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• W2016694525 abstract "Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from damaged DNA. Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA. The structure reveals that Fpg stabilizes the cFapydG nucleoside into an extrahelical conformation inside its substrate binding pocket. In contrast to the recognition of the 8-oxodG lesion, which is bound with the glycosidic bond in a syn conformation, the cFapydG lesion displays in the complex an anti conformation. Furthermore, Fpg establishes interactions with all the functional groups of the FapyG base lesion, which can be classified in two categories: (i) those specifying a purine-derived lesion (here a guanine) involved in the Watson-Crick face recognition of the lesion and probably contributing to an optimal orientation of the pyrimidine ring moiety in the binding pocket and (ii) those specifying the imidazole ring-opened moiety of FapyG and probably participating also in the rotameric selection of the FapydG nucleobase. These interactions involve strictly conserved Fpg residues and structural water molecules mediated interactions. The significant differences between the Fpg recognition modes of 8-oxodG and FapydG provide new insights into the Fpg substrate specificity. Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines such as 7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) from damaged DNA. Here, we report the crystal structure of the Fpg protein from Lactococcus lactis (LlFpg) bound to a carbocyclic FapydG (cFapydG)-containing DNA. The structure reveals that Fpg stabilizes the cFapydG nucleoside into an extrahelical conformation inside its substrate binding pocket. In contrast to the recognition of the 8-oxodG lesion, which is bound with the glycosidic bond in a syn conformation, the cFapydG lesion displays in the complex an anti conformation. Furthermore, Fpg establishes interactions with all the functional groups of the FapyG base lesion, which can be classified in two categories: (i) those specifying a purine-derived lesion (here a guanine) involved in the Watson-Crick face recognition of the lesion and probably contributing to an optimal orientation of the pyrimidine ring moiety in the binding pocket and (ii) those specifying the imidazole ring-opened moiety of FapyG and probably participating also in the rotameric selection of the FapydG nucleobase. These interactions involve strictly conserved Fpg residues and structural water molecules mediated interactions. The significant differences between the Fpg recognition modes of 8-oxodG and FapydG provide new insights into the Fpg substrate specificity. Reactive oxygen species generated in the cell during physiological processes or resulting from the cell exposure to exogenous chemical and physical agents can react with DNA to produce base lesions. The resulting DNA damages can interfere with both efficiency and fidelity of the DNA replication and transcription, thus participating in mutagenesis, carcinogenesis, and aging (1Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4342) Google Scholar). Oxidation of guanine in DNA generates a plethora of oxidative bases lesions of which 7,8-dihydro-8-oxoguanine (8-oxoG)1 and imidazole ring-opened purines (FapyG) are among the most abundant lesions (see Fig. 1) (2Cadet J. Berger M. Douki T. Ravanat J.L. Rev. Physiol. Biochem. Pharmacol. 1997; 131: 1-87PubMed Google Scholar, 3Ravanat J.-L. Di Mascio P. Martinez G.R. Medeiros M.H. Cadet J. J. Biol. Chem. 2000; 275: 40601-40604Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 4Breen A.P. Murphy J.A. Free Radic. Biol. Med. 1995; 18: 1033-1077Crossref PubMed Scopus (926) Google Scholar). FapyG lesions are also generated in DNA as by-products of N7-alkylated purines (5Chetsanga C.J. Lindahl T. Nucleic Acids Res. 1979; 6: 3673-3683Crossref PubMed Scopus (231) Google Scholar, 6Chetsanga C.J. Polidori G. Mainwaring M. Cancer Res. 1982; 42: 2616-2621PubMed Google Scholar, 7Chetsanga C.J. Frenette G.P. Carcinogenesis. 1983; 4: 997-1000Crossref PubMed Scopus (35) Google Scholar, 8Boiteux S. Bichara M. Fuchs R.P. Laval J. Carcinogenesis. 