J Biol. Chem. Research Article Revised: Feb. 28, 2010

1 1

STRUCTURAL BASIS OF O6-ALKYLGUANINE RECOGNITION BY A BACTERIAL ALKYLTRANSFERASE-LIKE DNA REPAIR PROTEIN *

James M. Aramini‡,1, Julie L. Tubbs§, Sreenivas Kanugula¶, Paolo Rossi‡, Asli Ertekin‡, Melissa Maglaqui‡, Keith Hamilton‡, Colleen T. Ciccosanti‡, Mei Jiang‡, Rong Xiao‡, Ta-tsen Soong||,

Burkhard Rost||, Thomas B. Acton‡, John K. Everett‡, Anthony E. Pegg¶, John A. Tainer§,†, and Gaetano T. Montelione‡,⊥ ,2

From the ‡Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854 and Northeast Structural Genomics Consortium, §Skaggs Institute for Chemical Biology and Department of Molecular

Biology, The Scripps Research Institute, La Jolla, California 92037, ¶Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, ||Department of Biochemistry and Molecular Biophysics,

Columbia University, New York, New York 10032 and Northeast Structural Genomics Consortium, †Life Sciences Division, Bioenergy and Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and ⊥Department of Biochemistry, Robert Wood Johnson Medical School, University

of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854 Running head: Structure of a Bacterial Alkyltransferase-like Protein

1,2To whom correspondence may be addressed: E-mail: jma@cabm.rutgers.edu; guy@cabm.rutgers.edu. Alkyltransferase-like proteins (ATLs) are a novel class of DNA repair proteins related to O6-alkylguanine-DNA alkyltransferases (AGTs) that tightly bind alkylated DNA and shunt the damaged DNA into the nucleotide excision repair (NER) pathway. Here we present the first structure of a bacterial ATL, from Vibrio parahaemolyticus (vpAtl). We demonstrate that vpAtl adopts an AGT-like fold, and that the protein is capable of tightly binding to O6-methylguanine (O6-mG) containing DNA and disrupting its repair by human AGT, a hallmark of ATLs. Mutation of highly conserved residues Y23 and R37 demonstrate their critical roles in a conserved mechanism of ATL binding to alkylated DNA. NMR relaxation data reveal a role for conformational plasticity in the guanine-lesion recognition cavity. Our results provide further evidence for the conserved role of ATLs in this primordial mechanism of DNA repair. O6-alkylguanine-DNA alkyltransferases (AGTs) are a large family (Pfam, PF01035; EC 2.1.1.63) of alkyl damage response proteins that reverse endogenous and exogenous alkylation at the O6 position of guanines, cytotoxic lesions which otherwise cause G•C to A•T mutations in DNA (1). Human AGT, also called MGMT, interferes with alkylating chemotherapies making it a target for anticancer drug design (1,2). AGTs are ubiquitous suicide enzymes that mediate the irreversible transfer of the alkyl group to a reactive

cysteine within a highly conserved PCHRV active site sequence motif by a direct reversal mechanism, featuring sequence-independent minor-groove binding to a helix-turn-helix motif and flipping of the damaged nucleotide (3,4). Alkyltransferase-like proteins (ATLs), thus far identified in prokaryotes and lower eukaryotes, constitute a new sub-class with sequence similarity to AGTs but lacking the critical cysteine alkyl receptor, which is most often replaced by a tryptophan (5,6). ATLs tightly bind a wide range of O6-alkylguanine adducts and block the repair of O6-mG by human AGT, but exhibit no alkyltransferase, glycosylase, or endonuclease activities (7-9). The recent first structural study of an ATL (10), from the fission yeast Schizosaccharomyces pombe (spAtl1) which lacks an AGT, provides strong evidence for a novel mechanism of DNA repair in which an ATL binds alkylated DNA in a manner analogous to AGTs, and the resulting non-enzymatic ATL•DNA complex triggers the NER pathway (10,11). Here, we present the solution NMR structure of the 100-residue ATL from Vibrio parahaemolyticus AQ3810 (Swiss-Prot entry A6B4U8_VIBPA; NESG id, VpR247; hereafter referred to as vpAtl), whose structure was solved as part of the Northeast Structural Genomics consortium (www.nesg.org) of the NIGMS Protein Structure Initiative. The vpAtl protein shares 47% sequence identity with spAtl1 and features a PWFRV active site sequence motif (Fig. 1A). We demonstrate that the structure of vpAtl is highly

