A model for 3-methyladenine recognition by 3-methyladenine DNA glycosylase I (TAG) from Staphylococcus aureus
The removal of chemically damaged DNA bases such as 3-methyladenine (3-MeA) is an essential process in all living organisms and is catalyzed by the enzyme 3-MeA DNA glycosylase I. A key question is how the enzyme selectively recognizes the alkylated 3-MeA over the much more abundant adenine. The crystal structures of native and Y16F-mutant 3-MeA DNA glycosylase I from Staphylococcus aureus in complex with 3-MeA are reported to 1.8 and 2.2 Å resolution, respectively. Isothermal titration calorimetry shows that protonation of 3-MeA decreases its binding affinity, confirming previous fluorescence studies that show that charge–charge recognition is not critical for the selection of 3-MeA over adenine. It is hypothesized that the hydrogen-bonding pattern of Glu38 and Tyr16 of 3-MeA DNA glycosylase I with a particular tautomer unique to 3-MeA contributes to recognition and selection.
Bacterial 3-methyladenine DNA glycosylase I (TAG; Forsyth et al., 2002; Ji et al., 2001) is ubiquitous in eubacteria (Supplementary Fig. S11; Drohat et al., 2002) but shows no sequence or structural similarity to mammalian 3-methyladenine DNA glycosylase (AAG; Lau et al., 2000). TAG belongs to the alkylpurine DNA glycosylase superfamily and hydrolyzes the N9–C1′ glycosylic bond between a 3-methyladenosine (3-MeA) nucleobase lesion and the deoxyribose ring (Riazuddin & Lindahl, 1978; Bjelland et al., 1993; Fig. 1a). 3-Methylation of adenine does not influence base pairing (Sedgwick et al., 2007); rather, the methyl group blocks replication by interfering with the interactions of DNA polymerase (Sedgwick et al., 2007; Engelward et al., 1996). Like the 8-oxoguanylate DNA glycosylases MutM and hOGG1 (Banerjee et al., 2005, 2006; Banerjee & Verdine, 2006; Blainey et al., 2006), TAG is thought to slide along the duplex until it encounters a lesion. TAG binds flipped-out 3-MeA and then cleaves the damaged base from the ribose. TAG from Staphylococcus aureus shares around 40% amino-acid sequence identity with the structurally characterized TAG enzymes from Salmonella typhi (Metz et al., 2007) and Escherichia coli (Drohat et al., 2002). The crystal structure of the S. typhi enzyme complexed with 3-MeA and abasic DNA (Metz et al., 2007) and an NMR structure of the E. coli enzyme complexed with 3-MeA (Cao et al., 2003) have been reported. Two absolutely conserved residues, Tyr16 and Glu38, were identified to form hydrogen bonds with 3-MeA and Trp46 stacks with 3-MeA (Cao et al., 2003; Metz et al., 2007). The methyl group does not appear to make extensive contacts. The crystal structure of the apo S. aureus enzyme has been reported (Oke et al., 2010). We wished to probe the basis of the discrimination between adenine and 3-MeA in the S. aureus enzyme.
Fluorescence binding measurements were performed as described by Cao et al. (2003) and Drohat et al. (2002). 2 µM TAG was titrated with 10–650 µM 3-MeA or adenine in 20 mM phosphate buffer pH 7.8 and 5.8; Figs. 2a and 2b). Isothermal titration calorimetry (ITC) experiments were carried out using a VP-ITC device (MicroCal) in the same buffer. 5 mM 3-MeA or 1.5 mM adenine solution was injected at 298 K into a sample cell containing ∼1.4 ml protein solution at 30–40 µM. Each titration consisted of a first 1 µl injection followed by up to 25 subsequent 10 µl injections or 48 subsequent 5 µl injections of the ligand as indicated. Calorimetric data were analyzed using the MicroCal ORIGIN software, fixing the stoichiometry as N = 1 (Figs. 2c and 2d; Supplementary Table S11).
Sitting-drop vapour-diffusion crystallization trials (1 µl protein solution plus 1 µl precipitant solution) were set up using a Cartesian Honeybee nanodrop crystallization robot which was integrated in a Hamilton-Thermo Rhombix system. The 3-MeA complexes of native and Y16F TAG were obtained by incubating TAG with 10 mM 3-MeA for 6 h before crystallization at 277 K. The complex crystals grew using a precipitant solution consisting of 0.1 M Tris–HCl pH 8.5, 1.8 M ammonium sulfate, 0.2 M Li2SO4 at 293 K as thin plates and grew to full size (0.2 × 0.2 × < 0.05 mm) in two to three weeks. Cryoprotectant solution was made by supplementing the crystallization precipitant solution with 20% glycerol. Crystals were mounted in Hampton Research cryoloops and rapidly cooled to 100 K prior to data collection.