1989; 10: 1905-1909Crossref PubMed Scopus (41) Google Scholar, 9Tudek B. Boiteux S. Laval J. Nucleic Acids Res. 1992; 20: 3079-3084Crossref PubMed Scopus (72) Google Scholar) (Fig. 1). 8-OxoG is a mutagenic DNA lesion because of alternative base-pairing possibility with dA, yielding G→T transversions in bacterial, yeast, and mammalian cells (10Tchou J. Kasai H. Shibutani S. Chung M.H. Laval J. Grollman A.P. Nishimura S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4690-4696Crossref PubMed Scopus (692) Google Scholar, 11Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 6321-6325Crossref PubMed Scopus (613) Google Scholar, 12Thomas D. Scot A.D. Barbey R. Padula M. Boiteux S. Mol. Gen. Genet. 1997; 254: 171-178Crossref PubMed Scopus (203) Google Scholar, 13Kamiya H. Nucleic Acids Res. 2003; 31: 517-531Crossref PubMed Scopus (232) Google Scholar). The FapyG lesion is potentially lethal and mutagenic (14Tudek B. J. Biochem. Mol. Biol. 2003; 36: 12-19Crossref PubMed Google Scholar). To avoid the deleterious effects of oxidative DNA damage, prokaryotes and eukaryotes have evolved the mechanism of DNA base excision repair initiated by the DNA glycosylases (15Lindahl T. Br. J. Cancer. 1987; 56: 91-95Crossref PubMed Scopus (28) Google Scholar). 8-OxoG and FapyG residues are recognized and excised by the same DNA glycosylases, the bacterial formamidopyrimidine-DNA glycosylases (Fpg or MutM) and its eukaryote functional homologue, the Ogg1 proteins (5Chetsanga C.J. Lindahl T. Nucleic Acids Res. 1979; 6: 3673-3683Crossref PubMed Scopus (231) Google Scholar, 10Tchou J. Kasai H. Shibutani S. Chung M.H. Laval J. Grollman A.P. Nishimura S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4690-4696Crossref PubMed Scopus (692) Google Scholar, 11Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 6321-6325Crossref PubMed Scopus (613) Google Scholar, 16Boiteux S. O'Connor T.R. Laval J. EMBO J. 1987; 6: 3177-3183Crossref PubMed Scopus (250) Google Scholar, 17Czeczot H. Tudek B. Lambert B. Laval J. Boiteux S. J. Bacteriol. 1991; 173: 3419-3424Crossref PubMed Google Scholar, 18van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci., U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (348) Google Scholar, 19Radicella J.P. Dherin C. Desmaze C. Fox M.S. Boiteux S. Proc. Natl. Acad. Sci., U. S. A. 1997; 94: 8010-8015Crossref PubMed Scopus (571) Google Scholar). Interestingly, the N7-Me-FapyG lesion is also efficiently repaired by DNA glycosylases that exhibit low and even undetectable capacity to release 8-oxoG such as the yeast Ntg1 and Ntg2 proteins (20Alseth I. Eide L. Pirovano M. Rognes T. Seeberg E. Bjoras M. Mol. Cell. Biol. 1999; 19: 3779-3787Crossref PubMed Google Scholar). Fpg and Ogg1 proteins belong to the DNA glycosylases/abasic (AP) lyase family because they initiate the removal of the base lesions by a cleavage of the glycosidic bond between the damaged base and its associated sugar, which is followed by subsequent scission of the DNA backbone at the resulting AP site by a β-elimination (Ogg1) or a β,δ-elimination (Fpg) reaction (18van der Kemp P.A. Thomas D. Barbey R. de Oliveira R. Boiteux S. Proc. Natl. Acad. Sci., U. S. A. 1996; 93: 5197-5202Crossref PubMed Scopus (348) Google Scholar, 21Bailly V. Verly W.G. O'Connor T. Laval J. Biochem. J. 1989; 262: 581-589Crossref PubMed Scopus (141) Google Scholar, 22O'Connor T.R. Laval J. Proc. Natl. Acad. Sci., U. S. A. 1989; 86: 5222-5226Crossref PubMed Scopus (140) Google Scholar). These enzymes use either the N-terminal amino group (Pro1 for Fpg) or the ϵ-amino group of an internal lysine (Lys249 for the human Ogg1) to perform a nucleophilic attack on the C1′ of the damaged nucleoside leading to an imino enzyme-DNA covalent complex, which is now established to be a common reaction intermediate for the glycosylase and lyase activities (23Zharkov D.O. Rieger R.A. Iden C.R. Grollman A.P. J. Biol. Chem. 1997; 272: 5335-5341Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 24Girard P.-M. Guibourt N. Boiteux S. Nucleic Acids Res. 1997; 25: 3204-3211Crossref PubMed Scopus (120) Google Scholar, 25Guibourt N. Castaing B. Auffret-van der Kemp P. Boiteux S. Biochemistry. 