Identification of a zinc finger domain in the human NEIL2 (Nei-like-2) protein.

The recently identified human NEIL2 (Nei-like-2) protein, a DNA glycosylase/AP lyase specific for oxidatively damaged bases, shares structural features and reaction mechanism with the Escherichia coli DNA glycosylases, Nei and Fpg. Amino acid sequence analysis of NEIL2 suggested it to have a zinc finger-like Nei/Fpg. However, the Cys-X2-His-X16-Cys-X2-Cys (CHCC) motif present near the C terminus of NEIL2 is distinct from the zinc finger motifs of Nei/Fpg, which are of the C4 type. Here we show the presence of an equimolar amount of zinc in NEIL2 by inductively coupled plasma mass spectrometry. Individual mutations of Cys-291, His-295, Cys-315, and Cys-318, candidate residues for coordinating zinc, inactivated the enzyme by abolishing its DNA binding activity. H295A and C318S mutants were also shown to lack bound zinc, and a significant change in their secondary structure was revealed by CD spectra analysis. Molecular modeling revealed Arg-310 of NEIL2 to be a critical residue in its zinc binding pocket, which is highly conserved throughout the Fpg/Nei family. A R310Q mutation significantly reduced the activity of NEIL2. We thereby conclude that the zinc finger motif in NEIL2 is essential for its structural integrity and enzyme activity.

Oxidative DNA damage has been implicated in mutagenesis and is suggested to be involved in the etiology of aging and many diseases including cancer (1,2). Repair of oxidatively damaged bases in all of the organisms occurs primarily via the DNA base excision repair pathway, which is initiated with the excision of damaged bases by DNA glycosylases (3). Until recently, only two DNA glycosylases, NTH1 (endonuclease III homolog) and OGG1 (8-oxoguanine DNA glycosylase), have been characterized in mammals, which are responsible for repair of oxidized pyrimidine and purine base lesions, respectively. Both OGG1 and NTH1, orthologs of the Escherichia coli glycosylase Nth, utilize an internal Lys residue as the active site nucleophile and carry out ␤-elimination at the abasic (AP) 1 site generated after base removal (4,5). However, E. coli has two other oxidized base-specific DNA glycosylases, namely MutM/Fpg and its paralog, Nei (6,7), which utilize the Nterminal Pro as the active site nucleophile (8) and carry out ␤␦-elimination at the AP site after excising the base lesion. We and others (9 -13) recently discovered and characterized two other mammalian DNA glycosylases and named these NEIL1 and NEIL2 (Nei-like-1 and -2), which are orthologs of E. coli Fpg/Nei (9 -13). Both NEILs use the N-terminal Pro as the active site and function as a DNA glycosylase/AP lyase to carry out ␤␦-elimination (9,10). The recombinant NEILs are active in excising a variety of oxidatively damaged bases but show significant differences in substrate preference. NEIL1 prefers reactive oxygen species-derived pyrimidines lesions and also efficiently removes FapyG and FapyA, the ring-opened oxidation products of purines (9,12). NEIL2 removes oxidized pyrimidine substrates from duplex DNA but is more efficient in excising oxidized bases when they are located in a DNA bubble structure (14).
NEIL2 shares overall identity of 32 and 27% with Fpg and Nei, respectively, and the key residues of the E. coli enzymes, particularly the N-terminal PE(L/G)P(E/L) motif, are completely conserved in NEIL2 (10). In contrast to the Nth family, the Fpg/Nei family utilizes two DNA binding motifs, a helixtwo-turn-helix (15) and a zinc finger motif (16). Fpg and Nei share significant homology with each other including the sequence of the zinc finger motif, which is of the C4 type (17). The zinc finger motifs are often involved in specific DNA recognition and have been identified in many DNA-binding proteins, transcription factors, and products of developmental control genes (18 -21). Furthermore, several proteins associated with DNA repair, such as Xeroderma Pigmentosum complementation group A, poly(ADP-ribose) polymerase, and replicationassociated protein A (RPA), have been shown to contain zinc finger domains (22)(23)(24)(25). In E. coli, the UvrA protein, which is involved in DNA damage recognition during nucleotide excision repair, also possesses zinc finger domain (26,27). Identification of zinc finger motifs in the ever-growing number of DNA-binding proteins is based primarily on the presence of conserved Cys or His residues and the spacing between them, which may be critical in recognition of specific double-stranded DNA sequences.