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.093591The latest version is at JBC Papers in Press. Published on March 8, 2010 as Manuscript M109.093591

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

2 2

analogous to the AGT fold and that this bacterial protein is capable of tightly binding to O6-mG containing DNA and disrupting its repair by human AGT. Site-directed mutagenesis experiments demonstrate the importance of highly conserved residues Y23 and R37 for binding to alkylated DNA. In addition, the NMR data further suggest that the O6-mG recognition cavity of bacterial ATLs exhibits some conformational flexibility, which may confer broader specificity for various alkyl guanine lesions. To our knowledge, this work represents the first structural characterization of a bacterial ATL.

EXPERIMENTAL PROCEDURES Complete details of the methods used in this work are provided in the supplemental information online. Sample Preparation - The cloning, expression, and purification of isotopically enriched protein samples of a 100-residue construct from the A79_1377 gene of Vibrio parahaemolyticus AQ3810 (NESG id, VpR247; vpAtl) plus C-terminal affinity tag (LEHHHHHH) was performed following standard protocols of the Northeast Structural Genomics (NESG) consortium (12). Samples of [U-13C,15N]- and [U-5%-13C,100%-15N]-vpAtl for NMR spectroscopy were concentrated by ultracentrifugation to 0.90 to 0.94 mM in 95% H2O / 5% 2H2O solution containing 20 mM MES, 200 mM NaCl, 10 mM DTT, 5 mM CaCl2 at pH 6.5. Analytical gel filtration chromatography, static light scattering (supplemental Fig. S1) and 1D 15N T1 and T2 relaxation data (supplemental Fig. S2) demonstrate that the protein is monomeric in solution under the conditions used in the NMR studies. Single residue mutations of vpAtl (Y23A, Y23F, R37A, and W54A) were cloned using the QuickChange site-directed mutagenesis kit (Stratagene), and expressed and purified following the same protocols used for the wild type protein. NMR Spectroscopy and Resonance Assignment - All NMR spectra were collected at 25 oC on Bruker AVANCE 600 and 800 MHz spectrometers equipped with 1.7-mm TCI and 5-mm TXI cryoprobes, respectively, and a Varian INOVA 600 MHz instrument with a 5-mm HCN cold probe, and referenced to internal DSS. Complete 1H, 13C, and 15N resonance assignments for vpAtl were determined using conventional

triple resonance NMR methods. Backbone assignments were made by combined use of AutoAssign 2.4.0 (13) and the PINE 1.0 server (14), and side chain assignment was completed manually. Histidine tautomeric states were elucidated by 2D 1H-15N heteronuclear multiple-quantum coherence (HMQC) spectroscopy (15). Resonance assignments were validated using the Assignment Validation Suite (AVS) software package (16) and deposited in the BioMagResDB (BMRB accession number 16272). Structure Determination and Validation - The solution NMR structure of vpAtl was calculated using CYANA 3.0 (17,18), and the 20 structures with lowest target function out of 100 in the final cycle calculated were further refined by restrained molecular dynamics in explicit water with CNS 1.2 (19,20). Structural statistics and global structure quality factors were computed using the PSVS 1.3 software package (21) and MolProbity 3.15 server (22). The global goodness-of-fit of the final structure ensembles with the NOESY peak list data was determined using the RPF analysis program (23). The final refined ensemble of 20 structures (excluding the C-terminal His6) was deposited in the Protein Data Bank (PDB entry 2KIF). All structure figures were made using PyMOL (http://www.pymol.org). 15N Relaxation Measurements - Residue specific longitudinal and transverse 15N relaxation rates (R1 and R2) as well as 1H-15N heteronuclear NOE values were obtained on [U-5%-13C,100%-15N]-vpAtl at a 15N Larmor frequency of 60.8 MHz using standard 2D gradient experiments (24). Generalized order parameters, S2, were computed from the backbone 15N relaxation and 1H-15N heteronuclear NOE data using the Modelfree 4.20 program (25,26) assuming an isotropic model for molecular motion. Human AGT competition assays - The inhibition of human AGT activity by spAtl1, vpAtl, and mutants of vpAtl was measured by adding purified hAGT to a preformed mixture of [3H]-methylated DNA and ATL and then assaying the mixture for alkyltransferase activity by determining the transfer of [3H]-methyl groups from O6-[3H]-methylguanine in DNA to purified human AGT protein (27). The assay mixture (1.0 ml), incubated at 37 °C for 15 min, contained 50 mM Tris-HCl (pH 7.6), 5 mM DTT, 50 µg hemocyanin, 0.1 mM EDTA, 15 µg of [3H]-methylated calf thymus DNA and different amounts of purified ATLs. Ten microliters of