Data for the native TAG–3-MeA complex were collected from a single crystal using 0.2° oscillations at a wavelength of 0.933 Å (ESRF beamline ID14-2) and were reduced using XDS (Kabsch, 2010). Data were collected from a single crystal of the Y16F TAG–3-MeA complex using an in-house Rigaku MicroMax-007 HF rotating-anode generator and Saturn 944 CCD detector. Data were reduced using HKL-2000 (Otwinowski & Minor, 1997) and POINTLESS (Evans, 2006; Potterton et al., 2003; Winn et al., 2011). Full details are given in Table 2. The E38Q mutant was also crystallized, but as no 3-MeA was located in the active site the structure is not described here; however, the structure has been deposited (PDB entry 4ai4 ).
The structures were solved with Phaser (McCoy et al., 2007) using the native apo structure (Oke et al., 2010; PDB entry 2jg6 ) as a search model. As the complex crystals grew in a different space group to the native crystals, a new free set of reflections was assigned for refinement. All structures were refined with REFMAC v.5.6.0117 (Murshudov et al., 2011); manual intervention employed Coot (Emsley & Cowtan, 2004). 3-MeA was added to the models when the Fo − Fc density was clear (Figs. 1c and 1d). MolProbity (Chen et al., 2010) was used for structure validation and Ramachandran analysis. TLS parameters were used in refinement. TLS groups were assigned using the TLSMD server (Painter & Merritt, 2006). Details of the refinement are given in Table 3.
The structure of the S. aureus TAG–3-MeA complex was determined to 1.8 Å resolution and that of the Y16F TAG–3-MeA complex to 2.22 Å resolution. The structure of the native 3-MeA complex is very similar to the crystal structure of the S. typhi TAG–3-MeA–abasic DNA complex (Metz et al., 2007) and the NMR structure of the E. coli TAG–3-MeA complex (Cao et al., 2003). Relative to apo TAG (Oke et al., 2010), Glu38 has rotated to make 2.7 Å contacts with the exocyclic N atom and N7 of 3-MeA. Tyr16 moves to make a 2.8 Å contact with the exocyclic N atom of 3-MeA (Fig. 3a). Trp46 stacks with the bound purine ring of 3-MeA, while Phe6, Tyr13 and Tyr21 make edge-on contacts. His41 rotates 80° to create space for 3-MeA to bind. The Y16F-mutant complex revealed that 3-MeA adopts a different orientation, although it preserves a bidentate hydrogen bond to Glu38 and a stacking interaction with Trp46 (Fig. 3b). This conformation is unlikely to be physiologically relevant, as it would require a very different orientation of the DNA to that observed in the S. typhi complex (Metz et al., 2007). Using a fluorescence assay, we measured 3-MeA binding (Fig. 2a), obtaining a similar result at pH 7.8 (Kd = 78 µM) to that for the E. coli enzyme at pH 7.5 (Kd = 42 µM; Cao et al., 2003). However, the assay is flawed for the S. aureus enzyme as the E38Q mutant gave the same result as for the native protein (Fig. 2b), which is physically unreasonable. ITC (Figs. 2c and 2d) showed clear differences between the native and mutant S. aureus enzymes (Y16F, Kd = 1.2 mM; E38Q, no binding) and gave Kd values of 220 µM at pH 7.8 and 471 µM at pH 5.8 for the native enzyme. We did not detect adenine binding.
3-Methyldeoxyadenosine is positively charged in DNA, whilst deoxyadenosine is neutral; simple charge–charge recognition was therefore the original explanation for the specificity of TAG (Labahn et al., 1996; Lau et al., 2000; Hollis et al., 2000). However, it has been shown that E. coli TAG binds 3-MeA but not adenine and binds protonated 3-MeA (pH 5.7) more weakly than neutral 3-MeA (pH 7.5) (Cao et al., 2003; Drohat et al., 2002), establishing that charge–charge recognition is not the sole explanation (Cao et al., 2003). We suggest that a particular hydrogen-bond pattern contributes to the selection of a specific but favoured (Sharma & Lee, 2002) neutral tautomer of 3-MeA (Fig. 3c) that is not available to adenosine (Fig. 3c) and that is disfavoured for protonated 3-MeA (Fig. 3c). Our hypothesis implies that there is an energetic penalty in reorganizing the hydrogen-bond network around Tyr16 to avoid a van der Waals clash (Fig. 3c). In DNA, 3-methyldeoxyadenosine can adopt a tautomer that has the same hydrogen arrangement as neutral 3-MeA and has positive charge (Fig. 3d), which is favoured at the active site (Metz et al., 2007). A clash of H atoms was observed between the amide of His136 and the amino group of adenine in human AAG and is used to preferentially select the damaged purine base (O'Brien & Ellenberger, 2004). Higher resolution data or neutron diffraction are required to further test the hypothesis for the TAG enzyme.