Here we show that NEIL2 possesses a single unusual CHCCtype zinc finger motif at its C terminus, which is distinct from that of Nei/Fpg, and that this motif is essential for maintaining the structural integrity and activity of NEIL2.

Expression of Wild-type (WT) and Mutant NEIL2 Polypeptides-The
WT full-length NEIL2 was cloned between the NdeI/XhoI sites of the expression plasmid pRSETB (Invitrogen) (10). The NEIL2 mutants (C291S, H295A, C315S, C318S, and R310A) were generated using a site-directed mutagenesis kit (Stratagene), and their authenticity was confirmed by direct DNA sequencing.
Log-phase cultures of E. coli DE884 mutM nei were transformed with expression plasmids of WT and mutant NEIL2 and then induced with 0.2 mM isopropyl-1-thio-␤-D-galactopyranoside at 16°C for 16 h. After centrifugation, the cell pellets were suspended in a lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 100 g/ml lysozyme, 5 mM dithiothreitol (DTT), and protease inhibitor mixture. After sonication and centrifugation, the supernatant was used for Western blot analysis or activity assay by trapping analysis (10).
Purification of Anti-NEIL2 Antibody and Western Blot Analysis-Polyclonal anti-NEIL2 antibodies were purified from rabbit antisera produced by Alpha Diagnostics (San Antonio, TX) by affinity chromatography on Sepharose 4B (Amersham Biosciences) covalently coupled to NEIL2. NEIL2-specific IgG was eluted with glycine-HCl, pH 2.8, and immediately neutralized with 0.1 volume of 1 M Tris base and stored at Ϫ80°C after concentration (Amicon) and dialysis in phosphate-buffered saline (PBS). Lysates of E. coli (2 g) expressing WT and mutants of NEIL2 were used for immunoblot analysis using ECL system as per manufacturer's protocol (Amersham Biosciences).
Analysis of Trapped NEIL2 Complexes-A 32 P-labeled duplex oligomer (100 fmol) containing 5-OHU⅐G was incubated with lysates (1 g) of E. coli expressing WT or mutant NEIL2 in 15 l of assay buffer in the presence of 25 mM NaCNBH 3 at 37°C for 30 min. The trapped complexes were then separated by SDS-PAGE (12% polyacrylamide) as described previously (10).
Purification of Wild-type and Mutant NEIL2-The recombinant WT NEIL2 polypeptide was purified as before (10). Two of the mutant proteins, namely C291S and H295A, were also purified similarly with some protocol modifications. After Polymin P precipitation, the ammonium sulfate-fractionated pellets were dialyzed in buffer A (25 mM Tris-HCl, pH 7.5, 10% glycerol, and 0.5 mM DTT) containing 100 mM NaCl and passed through 5-ml Q-and SP-Sepharose (Amersham Biosciences) columns attached in tandem, which were then washed with 120 mM NaCl. The mutant NEIL2 proteins in the flow-through were concentrated in an Amicon filter and loaded onto a 25-ml Superdex 75 column. The fractions eluted from Superdex were further purified by fast protein liquid chromatography on Mono Q using NaCl gradient (20 -200 mM) in buffer A. The NEIL2 mutants eluted at around 75 mM NaCl. The other two mutant proteins, C291S and C315S, were subcloned into a C-terminal His tag-containing vector, pET22b (Novagen). After induction and sonication as before, the cells were spun down at 13 K and the supernatant was loaded onto a Ni 2ϩ -nitrilotrioacetate-agarose column (Qiagen) previously equilibrated with buffer B (40 mM Tris-HCl, pH 7.5, and 1 M NaCl). After washing with buffer B, the His-tagged mutant proteins were eluted with an imidazole gradient (20 -200 mM imidazole in buffer B). After elution, the peak enzyme fractions were dialyzed against buffer C (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, and 10% glycerol). The enzymes were further purified by fast protein liquid chromatography on a Mono Q column like the untagged mutants. The arginine mutant R310Q was also Histagged in the C terminus and purified from the extract of plasmidbearing E. coli by affinity chromatography on Ni 2ϩ -nitrilotrioacetateagarose (Qiagen). After elution with 150 mM imidazole, the enzyme was dialyzed against 25 mM Tris-HCl, pH 7.5, and 50 mM NaCl and finally purified on a Mono S column. Purified preparations of WT or mutant NEIL2 proteins were never frozen and were stored at Ϫ20°C in PBS containing 50% glycerol.
