculation. From this set, 25 low energy structures were selected with distance restraint violations less than 0.5 Å and dihedral angle restraint violations less than 5o. Constraints between the protein ligands and the zinc ion were added using the procedure of Neuhaus et al. (15). The constraint values were based on tetrahedral coordination, using literature values for the HisN{epsilon},{delta}-Zn and CysS{gamma}-Zn bond lengths of 2.0 and 2.3 Å, respectively, which were obtained from EXAFS (extended x-ray absorption fine structure) measurements (16). The following additional distance constraints were used (15): His17N{epsilon} to His175N{delta}, 3.1 Å < d < 3.5 Å; His17N{epsilon} to Cys4(or Cys179)S{gamma} and His175N{delta} to Cys4(or Cys179)S{gamma}, 3.4 Å < d < 3.8 Å; His175N{delta} to CysS{gamma}, 3.4 Å < d < 3.8 Å; and Cys4S{gamma} to Cys179S{gamma}, 3.6 Å < d < 3.9 Å. The following ligand-zinc bond angles were used as constraints: His17N{epsilon}-Zn-His175N{delta} = His17N{epsilon}-Zn-CysS{gamma} = His175N{delta}-Zn-CysS{gamma} = Cys4S{gamma}-Zn-Cys179S{gamma} = Zn-CysS{gamma}-CysC{beta} = 109.5o, and Zn-HisN{epsilon}-HisC{epsilon} = Zn-HisN{delta}-HisC{epsilon} = 126o. The improper dihedral angle constraints were (Zn-HisN{epsilon}-HisC{epsilon}-HisN{delta}) = (Zn-HisN{delta}-HisC{epsilon}-HisN{epsilon}) = 180o. The zinc atom was given a charge of +2 in the calculations, and the default values for the bond and angle force constants in X-PLOR were employed (500 kcal/mol Å-2 and 70 kcal/mol Å-2, respectively).


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
TAG Has One Tight Zn2+ Binding Site—To determine whether TAG possesses a tight metal binding site, AAS measurements were performed (Table I). Two independently purified samples of TAG that had been dialyzed extensively against a neutral pH buffer containing excess chelating resin showed the presence of 1 molar eq of Zn2+ by AAS analysis (Table I). It was found that exhaustive dialysis of these same samples against a buffer that contained 40 µM CoCl2 (pH 7.5) still retained 1 eq of Zn2+, indicating that the site was inert to exchange under these conditions. Surprisingly, even extensive dialysis of the enzyme against a low pH buffer (pH 5.5) that contained 50 mM EDTA removed only about 0.8 eq of the bound metal. In fact, complete removal of Zn2+ required denaturation of the enzyme using 0.1 M HCl (Table I). These results indicate that previous reports of TAG not requiring a metal ion for activity were due to the very high binding affinity of Zn2+ for this site, which cannot be removed by standard chelating agents that are included in the purification buffers (6, 14). The previously reported dramatic loss in TAG activity below pH 6 may be attributed to metal ion loss and partial protein unfolding (17). We found that the apoTAG was unstable and aggregated extensively at pH 7.5 (data not shown).


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TABLE I

Zn2+ atomic absorption spectroscopy of TAG

 

Geometry and Ligands of the Zn2+ Site—One valuable method for determining the ligands and coordination geometry for a Zn2+ binding site in enzymes is to perform metal replacement with Co2+, a transition metal that gives diagnostic features in its UV-visible spectra (18, 19, 20). For example, thiolate-Co2+ bonds show a diagnostic charge transfer band at about 350 nm with a typical extinction coefficient of 900–1300 M-1cm-1/thiolate ligand (21, 22). Additional bands in the 576–670 nm range are indicative of the coordination geometry; tetrahedral coordination gives a strong signal (E >300 M-1cm-1), whereas pentacoordinate and octahedral sites have much lower molar absorptivities (50 < E < 225 M-1cm-1 and E < 30 M-1cm-1, respectively) (18, 23, 24, 25).