2000; 39: 1716-1724Crossref PubMed Scopus (22) Google Scholar). Fpg has been characterized through biochemical, functional, and genomic sequencing studies and more recently by crystal structure analysis (16Boiteux S. O'Connor T.R. Laval J. EMBO J. 1987; 6: 3177-3183Crossref PubMed Scopus (250) Google Scholar, 26Castaing B. Geiger A. Seliger H. Nehls P. Laval J. Zelwer C. Boiteux S. Nucleic Acids Res. 1993; 21: 2899-2905Crossref PubMed Scopus (101) Google Scholar, 27Tchou J. Bodepudi V. Shibutani S. Antoshechkin I. Miller J. Grollman A.P. Johnson F. J. Biol. Chem. 1994; 269: 15318-15324Abstract Full Text PDF PubMed Google Scholar, 28Duarte V. Gasparutto D. Jaquinod M. Cadet J. Nucleic Acids Res. 2000; 28: 1555-1563Crossref PubMed Scopus (102) Google Scholar, 29Sugahara M. Mikawa T. Kuramitsu T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (136) Google Scholar). The ability to study the recognition of damaged nucleotides by these enzymes at an atomic level became recently possible because of the progress of chemical synthesis, which now allows the design and synthesis of oligonucleotides containing defined lesions or even uncleavable lesion analogues at defined sites in DNA duplex (30Castaing B. Fourrey J.L. Hervouet N. Thomas M. Boiteux S. Zelwer C. Nucleic Acids Res. 1999; 27: 608-615Crossref PubMed Scopus (50) Google Scholar, 31Schärer O.D. Jiricny J. BioEssays. 2001; 23: 270-281Crossref PubMed Scopus (235) Google Scholar, 32Haraguchi K. Greenberg M.M. J. Am. Chem. Soc. 2001; 123: 8636-8637Crossref PubMed Scopus (61) Google Scholar, 33Wiederholt C.J. Delaney M.O. Greenberg M.M. Biochemistry. 2002; 41: 15838-15844Crossref PubMed Scopus (21) Google Scholar, 34Ober M. Linne U. Gierlich J. Carell T. Angew. Chem. Int. Ed. Engl. 2003; 42: 4947-4951Crossref PubMed Scopus (26) Google Scholar). Thus, crystal structures of several stable abortive Fpg/DNA complexes have been recently reported. Four different bacterial Fpg proteins have been used for structural studies: TtFpg from Thermus thermophilus, LlFpg from Lactococcus lactis, EcFpg from Escherichia coli, and BstFpg from Bacillus stearothermophilus. In addition to the structure of the free TtFpg (29Sugahara M. Mikawa T. Kuramitsu T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (136) Google Scholar), two classes of Fpg/DNA complexes have been described: Fpg bound to AP sites (35Serre L. Pereira de Jesus K. Boiteux S. Zelwer C. Castaing B. EMBO J. 2002; 21: 2854-2865Crossref PubMed Scopus (112) Google Scholar, 36Gilboa R. Zharkov D.O. Golan G. Fernandes A.S. Gerchman S.E. Matz E. Kycia J.H. Grollman A.P. Shoham G. J. Biol. Chem. 2002; 277: 19811-19816Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 37Fromme J.C. Verdine G.L. Nat. Struct. Biol. 2002; 9: 544-552PubMed Google Scholar) and Fpg bound to damaged bases such as 8-oxoG and dihydrouracil (DHU) (38Fromme J.C. Verdine G.L. J. Biol. Chem. 2003; 278: 51543-51548Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). These high resolution crystal structures allowed the depiction of the general strategy developed by Fpg to recognize and repair the damaged nucleoside. Fpg flips the recognized lesion out of the DNA helix through the major groove and stabilizes it in an extrahelical conformation inside the active site binding pocket. This nucleoside flipping out mechanism allows the exposure of the anomeric C1′-center of the damaged nucleoside (initially buried in DNA) to nucleophilic attack by the N-terminal Pro1 of the active site. The extrusion of the lesion is achieved by a strong torsion of DNA centered on the target site, which results from an intercalation in the minor groove of three conserved Fpg residues (Met75, Arg109, and Phe111 of LlFpg). The cytosine opposite the lesion is maintained by Arg109 in an intrahelical conformation preventing a local DNA collapse. In the present study, we report the crystallization and the structure determination at 1.8 Å resolution of a specific abortive complex between the L. lactis Fpg protein and a DNA double strand containing the synthetically stabilized carbocyclic FapydG (cFapydG) (34Ober M. Linne U. Gierlich J. Carell T. Angew. Chem. Int. Ed. Engl. 2003; 42: 