Incision Assay with 5-OHU-containing Bubble Oligomer-DNA strand cleavage at the abasic (AP) site after damaged base excision by NEIL2 occurs because of its intrinsic AP lyase activity. We have shown previously that NEIL2 has higher activity when the lesion is inside a bubble in an otherwise duplex oligomer (14). The strand incision by NEIL2 was used for its assay using an oligomer containing 5-OHU in the middle of unpaired 11-nt bubble (B11) as described previously (14). The 51-mer oligomer, 32 P-labeled at the 5Ј terminus of the lesioncontaining strand, was incubated with NEIL2 (WT and mutants) at 37°C for 15 min in a 15-l reaction mixture containing 40 mM Hepes, pH 7.5, 50 mM KCl, 100 g/ml bovine serum albumin, and 5% glycerol. After the reaction was stopped with 80% formamide and 20 mM NaOH, the cleaved oligomers were separated by denaturing gel electrophoresis in 15% polyacrylamide containing 7 M urea in 90 mM Tris borate, pH Electrophoretic Gel Mobility Shift Assay-The wild-type and mutant NEIL2 (C291S, H295A, C315S, C318S, and R310Q) were incubated with a 5Ј-32 P-labeled 5-OHU-containing bubble oligomer (5-OHU⅐B11) in 10 l of buffer containing 25 mM Hepes-KOH, pH 7.6, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 10 g/ml poly(dI-dC), and 12% glycerol at 20°C for 15 min followed by electrophoresis in 6% nondenaturing polyacrylamide gel containing 25 mM Tris-HCl, pH 7.5, 55 mM borate, and 0.6 mM EDTA, pH 7.4, at room temperature. The radioactivity in the shifted DNA protein complex was analyzed by PhosphorImager.
Quantitation of Zinc in NEIL2-The zinc content of wild-type and mutant NEIL2 polypeptides was determined by inductively coupled plasma mass spectrometry. The enzymes were dialyzed against PBS prior to analysis of Zn 2ϩ using a calibration curve with known amounts of Zn 2ϩ . The Zn content of the enzymes was corrected for contamination in the dialysis buffer.