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

3 3

purified hAGT (0.5 pmoles) were added to the above reaction mixture, and incubation was continued for 60 min at 37 °C and assayed for alkyltransferase activity. RESULTS Solution NMR structure of vpAtl. The structure of vpAtl adopts an AGT-like fold comprised of five alpha helices (α1, D2-H13; α2, Y23-G31; α3, Y35-L46; α4, G68-A80; α5, A92-K97) and two short antiparallel beta strands (β1, S21-T22; β2, V57-I58) in the core of the protein (Fig. 1B; supplemental Fig. S3). Structural statistics and a summary of the NMR data from this study are provided in Table 1 and supplemental Fig. S4, respectively. By analogy to homologs (vide infra), helices 2 and 3 comprise the helix-turn-helix (HTH) DNA-binding motif. A ConSurf (28) analysis of all ATLs from the AGT protein domain family (PF01035), reveals that highly conserved residues, demonstrated in spAtl1 (10) to be involved in flipping of the damaged alkyl guanine (Y23 and R37), interacting with the orphaned cytosine (R37) and binding to the alkyl moiety (Y23 and W54), are clustered in a partially occluded binding pocket (Fig. 1C). This face of the protein also features a quite positive electrostatic surface potential (29) due to several basic residues, in agreement with its role in DNA binding (Fig. 1D; supplemental Fig. S5). The binding pocket is flanked by the less conserved binding site loop (preceding helix 4) and C-terminal loop (between helices 4 and 5). In spAtl1 these loops are important for open-to-closed (free-to-bound) conformational changes and mediate interactions with proteins in the NER pathway, respectively (10). Two histidines (H13 in helix 1 and H38 flanking R37 in helix 3), largely unique to ATLs from Vibrio species, adopt neutral Nε2H tautomers under the conditions used in this study (supplemental Fig. S6). Backbone Dynamics of vpAtl. The internal dynamic properties of vpAtl were further investigated using backbone 15N relaxation and heteronuclear NOE experiments (24). Reduced 15N R2 relaxation rates and 1H-15N heteronuclear NOE values are observed for residues L46 to L52, which span the C-terminal end of helix 3 and the loop preceding the heart of the substrate binding site (Fig. 2A). These data result in reduced order parameters, S2, indicative of enhanced backbone motions within this binding pocket cap.