The UV-visible spectrum of Co2+-TAG (Fig. 2) is consistent with two sulfur ligands and a tetrahedral coordination geometry. Because of the extensive aggregation of apoTAG during the Co2+ spectroscopy, the concentration of the soluble protein in the cuvette could not be known precisely. However, comparing the ratio (R) of extinction coefficients of the 350 nm and 617 nm bands (R = E350/E617) after base-line correction still provides quantitative information on the number of sulfur ligands and the coordination geometry. The expected ratio for tetrahedral geometry is 1 < R < 4 for one thiolate ligand, and 2 < R < 9 for two thiolate ligands. The latter value encompasses the E350/E617 = 4 measured for TAG. This value differs considerably from the expected ratios for pentacoordinate and octahedral geometry for two thiolate ligands, which would be 8 <= R <= 52 and R >= 60, respectively. Although these estimates have some uncertainty, they are consistent with the strict conservation of 2 Cys and 2 His residues in the TAG family (Fig. 1), and the structural results described below.



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FIG. 2.
Absorption spectra of apo- and Co2+-TAG. A sample of TAG was prepared in which 80% of the bound Zn2+ was removed, 1 eq of Co2+ was then added, and the UV-visible spectrum was collected (solid line). The spectrum of apoTAG is shown as a broken line.

 

Electronic Properties of the Zn2+ Ligands Determined by Heteronuclear NMR Spectroscopy—Although the Co2+ replacement studies described above indicate the ligation of two sulfur atoms to the Zn2+, the identities of the remaining two ligands are not revealed by this method. Because our previous NMR structure and the homology comparison suggested that His17 and His175 were the remaining ligands (Fig. 1), we performed a 1H-15N LR-HSQC NMR experiment to investigate the electronic properties of these histidines (Fig. 3A; the histidine spin systems in Fig. 3A were obtained from three-dimensional 13C-edited aromatic and aliphatic NOESY experiments). This simple LR-HSQC experiment correlates the carbon-bound protons of the histidine rings with the imidazole nitrogen atoms and can unambiguously establish the tautomeric and protonation states of histidines in proteins (12, 26). The characteristic upside-down and sideways L-shaped patterns for the peaks in the two-dimensional spectrum for His175 and His17 and their well separated 15N chemical shifts indicate that these histidines are in the NE2-H and N{delta}1-H tautomeric forms, respectively, and that both are neutral (Fig. 3, A and B). Thus the nitrogens that are available to coordinate the zinc are the N{delta}1 of His175 and the N{epsilon}2 of His17. The 15N{delta}1 shift for His175 is by far the most deshielded nitrogen of all histidines in TAG, consistent with strong chelation to an electropositive Zn2+. The 15N{epsilon}2 chemical shift of the ligating nitrogen of His17 is also the most deshielded of the five histidines, also indicating strong chelation to the metal from this position. Similarly, the 13C{beta} chemical shifts of Cys4 and Cys179 are the most deshielded of the 8 cysteine residues of TAG, consistent with thiolate metal coordination from these side-chains (Fig. 3C).



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FIG. 3.
NMR studies of the zinc ligands. A, two-dimensional 1H-15N LR-HSQC spectrum of Zn2+-TAG at pH 6.6 and 20 °C. The individual histidine spin systems are connected by solid black lines. The cross-peaks of the residue His122, which are too weak to be observed using the current contour level, are marked with an asterisk. B, the 15N chemical shifts for the N{delta}1 and N{epsilon}2 side-chain atoms and the tautomeric states of the 5 histidine residues in TAG. C, the 13C chemical shifts for the {beta}-carbons of the 8 cysteine residues. The metal ligand is marked in bold characters in B and C.

 

Structure of Zn2+ Binding Site—Because a zinc binding site was entirely unanticipated in TAG, our previous NMR structure did not include any restraints to a Zn2+ atom (Protein Data Bank code: 1LMZ [PDB] ). Therefore, with unambiguous evidence for a Zn2+ site in hand, we further refined the solution structure using the same constraints employed previously but including distance and angle constraints between the Zn2+ and Cys4, His17, His175, and Cys179 (see "Experimental Procedures"). Inclusion of these constraints was justified on the basis of the UV-visible spectroscopy measurements, which indicated two sulfur ligands with tetrahedral geometry, and the LR-HSQC experiments, which indicated coordination of His17N{epsilon} and His175N{delta} to the zinc. The addition of the zinc ion and these new constraints did not increase the Lennard-Jones potential energy for the ensemble of 25 lowest energy structures, nor did it introduce new NOE violations. For the new ensemble, about 85% of the dihedral angles are in the most favored region, with 11% in the additionally allowed region. These statistics are within error of those reported previously; further structural statistics are reported in Table II.