Circular Dichroism Spectroscopy-All of the CD spectra were collected from proteins after dialysis and filtration in an AVIV 60DS spectrometer at 25°C. An average of three scans of the spectra (250 -200 nm) was used to obtain the final data. The molar ellipticity () was calculated using Equation 1, where obs is the observed ellipticity, MW is molecular weight, C is concentration (mg/ml), l is the path length of the cuvette in centimeters, and n refers to the number of residues. Protein concentrations were determined by the Bradford assay using bovine serum albumin as the standard. Molecular Modeling of 192-319 Residues of NEIL2-The sequence of NEIL2 with the potential DNA binding region (residues 192-319) was used as the seed sequence to search for a suitable template using BIOSERVER (meta server located at bioserv.cbs.cnrs.fr/). BIOSERVER submits to fold-recognition servers like 3D-PSSM (28), mGenThreader (29), Sam-T99 (30), and (Protein Data Bank (PDB)-BLAST. The results from different servers are parsed automatically, and the TITO program is used to evaluate most compatible template (31). The crystal structure of E. coli formamidopyrimidine-DNA glycosylase (Fpg/MutM, PDB code 1k82) (32) was selected as the most favorable template with a TITO score of Ϫ68,248 (PDB-BLAST score for this template: 1e Ϫ37 ; SAM-T99 score: 4.37e Ϫ42 ; 3DPSSM score: 2.7e Ϫ3 ; and mGenThreader score for 1ee8 template: 3e Ϫ4 ). The initial alignment was improved to minimize gaps in the ␤ or helix regions, and the final alignment had an identity of 30% to the template. Distance and dihedral constraints were extracted from the template using the geometry extraction program EXDIS, available in the homology modeling package MPACK (33)(34)(35). Structurally conserved regions or the fragments defined by excluding gaps in the pairwise alignment were used to extract geometric constraints. Upper and lower distance constraints were defined either by adding or subtracting a threshold of 0.25 Å to the actual distance for matching side-chain and main-chain (backbone) atoms. To position the side chains of Cys-291, His-295, Cys-315, and Cys-318, additional distance constraints among heavy side chain atoms were extracted from a CHCC-type zinc finger found in the DNA-binding domain of RAG1 (PDB code 1rmd, residues Cys-41, His-43, Cys-61, Cys-64). Upper-and lower-bound dihedral angle constraints were defined by adding or subtracting 5°. A total of 30 distance constraints per atom were extracted from the matching regions of the template. Models were generated using the distance geometry program DIAMOD. A few cycles of constrained energy minimization were applied using the program FANTOM, which minimizes constraint energies by successive application of quasi-Newton and Newton-Raphson minimizers (36), using the ECEPP/2 forcefield. The conformational energy of the model after energy minimization was Ϫ410 kcal/mol.

Expression and Activity of WT and Mutant NEIL2 in Crude E. coli Extracts-Sequence alignment of NEIL2 with Fpg and
Nei predicted that NEIL2 is a zinc finger protein with a CHCCtype motif near the C terminus. Cys-291, His-295, Cys-315, and Cys-318 are candidate residues for coordinating Zn 2ϩ (Fig. 1). This motif is distinct from the zinc finger motifs of Nei/Fpg, which are of the CCCC type (17). Single point mutants of NEIL2, C291S, H295A, C315S, and C318S were expressed in E. coli, and their expression was monitored by Western analysis ( Fig. 2A). The mutations did not affect the expression of NEIL2 in E. coli as indicated by the presence of a protein band of the predicted size in each induced bacterial lysate. In E. coli expressing the H295A mutant, a protein with slight slower migration was observed, which was not present (lane 2) in the control extract of E. coli, expressing the empty vector. Thus this band should be the H295A mutant of NEIL2. This analysis also underscored the strong specificity of the antibody.
All of the DNA glycosylases/AP lyases, regardless of their substrate preference, form transient Schiff bases with free AP site in DNA, which could be reduced with NaCNBH 3 (or NaBH 4 ) to form a stable "trapped complex" (37,38). We have shown earlier that NEIL2, similar to other MutM/Nei type enzymes, is inactivated when the N-terminal Pro, the active site nucleophile, is blocked or eliminated (10). Fig. 2B shows SDS-PAGE separation of 32 P-labeled trapped complexes generated with extracts of mutM nei E. coli harboring WT or mutant NEIL2 expression plasmids after incubation of a 5-OHU⅐G-containing oligomer. Because the mobility of such complexes reflects the size of the DNA glycosylase when the same oligomer substrate is used, it is evident that, in control E. coli lacking Fpg and Nei, only endogenous Nth formed a major trapped complex (lane 3). Crude bacterial lysates harboring WT NEIL2 formed a trapped complex of the same size as the purified recombinant NEIL2 (lane 2) used as a marker. However, the lack of such trapped complexes with crude lysates expressing various mutant NEIL2 proteins (lanes 5-8) suggests that the mutants are inactive as AP lyases. This loss of activity is probably the result of a loss of the zinc finger motif, critical either for structural integrity or the DNA binding activity of this enzyme.