Moreover, there are a handful of weak/missing backbone amide NMR resonances in the protein, consistent with conformational exchange broadening. Mapping these effects onto the structure of vpAtl clearly reveals that several conformationally dynamic residues cluster around the substrate binding pocket (Fig. 2B). This plasticity provides a structural basis for the broad range of guanine lesions that can be recognized by ATLs (8,11); flexibility in the recognition cavity provides a capacity for molding the binding site around various alkyl guanine lesions besides O6-mG. Comparison to Related Structures. The solution structure of vpAtl is structurally very similar to the recent crystal structures of bound and free spAtl1 [Dali (30) Z-scores: 3GYH, 16.1; 3GX4, 15.7; 3GVA, 15.0; Cα RMSDs: 3GYH, 1.7 Å; 3GX4, 1.8 Å; 3GVA, 1.9 Å] (10) as well as to structures of the C-terminal domains of human AGT (3,4,31,32), Escherichia coli Ada-C (33), and archaeal (34,35) AGTs (Dali Z-scores ranging from 5.4 to 11.1) which share < 30% sequence identity with vpAtl. Considering the metric of modeling leverage (36), an important measure of the new structural information provided by a protein structure, the vpAtl structure has a novel leverage value of 26 models and total modeling leverage value of 1,493 structural models (UniProt release 12.8; PSI Blast E < 10-10). Of these, 17 sequences are putative ATLs from eukaryotes including the recently identified ATL from sea anenome (10). The vpAtl structure most closely resembles the closed (DNA-bound) form of spAtl1, and conserved residues important for damage recognition and binding superimpose well in the structures (Fig. 3A). There are, however, subtle differences between the structures. Both the binding site and C-terminal loops are shorter in vpAtl and the binding pocket in vpAtl is partially buried by residues from the binding site loop (S65 and L66) and the binding pocket cap (L46-P53), resulting in a much smaller binding pocket (<Area> (Å2) = 185 ± 61) than that observed for spAtl1 (10). However, as discussed above, the 15N relaxation and 1H-15N heteronuclear NOE data along with weak/missing backbone amide resonances, indicate that there are backbone conformational dynamics within the binding pocket cap (Fig. 2), suggesting that vpAtl may adopt conformations in which the substrate binding pocket is more exposed.

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

4 4

Inhibition of hAGT-mediated DNA repair by vpAtl. Like the ATL proteins from E. coli, S. pombe and T. thermophilus (7-9), vpAtl does not exhibit alkyltransferase activity (data not shown). However, a common trait of ATLs is their ability to tightly bind O6-alkylguanine DNA with up to subnanomolar affinity (10), and prevent its repair by AGTs. The inhibition of human AGT O6-mG repair by vpAtl and spAtl1 is shown in Fig. 3B. In this assay, varying amounts of ATL are preincubated with [3H]-methylated DNA, followed by incubation with human AGT and measurement of the amount of radiolabel transferred to the AGT (27). As expected for an ATL, vpAtl strongly inhibits O6-mG repair by hAGT when present in molar excess and exhibits a similar affinity for alkylated DNA compared to spAtl1. The DNA binding roles of selected conserved residues in vpAtl, namely Y23, R37, and W54, were further examined using this competition assay (Fig. 3C,D). Replacing either Y23 or R37 with alanine yields mutants that have no effect on hAGT activity, meaning that their ability to bind alkylated DNA is severely impaired. On the other hand, the Y23F mutant is still capable of blocking repair of methylated DNA by hAGT, albeit not as effectively as wild type vpAtl (Fig. 3C). These data are consistent with the requirement of a bulky aromatic residue at the position of Y23 to flip the damaged guanine base into the binding pocket, and the function of R37 to intercalate the DNA and hydrogen bond to the orphaned cytosine (10). Similar effects were observed in analogous mutagenesis experiments on human AGT (37,38). Finally, swapping W54 for an alanine (W and A are present in ≈ 89% and ≈ 9%, respectively, of ATLs; Pfam 23.0) results in an ATL that also exhibits some affinity for methylated DNA, although less than that for wild type vpAtl (Fig. 3D); a similar effect was observed for the ATL from E. coli (7). Overall, the relative affinities of the ATLs and mutants studied here for O6-mG DNA follow the trend: spAtl1 ≥ vpAtl > [Y23F]-vpAtl > [W54A]-vpAtl >> [Y23A]-vpAtl ≈ [R37A]-vpAtl. DISCUSSION The results on vpAtl presented here demonstrate a high degree of structural conservation between bacterial and yeast ATLs, and that vpAtl exhibits the hallmark biochemical behavior of ATLs. Furthermore, mutation of