4947-4951Crossref PubMed Scopus (26) Google Scholar). The synthetic stabilization of the lesion is essential because the lesion has a high tendency to anomerize and to decompose under DNA synthesis conditions, which compromises any attempts to prepare oligonucleotides containing this lesion with a purity as required for biomolecule crystallization. Our crystallization efforts with the stabilized lesions enabled us to obtain the first x-ray structure in which the Fapy lesion is observed in a complex with a DNA repair enzyme. Surprisingly, our structure reveals a FapyG recognition mode of Fpg quite different from that of 8-oxoG. DNA and Protein—The preparation of a single-stranded oligonucleotide containing the cFapydG residue has been described recently (34Ober M. Linne U. Gierlich J. Carell T. Angew. Chem. Int. Ed. Engl. 2003; 42: 4947-4951Crossref PubMed Scopus (26) Google Scholar). After purification, the modified oligonucleotide CTCTTT(cFapydG)TTTCTCG was annealed with the complementary strand GCGAGAAACAAAGA to form a 14-mer duplex. To obtain the ΔP1-LlFpg mutant protein, the deletion of Pro1 was achieved using the QuikChange® site-directed mutagenesis kit (Stratagene) with the pMAL-LP1G recombinant plasmid as DNA template (39Pereira de Jesus K. Serre L. Hervouet N. Bouckson-Castaing V. Zelwer C. Castaing B. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 679-682Crossref PubMed Scopus (9) Google Scholar). The resulting recombinant plasmid pMAL-LDP1 is used to produce the ΔP1-LlFpg mutant. ΔP1-LlFpg is purified as described for P1G-LlFpg (35Serre L. Pereira de Jesus K. Boiteux S. Zelwer C. Castaing B. EMBO J. 2002; 21: 2854-2865Crossref PubMed Scopus (112) Google Scholar). Apparent dissociation constants (Kd(app)) were determined by electrophoresis mobility shift assay as described previously (30Castaing B. Fourrey J.L. Hervouet N. Thomas M. Boiteux S. Zelwer C. Nucleic Acids Res. 1999; 27: 608-615Crossref PubMed Scopus (50) Google Scholar). Crystallization, Data Collection, and Structure Determination—cFapydG:dC-containing 14-mer DNA duplex was mixed with ΔP1-LlFpg in 1.3 molar excess of DNA and adjusted to a final protein/DNA complex concentration of 2-5 mg/ml. Using the sparse-matrix crystallization screening kit (Hampton Research), crystallization was performed at 20 °C by the hanging drop vapor diffusion method. Under several crystallization conditions, crystals appeared within few days. Best crystallization conditions were found for drops containing a 1/1 (v/v) ratio of the protein/DNA complex and a solution of 0.1 m Hepes/NaOH, pH 7.0, 1.3-1.5 m sodium citrate equilibrated to the same solution. Crystals suitable for a complete x-ray diffraction study were soaked in a cryoprotecting solution and frozen in liquid nitrogen. X-ray diffraction data were collected at 100 K on a Quantum ADSC-Q4 charge-coupled device detector at the ID14-EH2 beam line of the European Synchrotron Radiation Facility. The diffraction patterns were processed with MOSFLM (40Leslie A.G. Brick P. Wonacott A.J. CCP4 Newsletter. 1996; 18: 33-39Google Scholar) and scaled with SCALA from the CCP4 package (41Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). The phase problem was solved by the molecular replacement method using the program AMoRe (42Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar) with the coordinates of the LlFpg-1,3-propanediol complex as a search model (Protein Data Bank accession number 1NNJ). A random sample of 5% of reflections in the data set were excluded from the refinement and used for Rfree calculation. The Dundee PRODRG2 server (43van Aalten D.M.F. Bywater R. Findlay J.B.C. Hendlich M. Hooft R.W.W. Vriend G. J. Comput. Aided Mol. Des. 1996; 10: 255-262Crossref PubMed Scopus (569) Google Scholar) was used to generate molecular topology files of the cFapydG nucleobase. In an attempt to avoid model bias, the initial model refinement was carried out using CNS (44Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges N. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). Several cycles of simulated annealing, energy minimization, B-factor refinement, and annealed omit map calculation were interspersed with manual rebuilding using TURBO-FRODO (45Roussel A. Cambillau C. Silicon Graphics Silicon Graphics Geometry Partners Directory. 