DNA Binding and Incision Activity of Purified WT and Mutant NEIL2-Wild type and NEIL2 mutants (C291S, C315S, C318S, H295A, and R310Q) were purified to apparent homogeneity (Fig. 3). Surprisingly, the H295A mutant migrated abnormally and ran more slowly than the WT or the other mutants. We have shown earlier that NEIL2 is active in excising several cytosine-derived lesions and has robust activity for 5-OHU in bubble DNA (14). Purified WT and mutant NEIL2 polypeptides were tested for DNA incision assays with a 5-OHU-containing bubble oligomer (5 OHU⅐B11). The expected ␤␦-elimination product, i.e. cleaved 5-OHU-containing strand with 3Ј-phosphate termini, was observed with the WT NEIL2 protein (14). However, neither mutant NEIL2 generated the oligomer fragment to a detectable extent (Fig. 4A). The WT and mutant proteins were also assayed for their DNA binding activity by gel mobility shift assay with 5 OHU containing B11 oligomer. Again the DNA protein complex was observed only with WT NEIL2 (lane 2) but not the mutant proteins (Fig. 4B,  lanes 3-6).
Zinc Content of Wild-type and Mutant NEIL2-Purified WT and C318S and H295A mutant NEIL2 were analyzed for zinc content by inductively coupled plasma mass spectrometry. The WT NEIL2 protein contained 0.97 Ϯ 0.086 mol zinc/mol protein. In contrast, C318S and H295A mutants contained Ͻ0.1 mol of zinc/mol protein (Table I).
Structural Alterations in H295A and C318S NEIL2 Mutants-The presence of zinc was shown to be essential for the folding and stability of many classical Zn 2ϩ finger proteins. Based on our results that Cys-291, His-295, Cys-315, and Cys-318 residues are responsible for coordinating Zn 2ϩ , we examined the effect of mutation in these residues on the secondary structure of the protein by analyzing the CD spectra of purified WT and two mutant NEIL2 (H295A and C318S) proteins. The far-UV CD spectrum (Fig. 5) shows that WT NEIL2 has distinct secondary structure as indicated by the minima in molar ellipticity at 208 and 222 nm. However, the CD spectra of both the H295A and C318S proteins showed strong reduction in the mean residue ellipticity. These results indicate gross structural changes induced by mutations at His-295 or Cys-318 of NEIL2.
Molecular Model of NEIL2 Residues 192-319 -A homology model of C-terminal residues 192-319 of NEIL2 was built using E. coli Fpg (PDB code 1k82) (32). The structural alignment used for the modeling is represented in Fig. 6A. The final model (Fig. 6B) showed a backbone root mean square deviation value of 0.55 Å to the template. The models consist of helices formed by residues 203-208, 216 -220, 231-241, and 254 -271, a 3   helix by residues 249 -251, and a ␤-sheet consisting of two ␤-strands formed by residues 300 -302 and 312-314. The model has helix-two turn-helix motif formed by four helices and a novel ␤-hairpin CHCC-type zinc finger. The CHCC-type zinc finger is formed by residues Cys-291, His-295, Cys-315, and Cys-318. Mutations in any one of these residues abolish the function of NEIL2. The ␤-hairpin zinc finger provides a necessary structural framework to position the conserved Arg-310 that may take part in the catalytic reaction. By superimposing the model on the co-crystal structure of DNA bound to E. coli Fpg (PDB code 1k82), we found that the conserved Arg-310 (Fig. 6C) is placed in a position similar to Arg-258 in E. coli Fpg (32). Thus our model of human NEIL2 provides a mechanistic insight into the tertiary structure of the protein. The model is available at www.rcsb.org (PDB code 1vzp). Requirement of Arg-310 for Enzyme Activity-To clarify the role of the conserved Arg residue identified by molecular modeling (Fig. 6C), we constructed and characterized the NEIL2 site-directed mutant, R310Q. The purified R310Q protein showed greatly diminished activity relative to the WT NEIL2 with a 5-OHU containing bubble DNA substrate (Fig. 7A) and also decreased DNA binding (Fig. 7B). Thus these results further confirm that the zinc finger positions the conserved Arg-310 correctly in the active site pocket of NEIL2.