highly conserved residues Y23 or R37 to alanine abolishes the ability of vpAtl to block methylated DNA repair by human AGT, and the affinity of the Y23F mutant for methylated DNA demonstrates the necessity of a bulky aromatic residue in this position. To our knowledge, this is the first mutagenesis study examining these critical residues in ATLs and their roles in binding to O6-mG DNA and blocking human AGT activity. Taken together, our structural and mutagenesis results provide strong evidence for a conserved mechanism of ATL binding to alkylated DNA mediated by critical tyrosine and arginine residues and involving the flipping out of the damaged base (10). Like several other organisms, including E. coli, V. parahaemolyticus possesses genes for both ATL and AGT. It was recently shown that repair of O6-alkylguanine lesions in E. coli is segregated between the direct repair (AGT) and ATL-coupled NER pathways on the basis of the size of the alkyl group, with the latter in charge of repairing O6-alkylguanine adducts larger in size than a methyl group, which are poor substrates for AGTs (11). Moreover, the high structural similarity between free vpAtl and free and bound spAtl1 suggests that vpAtl also mediates O6-alkylguanine repair by recruitment of proteins involved in NER. Hence, we postulate that vpAtl is also capable of interacting with a broad range of O6-alkylguanine substrates and likely mediates an analogous cross-talk between alkyltransferase and NER repair pathways. In this scenario, vpAtl would function to channel bulkier O6-alkylguanine lesions into the NER pathway. Conformational dynamics within the recognition cavity of apo ATL, revealed for the first time by this structural NMR study, confer functional plasticity that may be essential for providing its wider range of guanine lesion specificity. In light of the recent structural and biochemical characterization of S. pombe Atl1 and the predicted occurrence of ATLs in archaea (10), our results for vpAtl also provide further support for the hypothesis that ATLs are an ancient class of non-enzymatic proteins at the interface of the base and nucleotide excision repair pathways for DNA repair. These results thus help provide a unified understanding of DNA damage responses, which has been a major goal for the structural biology of DNA repair since the discovery of base and nucleotide excision repair pathways (39).

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

5 5

REFERENCES

1. Tubbs, J. L., Pegg, A. E., and Tainer, J. A. (2007) DNA Repair 6, 1100-1115. 2. Verbeek, B., Southgate, T. D., Gilham, D. E., and Margison, G. P. (2008) Br. Med. Bull. 85, 17-33. 3. Daniels, D. S., Woo, T. T., Luu, K. X., Noll, D. M., Clarke, N. D., Pegg, A. E., and Tainer, J. A.

(2004) Nature Struct. Mol. Biol. 11, 714-720. 4. Duguid, E. M., Rice, P. A., and He, C. (2005) J. Mol. Biol. 350, 657-666. 5. Margison, G. P., Butt, A., Pearson, S. J., Wharton, S., Watson, A. J., Marriott, A., Caetano, C. M. P.

F., Hollins, J. J., Rukazenkova, N., Begum, G., Santibáñez-Koref, M. F. (2007) DNA Repair 6, 1222-1228.

6. Reissner, T., Schorr, S., and Carell, T. (2009) Angew. Chem. Int. Ed. 48, 7293-7295. 7. Pearson, S. J., Ferguson, J., Santibanez-Koref, M., and Margison, G. P. (2005) Nucleic Acids Res.

33, 3837-3844. 8. Pearson, S. J., Wharton, S., Watson, A. J., Begum, G., Butt, A., Glynn, N., Williams, D. M.,

Shibata, T., Santibáñez-Koref, M. F., and Margison, G. P. (2006) Nucleic Acids Res. 34, 2347-2354.

9. Morita, R., Nakagawa, N., Kuramitsu, S., and Masui R (2008) J. Biochem. 144, 267-277. 10. Tubbs, J. L., Latypov, V., Kanugula, S., Butt, A., Melikishvili, M., Kraehenbuehl, R., Fleck, O.,

Marriott, A., Watson, A. J., Verbeek, B., McGown, G., Thorncroft, M., Santibanez-Koref, M. F., Millington, C., Arvai, A. S., Kroeger, M. D., Peterson, L. A., Williams, D. M., Fried, M. G., Margison, G. P., Pegg, A. E., and Tainer, J. A. (2009) Nature 459, 808-813.