86. Silicon Graphics Corp., Mountain View, CA1991: 81Google Scholar). Then, the structure was further refined by the maximum-likelihood method using REFMAC 5 (46Murshudov G.N. Vagin A.A. Lebedev A. Wilson K.S. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 247-255Crossref PubMed Scopus (1010) Google Scholar). Water molecules were picked up from the automatic protocol of ARP/wARP (47Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (480) Google Scholar) on the basis of the peak heights and distance criteria. During the last refinement steps, anisotropic B factor parameters were introduced for the zinc atom. The final refined model consists of 265 protein residues (electron density was not observed for the flexible loop comprised of residues 220-224), 28 nucleotide residues, 1 glycerol molecule, 1 zinc ion, and a total of 431 water molecules. 6 residues were refined in two alternate conformations, and 11 protein side chains were not completely modeled. Data collection and model refinement statistics are summarized in Table I.Table IData collection and refinement statisticsData collection statisticsRadiation sourceESRF ID14-EH2Wavelength (Å)0.933Total observations200299Unique reflections55781Completeness (%)aValues in parentheses refer to data in the highest resolution shell.99.3 (99.3)RedundancyaValues in parentheses refer to data in the highest resolution shell.3.6 (3.6)RsymaValues in parentheses refer to data in the highest resolution shell.bRsym=Σ|I−〈I〉|ΣI where I is the observed intensity and 〈I〉 is the average intensity from multiple observations of the symmetry-related reflections.4.8 (23.4)I/σaValues in parentheses refer to data in the highest resolution shell.8.7 (3.1)Refinement and model statisticsResolution (Å)20-1.8Number of reflections used52844Rwork (%)cR=Σ‖Fobs|−|Fcalc‖/Σ|Fobs|⋅Rfree is the R-value for a subset of 5% of the reflection data, which were not included in the crystallographic refinement.17.9Rfree (%)cR=Σ‖Fobs|−|Fcalc‖/Σ|Fobs|⋅Rfree is the R-value for a subset of 5% of the reflection data, which were not included in the crystallographic refinement.20.7Average B valuesAll atoms (Å2)29.04Protein atoms (Å2)24.85DNA atoms (Å2)36.88Water atoms (Å2)39.53Root mean square deviation from idealityBond lengths (Å)0.018Bond angles (°)1.709Torsion angles (°)5.737No. of atomsProtein2157DNA568Glycerol6Water431a Values in parentheses refer to data in the highest resolution shell.b Rsym=Σ|I−〈I〉|ΣI where I is the observed intensity and 〈I〉 is the average intensity from multiple observations of the symmetry-related reflections.c R=Σ‖Fobs|−|Fcalc‖/Σ|Fobs|⋅Rfree is the R-value for a subset of 5% of the reflection data, which were not included in the crystallographic refinement. Open table in a new tab Accession Numbers—The atomic coordinates of the LlFpg/FapydGDNA complex have been deposited in the Protein Data Bank under the accession number 1TDZ. Crystals of a catalytic defective LlFpg mutant (ΔP1-LlFpg in which Pro1 has been deleted) bound to a cFapydG-containing 14-mer DNA duplex have been obtained (see “Experimental Procedures”). ΔP1-LlFpg binds to cFapydG-containing DNA with high affinity similar to that of wild type LlFpg (Kd(app) of 3 and 2.6 nm, respectively). The chemical modification present in the cFapydG lesion analogue (replacement of the 2′-deoxyribose sugar backbone by the cyclopentane skeleton) conserves all the functional groups and therefore the base-pairing properties of the natural FapydG lesion (34Ober M. Linne U. Gierlich J. Carell T. Angew. Chem. Int. Ed. Engl. 2003; 42: 4947-4951Crossref PubMed Scopus (26) Google Scholar). The structure of ΔP1-LlFpg/cFapydG-DNA was solved by molecular replacement using as a search model the P1G-LlFpg/1,3-propanediol-DNA coordinates (Protein Data Bank accession number 1NNJ) and was refined to 1.8 Å resolution. The final model consists of one polypeptide chain including residues 2 to 219 and 225 to 271 (the residues 220 to 224 of the αF-β9 loop are missing in the electron density map of the final