DISCUSSION
Zinc is an essential trace element and the second most abundant metal in mammalian cells. The adult human body contains ϳ3 g of Zn 2ϩ (39). It plays key roles in the maintenance of chromatin structure and also in nucleic acid metabolism as a structural component of enzymes in DNA replication, transcription, and DNA repair (40). The binding of zinc stabilizes the folded conformations of protein domains so that they may facilitate interactions with other macromolecules such as DNA. The lack of redox activity for the zinc ion and its binding and exchange kinetics may also be important in the use of zinc for specific functional roles, unlike other transition metal ions that might engage in free radical generation, leading to carcinogenic oxidative damage in cells (41). The peptide motifs containing bound zinc and named "zinc fingers" in proteins were first FIG. 6. Model of residues 192-319 of NEIL2. A, structural alignment for NEIL2 and E. coli Fpg (PDB code 1K82) template generated by 3DPSSM. Identical residues are indicated by asterisks. Residues that participate in coordination of zinc ion are highlighted in red and boldface. B, ribbon diagram of a model of C-terminal region of NEIL2 generated using MOLMOL (49). The helices (shown in green) form the helix two-turn helix motif that binds to DNA. CHCC-type ␤-hairpin zinc finger is highlighted with coordinating residues Cys-291, His-295, Cys-315, and Cys-318. C, ribbon diagram of model of C-terminal region of NEIL2 with DNA generated with MOLMOL. The helices (shown in blue) form the helix two-turn helix motif that binds to DNA. CHCC-type ␤-hairpin zinc finger is highlighted with coordinating residues Cys-291, His-295, Cys-315, and Cys-318. The zinc ion stabilizes ␤-strands (shown in green) that is critical to position of the catalytic residue Arg-310, which is conserved in E. coli Fpg.
identified approximately 20 years ago during the investigation of eukaryotic transcription factors. Since then, Ͼ10 different classes of zinc finger motifs have been discovered and characterized, many for their ability to bind nucleic acids in a sequence-specific manner and others for specifically mediating protein-protein interactions (42). Unlike the typical mode of Zn 2ϩ coordination within the catalytic center of enzymes, tetrahedral coordination of Zn 2ϩ in zinc fingers characteristically involves 2-4 potentially redox-reactive sulfhydryl groups (cysteine). It is estimated that zinc finger proteins constitute up to 1% of all human gene products with each of these proteins containing from 1 to 30 repeats of cysteine (ϩ histidine)-containing zinc finger motifs.
NEIL1 and NEIL2 are two orthologs of E. coli Fpg/Nei, which were shown to be zinc finger proteins. Although NEIL1 and NEIL2 have significant functional overlap and use the same reaction chemistry as Fpg and Nei, only NEIL2 possesses a potential zinc finger motif. Near the C terminus, it contains a unique sequence with three Cys and one His residues in an unusual zinc finger configuration (Fig. 1) that is not homologous to the zinc finger motifs of Nei/Fpg. In this study, we set out to confirm that NEIL2 is indeed a zinc finger protein and then examine the role of this motif in the structure and function of this glycosylase.