11. Mazon, G., Philippin, G., Cadet, J., Gasparutto, D., and Fuchs, R. P. (2009) DNA Repair 8, 697-703.

12. Acton, T. B., Gunsalus, K. C., Xiao, R., Ma, L.-C., Aramini, J., Baran, M. C., Chiang, Y.-W., Climent, T., Cooper, B., Denissova, N. G., Douglas, S. M., Everett, J. K., Ho, C. K., Macapagal, D., Rajan, P. K., Shastry, R., Shih, L.-Y., Swapna, G. V. T., Wilson, M., Wu, M., Gerstein, M., Inouye, M., Hunt, J. F., and Montelione, G. T. (2005) Methods Enzymol. 394, 210-243.

13. Moseley, H. N. B., Monleon, D., and Montelione, G. T. (2001) Methods Enzymol., 339, 91-108. 14. Bahrami, A., Assadi, A. H., Markley, J. L., and Eghbalnia, H. R. (2009) PLoS Comput. Biol. 5,

e1000307. 15. Pelton, J. G., Torchia, D. A., Meadow, N. D., and Roseman, S. (1993) Protein Sci. 2, 543-558. 16. Moseley, H. N. B., Sahota, G., and Montelione, G. T. (2004) J. Biomol. NMR 28, 341-355. 17. Güntert, P., Mumenthaler, C., and Wüthrich, K. (1997) J. Mol. Biol. 273, 283-298. 18. Herrmann, T., Güntert, P., and Wüthrich, K. (2002) J. Mol. Biol. 319, 209-227. 19. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W.,

Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. D54, 905-921.

20. Linge, J. P., Williams, M. A., Spronk, C. A. E. M., Bonvin, A. M. J. J., and Nilges, M. (2003) Proteins 50, 496-506.

21. Bhattacharya, A., Tejero, R., and Montelione, G. T. (2007) Proteins 66, 778-795. 22. Davis, I. W., Leaver-Fay, A., Chen, V. B., Block, J. N., Kapral, G. J., Wang, X., Murray, L. W.,

Arendall III, W. B., Snoeyink, J., Richardson, J. S., and Richardson, D. C. (2007) Nucleic Acids Res. 35, W375-W383.

23. Huang, Y. J., Powers, R., and Montelione, G. T. (2005) J. Am. Chem. Soc. 127, 1665-1674. 24. Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G., Shoelson, S. E.,

Pawson, T., Forman-Kay, J. D., and Kay, L. E. (1994) Biochemistry 33, 5984-6003. 25. Mandel, A. M., Akke, M., and Palmer III, A. G. (1995) J. Mol. Biol. 246, 144-163. 26. Palmer III, A. G., Rance, M., and Wright, P. E. (1991) J. Am. Chem. Soc. 113, 4371-4380. 27. Kanugula, S., and Pegg, A. E. (2001) Environ. Mol. Mutagen. 38, 235-243. 28. Glaser, F., Pupko, T., Paz, I., Bell, R. E., Bechor-Shental, D., Martz, E., and Ben-Tal, N. (2003)

Bioinformatics 19, 163-164. 29. Rocchia, W., Alexov, E., and Honig, B. (2001) J. Phys. Chem. B 105, 6507–6514. 30. Holm, L., Kääriäinen, S., Rosenström, P., and Schenkel, A. (2008) Bioinformatics 24, 2780-2781.

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

6 6

31. Daniels, D. S., Mol, C. D., Arvai, A. S., Kanugula, S., Pegg, A. E., and Tainer, J. A. (2000) EMBO J. 19, 1719-1730.

32. Wibley, J. E. A., Pegg, A. E., and Moody, P. C. E. (2000) Nucleic Acids Res. 28, 393-401. 33. Moore, M. H., Gulbis, J. M., Dodson, E. J., Demple, B., and Moody, P. C. E. (1994) EMBO J. 13,

1495-1501. 34. Hashimoto, H., Inoue, T., Nishioka, M., Fujiwara, S., Takagi, M., Imanaka, T., and Kai, Y. (1999)