model), one 14-mer DNA duplex, 431 solvent atoms, one zinc atom, and one glycerol molecule per complex (Table I). The global structures of the DNA and the protein in the complex are identical to those of previous LlFpg/AP-DNA structures (the root mean square deviation calculated on all C-α protein atoms is 0.282 Å). The 14-mer DNA duplex adopts a bent structure centered on the damaged nucleoside (Fig. 2A). The DNA backbone at the target site is unchanged because the (-p-O-C5′-C4′-C3′-O-p-) skeleton of the cFapydG cyclopentane ring fits perfectly the one of the 1,3-propanediol AP site analogue (data not shown). An additional and defined electron density in the active site corresponds to the cFapydG nucleoside (Fig. 2B). The FapydG-binding Pocket—The cFapydG lesion is locked by the enzyme in an extra-helical conformation inside a large protein cavity in which 8-oxodG is also observed (38Fromme J.C. Verdine G.L. J. Biol. Chem. 2003; 278: 51543-51548Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The cFapydG nucleoside is stabilized in this pocket with the glycosidic bond in anti-conformation. It is now accepted that the base flipping mechanism is a common strategy used by DNA glycosylases to expose the C1′ of the initially buried damaged nucleoside to the nucleophilic attack of a water molecule (for monofunctional enzymes) or an enzyme reactive amino group (for bifunctional enzymes) (31Schärer O.D. Jiricny J. BioEssays. 2001; 23: 270-281Crossref PubMed Scopus (235) Google Scholar). Contrary to the relatively small, preformed, and rigid pocket of the human Ogg1, the Fpg binding pocket is defined by a very large cavity closed on one side by the DNA intercalated Fpg triad Arg109, Phe111, and Met75 (35Serre L. Pereira de Jesus K. Boiteux S. Zelwer C. Castaing B. EMBO J. 2002; 21: 2854-2865Crossref PubMed Scopus (112) Google Scholar), which results in the isolation of the damaged nucleoside from the remainder DNA backbone, thus preventing its reinsertion into the DNA grooves (Fig. 3). The lesion binding pocket is on the other side enlarged and extends to the outer side of the protein by a wide opening accessible to water molecules (Fig. 3). The inner surface of the binding pocket surrounding the damaged nucleoside is formed by residues belonging to several domains of the protein: the helix αA and the β4-β5 loop of the N-terminal domain and the structured part of αF-β9 loop located between the H2TH motif and the zinc finger in the C-terminal domain of the enzyme (29Sugahara M. Mikawa T. Kuramitsu T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (136) Google Scholar, 35Serre L. Pereira de Jesus K. Boiteux S. Zelwer C. Castaing B. EMBO J. 2002; 21: 2854-2865Crossref PubMed Scopus (112) Google Scholar). The precise architecture of the binding pocket is believed to be modulated by the dynamic part of the αF-β9 (residues 220-224, a brown dashed line in Fig. 2A). As it has been already observed for AP sites and 8-oxodG, the cyclopentane skeleton of cFapydG mimicking the ribofuranoside is not directly contacted by the enzyme in the binding pocket. However, its conformation is driven by numerous interactions between protein residues and the DNA phosphodiester backbone of the damaged strand (35Serre L. Pereira de Jesus K. Boiteux S. Zelwer C. Castaing B. EMBO J. 2002; 21: 2854-2865Crossref PubMed Scopus (112) Google Scholar, 37Fromme J.C. Verdine G.L. Nat. Struct. Biol. 2002; 9: 544-552PubMed Google Scholar). Among these interactions, the most remarkable ones are those established by Arg260 (situated in the β9-β10 loop of the Fpg zinc finger) with the two phosphate groups bordering the lesion and Tyr238 and Lys57, which contact the phosphates at the 5′- and 3′-side of the damaged nucleoside, p0 and p-1, respectively (Fig. 4A). Arg260, Tyr238, and Lys57 cooperate to constrain the bent DNA structure at the target site. One of the π-faces of the heterocycle of cFapyG is partly covered by the side chain of Ile219 whereas the other π-face is exposed to water molecules restrained in the binding pocket (Fig. 4B). A similar situation is observed for Fpg bound to 8-oxodG (38Fromme J.C. Verdine G.L. J. Biol. Chem. 2003; 278: 51543-51548Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). For most DNA glycosylases, the extrahelical base binding pocket is designed both to be compatible with the shape of the damaged nucleoside and to allow the formation of specific hydrogen bonds between the various donor and acceptor groups of the damaged base and the enzyme (48Jiang Y.L. Stivers J.T. Biochemistry. 