We used site-directed mutagenesis to confirm the requirement for the three Cys and one His residues predicted to coordinate with zinc in the putative zinc finger domain of NEIL2. The DNA-trapping assay is a fast and definitive method for assessing the base excision activity of DNA glycosylase/AP lyases. Because trapped complexes with a radiolabeled oligomer can be separated by SDS-PAGE that can identify glycosylases based on their mobility (43), we tested whether any of the mutant proteins has DNA glycosylase/AP lyase activity. The absence of trapped complexes with all four mutants confirmed that mutation of any one of these Cys and His residues totally abolished the enzymatic activity (Fig. 2). This is consistent with a critical role for the zinc finger in enzymatic function, as was also observed for Fpg and Nei (44,45). We then tested whether the loss of enzymatic activity was due to loss of DNA binding activity. We carried out electrophoretic mobility shift assay with purified WT and mutant proteins using a 5-OHU-containing bubble substrate oligomer. All of the cysteine mutants and the single histidine mutant failed to bind substrate DNA, indicating that the zinc finger motif is essential for DNA binding. Subsequent studies confirmed that the purified mutant NEIL2s have no strand incision activity with the substrate oligomer (Fig. 4). Finally, mass spectroscopic analysis showed that the WT NEIL2 contained 1 mol of zinc per mol of protein, whereas the mutants (C318S and H295A) lacked bound Zn 2ϩ (Table I). This provided the strongest evidence that the candidate Cys and His residues are indeed responsible for zinc coordination and that the mutation of even one residue abolishes this coordination. This raised the question as to whether the loss of Zn 2ϩ induces any change in the secondary structure of NEIL2. The CD spectrum clearly showed that the secondary structures of the C318S and H295A mutants were drastically altered (Fig. 5). Mutations in these residues are likely to prevent proper folding of the ␤-strands into a hairpin motif (Fig. 6B), and hence the geometry of the critical conserved residue Arg-310 (predicted from the model) may not be situated correctly. Mutation of Arg-310 strongly reduced NEIL2 activity without significantly affecting DNA binding (Fig. 7), unlike the mutations in zinc-coordinating residues. This strongly suggests that Arg-310 in NEIL2, positioned by the zinc binding pocket, is highly conserved as in the Fpg/Nei family and performs an indispensable catalytic role similar to that of Arg-258 in E. coli Fpg (32) or Arg-253 in E. coli Nei (46). In both case, this residue participates in protonation of 5Ј-phosphate. It was recently shown that the conserved Arg is also present in the other mammalian nei homolog, NEIL1 (47). Although mammalian NEIL1 has similar folds as the bacterial Fpg/Nei, it has an unusual motif near the C terminus. This structural motif mimics the ␤-hairpin zinc finger found in members of the Fpg/Nei class (including NEIL2) but lacks the loops harboring the canonical zinc-binding residues and therefore does not coordinate zinc. Interestingly, the critical Arg (Arg-277 in NEIL1) is positioned in the loop connecting the two ␤-strands of the zinc-less finger and its mutation to Ala showed a strong reduction of glycosylase activity (47). We have shown here that the R310Q mutant of NEIL2 also has markedly reduced glycosylase activity. Thus the conserved Arg is positioned critically for glycosylase activity in both the zinc-containing (NEIL2 and Fpg/Nei) and zinc-less fingers (NEIL1).
Thus our studies reveal that the localized destabilization of the zinc finger motif in NEIL2 affected the conformation of the whole polypeptide. The structural perturbation in the mutants was also reflected during purification. The WT NEIL2 binds strongly to the SP resin during fast protein liquid chromatography. In contrast, the zinc finger mutants did not bind to SP and bound weakly to the Q column. Taken together, our results provide definitive evidence for the identity of zinc-coordinating residues and show that the zinc finger motif is integral to the structure and function of NEIL2. We may note in passing that the nonconsensus (CHCC-type) motif is rather uncommon among the zinc finger proteins involved in DNA metabolism.
Further work with high resolution x-ray diffraction analysis will provide a more detailed understanding regarding specific residues involved in maintenance of the zinc finger domain and overall stability of the protein. A larger implication of this study involves public health issues such as malnutrition and hence lower zinc levels in the diet (48), or exposure to chemical toxins or radiation, which induce oxidative stress, could inactivate NEIL2, leading to reduced repair of oxidative damage of the genome.