J. Mol. Biol. 292, 707-716. 35. Roberts, A., Pelton, J. G., and Wemmer, D. E. (2006) Magn. Reson. Chem. 44, S71-S82. 36. Liu, J., Montelione, G. T., and Rost, B. (2007) Nature Biotechnol. 25, 849-851. 37. Kanugula, S., Goodtzova, K., Edara, S., and Pegg, A. E. (1995) Biochemistry 34, 7113-7119. 38. Goodtzova, K., Kanugula, S., Edara, S., and Pegg, A. E. (1998) Biochemistry 37, 12489-12495. 39. Huffman, J. L., Sundheim, O., and Tainer, J. A. (2005) Mutation Res. 577, 55-76. Supplemental information is available at online at http://www.jbc.org. Acknowledgements We thank Rachel L. Belote, G.V.T. Swapna, and Markus Fischer for valuable scientific discussion. This work was supported by grants from the National Institute of General Medical Sciences Protein Structure Initiative U54-GM074958 (GTM), and National Institutes of Health grants CA097209 (JAT, AEP) and CA018137 (SK). Abbreviations The abbreviations used are: AGT, O6-alkylguanine-DNA alkyltransferase; ATL, alkyltransferase-like protein; DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; DTT, dithiothreitol; HSQC, heteronuclear single quantum coherence; MES, 2-(N-morpholino)ethanesulfonic acid; NER, nucleotide excision repair; NOE, nuclear Overhauser effect; RMSD, root mean square deviation.

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

7 7

FIGURE LEGENDS

Fig. 1. Solution NMR structure of vpAtl. (A) Structure-based sequence alignment of vpAtl, spAtl1, and hAGT (residues 91-176). The sequence numbering for vpAtl and the secondary structural elements found in its solution NMR structure are shown above the alignment. Identical residues are shown in red. Residues in the HTH and active site sequence motifs are boxed in blue and yellow, respectively. Key highly conserved, functionally important residues (Y23, R37, and W54 in vpAtl) are denoted by triangles below the alignment. (B) Stereoview into the putative alkyl binding site in the lowest energy (CNS) conformer from the final solution NMR structure ensemble of vpAtl. The α-helices and β-strands are shown in cyan and magenta, respectively. Side chains of Y23, R37, and W54 are shown in red, blue and yellow, respectively. (C) ConSurf (28) image showing the conserved residues in the alkyl binding site of vpAtl (same view as in B). Residue coloring ranges from magenta (highly conserved) to cyan (variable), and reflects the degree of residue conservation across ATL sequences extracted from the entire O6-alkylguanine-DNA methyltransferase protein domain family (PF01035, Pfam 23.0). (D) DelPhi (29) electrostatic surface potential of vpAtl showing negative (red), neutral (white), and positive (blue) charges.

Fig. 2. Backbone dynamics of vpAtl. (A) Plots of backbone amide 15N R1 and R2 relaxation rates, 1H-15N heteronuclear NOEs, and generalized order parameters, S2, versus residue number obtained on [U-5%-13C,100%-15N]-vpAtl at a 15N Larmor frequency of 60.8 MHz. Order parameters were computed using the Modelfree 4.20 program (25,26) assuming an isotropic model, yielding an overall rotational correlation time, τc, of 7.9 ns. (B) Backbone dynamics of vpAtl mapped onto its structure. Residues with S2 ≤ 0.7, indicative of enhanced backbone flexibility, are in red, and residues with weak or missing 1H-15N HSQC resonances are colored yellow. Prolines are shown in grey and the substrate binding pocket and binding pocket cap are indicated.

Fig. 3. Conserved structure and DNA binding properties of vpAtl. (A) Overlay of the vpAtl structure (cyan) and the crystal structure of spAtl1 bound to O6-mG-DNA (PDB entry, 3GX4; magenta) (10). The side chains of Y23, R37 and W54 are shown in red, blue and yellow, respectively, and the O6-mG in the bound spAtl1 structure is shown in grey. (B) Percent activity of hAGT as a function of ATL concentration for vpAtl (triangles) and spAtl1 (circles). (C) Effect of mutating Y23 in vpAtl on hAGT activity; wild type vpAtl (black), [Y23A]-vpAtl (red triangles), [Y23F]-vpAtl (red circles). (D) Effect of mutating R37 and W54 in vpAtl on hAGT activity; wild type vpAtl (black), [R37A]-vpAtl (blue), and [W54A]-vpAtl (gold).