The work was funded by the BBSRC SPoRT initiative (BB_BBS/B/14426).
Banerjee, A., Santos, W. L. & Verdine, G. L. (2006). Science, 311, 1153–1157. Web of Science CrossRef PubMed CAS
Banerjee, A. & Verdine, G. L. (2006). Proc. Natl Acad. Sci. USA, 103, 15020–15025. Web of Science CrossRef PubMed CAS
Banerjee, A., Yang, W., Karplus, M. & Verdine, G. L. (2005). Nature (London), 434, 612–618. Web of Science CrossRef PubMed CAS
Bjelland, S., Bjørås, M. & Seeberg, E. (1993). Nucleic Acids Res. 21, 2045–2049. CrossRef CAS PubMed Web of Science
Blainey, P. C., van Oijen, A. M., Banerjee, A., Verdine, G. L. & Xie, X. S. (2006). Proc. Natl Acad. Sci. USA, 103, 5752–5757. Web of Science CrossRef PubMed CAS
Cao, C., Kwon, K., Jiang, Y. L., Drohat, A. C. & Stivers, J. T. (2003). J. Biol. Chem. 278, 48012–48020. Web of Science CrossRef PubMed CAS
Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12–21. Web of Science CrossRef CAS IUCr Journals
Drohat, A. C., Kwon, K., Krosky, D. J. & Stivers, J. T. (2002). Nature Struct. Biol. 9, 659–664. Web of Science CrossRef PubMed CAS
Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126–2132. Web of Science CrossRef CAS IUCr Journals
Engelward, B. P., Dreslin, A., Christensen, J., Huszar, D., Kurahara, C. & Samson, L. (1996). EMBO J. 15, 945–952. CAS PubMed Web of Science
Evans, P. (2006). Acta Cryst. D62, 72–82. Web of Science CrossRef CAS IUCr Journals
Forsyth, R. A. et al. (2002). Mol. Microbiol. 43, 1387–1400. Web of Science CrossRef PubMed CAS
Hollis, T., Ichikawa, Y. & Ellenberger, T. (2000). EMBO J. 19, 758–766. Web of Science CrossRef PubMed CAS
Ji, Y., Zhang, B., Van Horn, S. F., Warren, P., Woodnutt, G., Burnham, M. K. & Rosenberg, M. (2001). Science, 293, 2266–2269. Web of Science CrossRef PubMed CAS
Kabsch, W. (2010). Acta Cryst. D66, 125–132. Web of Science CrossRef CAS IUCr Journals
Labahn, J., Schärer, O. D., Long, A., Ezaz-Nikpay, K., Verdine, G. L. & Ellenberger, T. E. (1996). Cell, 86, 321–329. CrossRef CAS PubMed Web of Science
Lau, A. Y., Wyatt, M. D., Glassner, B. J., Samson, L. D. & Ellenberger, T. (2000). Proc. Natl Acad. Sci. USA, 97, 13573–13578. Web of Science CrossRef PubMed CAS
McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674. Web of Science CrossRef CAS IUCr Journals
Metz, A. H., Hollis, T. & Eichman, B. F. (2007). EMBO J. 26, 2411–2420. Web of Science CrossRef PubMed CAS
Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355–367. Web of Science CrossRef CAS IUCr Journals
O'Brien, P. J. & Ellenberger, T. (2004). J. Biol. Chem. 279, 9750–9757. Web of Science PubMed CAS
Oke, M. et al. (2010). J. Struct. Funct. Genomics, 11, 167–180. CrossRef CAS PubMed
Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326. CrossRef CAS Web of Science
Painter, J. & Merritt, E. A. (2006). J. Appl. Cryst. 39, 109–111. Web of Science CrossRef CAS IUCr Journals
Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. (2003). Acta Cryst. D59, 1131–1137. Web of Science CrossRef CAS IUCr Journals
Riazuddin, S. & Lindahl, T. (1978). Biochemistry, 17, 2110–2118. CrossRef CAS PubMed Web of Science
Sedgwick, B., Bates, P. A., Paik, J., Jacobs, S. C. & Lindahl, T. (2007). DNA Repair, 6, 429–442. Web of Science CrossRef PubMed CAS
Sharma, S. & Lee, J. K. (2002). J. Org. Chem. 67, 8360–8365. Web of Science CrossRef PubMed CAS
Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. Web of Science CrossRef CAS IUCr Journals
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