2002; 41: 11236-11247Crossref PubMed Scopus (57) Google Scholar, 49Abner C.W. Lau A.Y. Ellenberger T. Bloom L.B. J. Biol. Chem. 2001; 276: 13379-13387Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 50Connor E.E. Wyatt M.D. Chem. Biol. 2002; 9: 1033-1041Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 51Kavli B. Slupphaug G. Mol C.D. Arvai A.S. Peterson S.B. Tainer J.A. Krokan H.E. EMBO J. 1996; 15: 3442-3447Crossref PubMed Scopus (152) Google Scholar). Fpg uses H-bonds and van der Waals contacts to bind the base lesion. However, some DNA glycosylases such as AlkA and AAG are devoid of any hydrogen bonding but use exclusively π-π and π-cation interactions. In these enzymes, aromatic protein residues surrounding the cationic alkylated base are the key discriminatory elements (52Labahn J. Scharer O.D. Long A. Ezaz-Nikpay K. Verdine G.L. Ellenberger T.E. Cell. 1996; 86: 321-329Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Similarly, hOgg1 utilizes Phe319 and Cys253 to stack toward both π-faces of 8-oxoG, sandwiching the damaged base in the active site (53Bruner S.D. Norman D.P.G. Verdine G.L. Nature. 2000; 403: 859-866Crossref PubMed Scopus (826) Google Scholar). To summarize, Fpg has selected H-bond interactions to achieve the specific recognition of cFapydG and 8-oxodG rather than a π-cation interaction strategy. Features of FapyG-specific Recognition—Several LlFpg residues cooperate to constrain cFapydG in the extrahelical base-binding pocket: Glu2 and Glu5 of the helix αA at the N terminus of the enzyme, Met75 and Glu76 of the β4-β5 loop, and Ser217, Ile219, and Tyr238 of the αF-β9 loop (Fig. 4). Except for Glu76, all of these residues are strictly conserved in the primary structure of the Fpg family (Fig. 5). Fpg recognizes the Watson-Crick face of FapyG through hydrogen-bonding contacts involving four residues: Glu2, Glu5, Ser217, and Ile219 (Fig. 4). Especially, the main chain amide of Ile219 recognizes the O6-carbonyl of FapyG, whereas the main chain carbonyl of Ser217 forms simultaneously hydrogen-bonds with both N1 and N2 of FapyG. The N2 of the damaged base is also hydrogen bonded by the carboxyl side chain of Glu5. Through two hydrogen interactions with a tightly bound water molecule (wat33 with B factor 22.43), the amide NH and the carboxyl O groups of the main chain and side chain of Glu2, respectively, cooperate with Ser217 and Glu5 to recognize N2 of FapyG (Fig. 4). Glu2 is also involved in the recognition of the N3 functional group of FapyG via wat33. This structural water molecule has been already observed in the binding pocket of LlFpg bound to the AP site (Protein Data Bank accession number 1NNJ) and in the borohydride-trapped complex of BstFpg covalently bound to the AP site (37Fromme J.C. Verdine G.L. Nat. Struct. Biol. 2002; 9: 544-552PubMed Google Scholar). From previous studies, Glu2 was shown to play a major role in DNA glycosylases activity (54Lavrukhin O.V. Lloyd R.S. Biochemistry. 2000; 39: 15266-15271Crossref PubMed Scopus (48) Google Scholar) and to interact with the C4′-OH of the furanose ring-opened deoxyribose of the imino enzyme-DNA intermediate, the common transition state of the glycosylase and lyase processes (37Fromme J.C. Verdine G.L. Nat. Struct. Biol. 2002; 9: 544-552PubMed Google Scholar, 55Zharkov D.O. Golan G. Gilboa R. Fernandes A.S. Gerchman S.E. Kycia J.H. Rieger R.A. Grollman A.P. Shoham G. EMBO J. 2002; 21: 789-800Crossref PubMed Scopus (154) Google Scholar). Surprisingly, the mutation E2Q" @default.
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• W2016694525 title "Structural Basis for the Recognition of the FapydG Lesion (2,6-Diamino-4-hydroxy-5-formamidopyrimidine) by Formamidopyrimidine-DNA Glycosylase" @default.
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