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

8 8

Table 1. Summary of NMR and structural statistics for vpAtla Completeness of resonance assignmentsb

Backbone (%) 97.6 Side chain (%) 98.3 Aromatic (%) 100 Stereospecific methyl (%) 100 Conformationally-restricting constraintsc Distance constraints Total 2448

intra-residue (i = j) 621 sequential (|i- j| = 1) 547 medium range (1 < |i – j| < 5) 493 long range (|i – j| ≥ 5) 787 distance constraints per residue 24.2

Dihedral angle constraints 125 Hydrogen bond constraints total 62

long range (|i – j| ≥ 5) 8 Number of constraints per residue 26.1 Number of long range constraints per residue 7.9 Residual constraint violations c Average number of distance violations per structure 0.1 – 0.2 Å 1.55

0.2 – 0.5 Å 0.1 > 0.5 Å 0 average RMS distance violation / constraint (Å) 0.01 maximum distance violation (Å) 0.31 Average number of dihedral angle violations per structure 1 – 10° 1.7

> 10° 0 average RMS dihedral angle violation / constraint (degree) 0.37 maximum dihedral angle violation (degree) 4.60 RMSD from average coordinates (Å) c,d backbone atoms 0.5 heavy atoms 0.8 Procheck Ramachandran statistics c,d most favored regions (%) 91.5 additional allowed regions (%) 8.5 generously allowed (%) 0.0 disallowed regions (%) 0.0

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

9 9

Table 1 (Cont’d). Summary of NMR and structural statistics for vpAtla

MolProbity Ramachandran statistics e

favored regions (%) 95.4 allowed (%) 100.0 number of outliers 1 Global quality scores c

Raw Z-score Verify3D 0.48 0.32 ProsaII 0.91 1.08 Procheck(phi-psi) d -0.13 -0.20 Procheck(all) d -0.02 -0.12 Molprobity clash 18.92 -1.72 RPF Scoresf Recall 0.974 Precision 0.937 F-measure 0.955 DP-score 0.835 a Structural statistics were computed for the ensemble of 20 deposited structures (PDB entry,

2KIF). b Computed using AVS software (16) from the expected number of peaks, excluding: highly

exchangeable protons (N-terminal, Lys, and Arg amino groups, hydroxyls of Ser, Thr, Tyr), carboxyls of Asp and Glu, non-protonated aromatic carbons, and the C-terminal His6 tag.

c Calculated using PSVS 1.3 program (21). Average distance violations were calculated using the sum over r-6.

d Ordered residue ranges [S(phi) + S(psi) > 1.8] : 2-32,35-47,50-100. e Calculated for all residues in the ensemble using the MolProbity 3.15 server (22). f RPF scores (23) reflecting the goodness-of-fit of the final ensemble of structures (including

disordered residues) to the NMR data.

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

10 10

Figure 1.

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

11 11

Figure 2.

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

J Biol. Chem. Research Article Revised: Feb. 28, 2010

12 12

Figure 3.

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from

Gaetano T. MontelioneBurkhard Rost, Thomas B. Acton, John K. Everett, Anthony E. Pegg, John A. Tainer andMaglaqui, Keith Hamilton, Colleen T. Ciccosanti, Mei Jiang, Rong Xiao, Ta-tsen Soong,

James M. Aramini, Julie L. Tubbs, Sreenivas Kanugula, Paolo Rossi, Asli Ertekin, MelissaDNA repair protein

-alkylguanine recognition by a bacterial alkyltransferase-like6Structural basis of O

published online March 8, 2010J. Biol. Chem. 

  10.1074/jbc.M109.093591Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2010/03/08/M109.093591.DC1

by guest on July 4, 2019http://w

ww

.jbc.org/D

ownloaded from