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TABLE II

Structural statistics for 3-methyladenine DNA glycosylase 1 (TAG)

 

The new NMR structure containing Zn2+ is depicted as a ribbon diagram in Fig. 4A and confirms that the Zn2+ binding site tethers the amino and carboxyl termini of TAG. This structure is nearly identical to the deposited TAG structure with r.m.s.d. values of 0.66 Å for backbone atoms and 0.86 Å for all heavy atoms for residues 11–174. The 3-methyladenine binding site, marked with an asterisk in Fig. 4A, is removed from the metal site, precluding any direct involvement of the metal in catalysis. However one metal ligand, His175, is involved in a strong H-bond to the carbonyl oxygen of the active site group, Trp21 (His175 {delta}(NH{epsilon}2) = 14.75 ppm), suggesting that the zinc could indirectly organize the structure of the active site. The detailed structure of the Zn2+ binding site is shown in Fig. 4B (27).



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FIG. 4.
NMR solution structure. A, three-dimensional structure of TAG including restraints to a Zn2+ ion. The signature HhH structural motif is highlighted in green, and the remaining elements of the conserved helical domain of the HhH glycosylase fold are colored blue. The structure elements that are unique to TAG are shown in orange-red, and the 3-methyladenine binding pocket is indicated with an asterisk. B, the tetrahedral Zn2+ binding site of TAG.

 

Role of the Zinc Snap Motif in the Helix-Hairpin-Helix Superfamily—The Zn2+ binding site in TAG is distinct from zinc finger motifs found in bacteria to eukaryotes (28), as well as the zinc binding motifs found in the bacterial 8-oxoguanine glycosylase, MutM (8, 9). The four Cys zinc finger motif in MutM is in the arrangement CX2CX16CX2C and serves to stabilize two {beta}-strands that deliver three polar side chains to their interactions with the phosphodiester backbone (9). In contrast, the zinc motif in TAG consists of two sets of CXnH submotifs, which are separated by 143–151 amino acid residues. This arrangement differs significantly from zinc finger motifs, which cluster in the region of a turn or a loop. The split zinc binding motif, with ligands donated from both termini of TAG, acts as a "snap" for closing the ends of the protein. The zinc binding site is distant from the previously modeled DNA binding region of TAG (4) and serves to stabilize tertiary structure, but it is not involved directly in DNA binding. Given the small size of the domain, the zinc ion provides a critical stabilizing element. This is supported by our observation that the apoprotein appears to be poorly folded, leading to aggregation and eventual precipitation. Difficulties in preparation of the TAG mutant, H17A, because of low expression levels and aggregation (data not shown), are consistent with the conclusion that the Zn+2 binding motif is important for stabilization of the protein. Structural comparisons between TAG and other HhH glycosylases show that all other family members have appendages to their amino and carboxyl termini that interact extensively and are likely to contribute to stabilizing the HhH fold. Therefore, the newly revealed zinc snap motif allows the TAG family to efficiently stabilize the HhH structure without additional protein scaffolding.


    FOOTNOTES
 
The atomic coordinates and structure factors (code 1NKU [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by National Institutes of Health Grant GM46835. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205-2185. Tel.: 410-502-2758; Fax: 410-955-3023; E-mail: jstivers{at}jhmi.edu.

1 The abbreviations used are: TAG, 3-methyladenine DNA glycosylase I; HhH, helix-hairpin-helix; AAS, atomic absorption spectroscopy; NOE, nuclear Overhauser effect; r.m.s.d., root-mean-square deviation; MES, 4-morpholineethanesulfonic acid; NOES, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence. Back

2 us.expasy.org/tools/protparam.html. Back

3 nmr.cit.nih.gov/xplor-nih. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Albert S. Mildvan for assistance with the atomic absorption spectroscopy analysis and Daniel Krosky and Lauren Morgans for helpful comments on the manuscript.



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 ABSTRACT
 INTRODUCTION
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 RESULTS AND DISCUSSION
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