Structural basis for enzymatic excision of N1-methyladenine and N3-methylcytosine from DNA
2007; Springer Nature; Volume: 26; Issue: 8 Linguagem: Inglês
10.1038/sj.emboj.7601662
ISSN1460-2075
AutoresIngar Leiros, Marivi P Nabong, Kristin Grøsvik, Jeanette Ringvoll, Gyri T. Haugland, Lene Uldal, Karen Reite, Inger K. Olsbu, Ingeborg Knævelsrud, Elin Moe, Ole A. Andersen, Nils-Kåre Birkeland, Peter Ruoff, Arne Klungland, Svein Bjelland,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle29 March 2007free access Structural basis for enzymatic excision of N1-methyladenine and N3-methylcytosine from DNA Ingar Leiros Ingar Leiros The Norwegian Structural Biology Centre, University of Tromsø, Tromsø, Norway Search for more papers by this author Marivi P Nabong Marivi P Nabong Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Kristin Grøsvik Kristin Grøsvik Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Jeanette Ringvoll Jeanette Ringvoll Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Gyri T Haugland Gyri T Haugland Department of Biology, University of Bergen, Bergen, Norway Search for more papers by this author Lene Uldal Lene Uldal Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Karen Reite Karen Reite Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Inger K Olsbu Inger K Olsbu Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Ingeborg Knævelsrud Ingeborg Knævelsrud Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Department of Biology, University of Bergen, Bergen, Norway Search for more papers by this author Elin Moe Elin Moe The Norwegian Structural Biology Centre, University of Tromsø, Tromsø, Norway Search for more papers by this author Ole A Andersen Ole A Andersen The Norwegian Structural Biology Centre, University of Tromsø, Tromsø, Norway Search for more papers by this author Nils-Kåre Birkeland Nils-Kåre Birkeland Department of Biology, University of Bergen, Bergen, Norway Search for more papers by this author Peter Ruoff Peter Ruoff Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Arne Klungland Arne Klungland Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Svein Bjelland Corresponding Author Svein Bjelland Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Ingar Leiros Ingar Leiros The Norwegian Structural Biology Centre, University of Tromsø, Tromsø, Norway Search for more papers by this author Marivi P Nabong Marivi P Nabong Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Kristin Grøsvik Kristin Grøsvik Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Jeanette Ringvoll Jeanette Ringvoll Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Gyri T Haugland Gyri T Haugland Department of Biology, University of Bergen, Bergen, Norway Search for more papers by this author Lene Uldal Lene Uldal Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Karen Reite Karen Reite Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Inger K Olsbu Inger K Olsbu Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Ingeborg Knævelsrud Ingeborg Knævelsrud Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Department of Biology, University of Bergen, Bergen, Norway Search for more papers by this author Elin Moe Elin Moe The Norwegian Structural Biology Centre, University of Tromsø, Tromsø, Norway Search for more papers by this author Ole A Andersen Ole A Andersen The Norwegian Structural Biology Centre, University of Tromsø, Tromsø, Norway Search for more papers by this author Nils-Kåre Birkeland Nils-Kåre Birkeland Department of Biology, University of Bergen, Bergen, Norway Search for more papers by this author Peter Ruoff Peter Ruoff Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Arne Klungland Arne Klungland Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Svein Bjelland Corresponding Author Svein Bjelland Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway Search for more papers by this author Author Information Ingar Leiros1,‡, Marivi P Nabong2,3,‡, Kristin Grøsvik3, Jeanette Ringvoll2, Gyri T Haugland4, Lene Uldal2, Karen Reite2, Inger K Olsbu3, Ingeborg Knævelsrud3,4, Elin Moe1, Ole A Andersen1, Nils-Kåre Birkeland4, Peter Ruoff3, Arne Klungland2 and Svein Bjelland 3 1The Norwegian Structural Biology Centre, University of Tromsø, Tromsø, Norway 2Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, University of Oslo, Rikshospitalelt-Radiumhospitalet HF, Oslo, Norway 3Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway 4Department of Biology, University of Bergen, Bergen, Norway ‡These authors contributed equally to this work *Corresponding author. Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Kristine Bonnevies rd 30, N-4036 Stavanger, Norway. Tel.: +47 51831884; Fax: +47 51831750; E-mail: [email protected] The EMBO Journal (2007)26:2206-2217https://doi.org/10.1038/sj.emboj.7601662 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info N1-methyladenine (m1A) and N3-methylcytosine (m3C) are major toxic and mutagenic lesions induced by alkylation in single-stranded DNA. In bacteria and mammals, m1A and m3C were recently shown to be repaired by AlkB-mediated oxidative demethylation, a direct DNA damage reversal mechanism. No AlkB gene homologues have been identified in Archaea. We report that m1A and m3C are repaired by the AfAlkA base excision repair glycosylase of Archaeoglobus fulgidus, suggesting a different repair mechanism for these lesions in the third domain of life. In addition, AfAlkA was found to effect a robust excision of 1,N6-ethenoadenine. We present a high-resolution crystal structure of AfAlkA, which, together with the characterization of several site-directed mutants, forms a molecular rationalization for the newly discovered base excision activity. Introduction Enzymatic transmethylation reactions using S-adenosylmethionine as a donor to methylate certain DNA base positions are widespread among organisms. 5-Methylcytosine (m5C) is a minor but significant component of eukaryotic DNA, residing in CpG sequences throughout the genome (Vanyushin et al, 1970). It is considered to play an important regulatory role including gene silencing. In the majority of prokaryotes, m5C and N6-methyladenine protect genomic DNA from digestion by their own restriction endonucleases, and are also involved in repair, replication and expression. Because of its resistance to deamination-induced mutagenesis, N4-methylcytosine replaces m5C in the most thermophilic organisms (Ehrlich et al, 1987). However, erroneous non-enzymatic methylation of DNA (and other macromolecules) by S-adenosylmethionine, as well as by other cofactors, for example, N5-methyltetrahydrofolic acid, also occurs at a slow rate (Barrows and Magee, 1982; Rydberg and Lindahl, 1982). In double-stranded DNA (dsDNA), the major product formed by these reactions is the relatively innocuous N7-methylguanine, followed by N3-methyladenine (m3A). The latter is an important lethal lesion, possibly owing to protrusion of the N3-methyl group into the minor groove of the DNA double helix, thereby blocking DNA replication at the site of the lesion. These and other minor products, such as the similarly cytotoxic N3-methylguanine (m3G) and the O2-alkylpyrimidines, are excised from DNA in vivo by methylpurine-DNA glycosylase (MPG) enzymes (Sedgwick, 2004), leaving behind an apurinic/apyrimidinic (AP) site. This reaction initiates the so-called base excision repair (BER) pathway (Friedberg et al, 2006), which is predominantly completed by reinsertion of a single nucleotide by the activity of a 5′-acting AP endonuclease, DNA deoxyribophosphodiesterase, DNA polymerase and DNA ligase. O6-alkylguanine and O4-alkylthymine have long been regarded as the most mutagenic lesions owing to their ability to base-pair with thymine and guanine, respectively. They are corrected through direct damage reversal by methyl transfer to a cysteine residue in a DNA alkyltransferase (Sedgwick, 2004). In adenine and cytosine, the N1- and N3-positions are, respectively, the nucleophilic centers most reactive to alkylating agents. However, since base pairing protects these positions from methylation in dsDNA, the highly toxic N1-methyladenine (m1A) and both mutagenic and toxic N3-methylcytosine (m3C) (Delaney and Essigmann, 2004) are major products in single-stranded DNA (ssDNA) only (Beranek, 1990). Repair of m1A in DNA has eluded scientists for decades (Sedgwick, 2004), but it was recently reported to be carried out by a novel mechanism: oxidative demethylation. This is catalyzed by the AlkB protein and homologues in Escherichia coli and mammals, respectively (Duncan et al, 2002; Falnes et al, 2002; Trewick et al, 2002; Aas et al, 2003). However, some organisms, including the archaeons, lack AlkB homologues, which prompted the question whether other enzymatic mechanisms are involved in m1A repair. We report here that m1A and m3C, as well as the alkylated base analogue 1,N6-ethenoadenine (εA), are all excised with high efficiency from DNA by the MPG enzyme AfAlkA (Birkeland et al, 2002) and are consequently repaired through the BER pathway in the hyperthermophilic archaeon Archaeoglobus fulgidus. We present a high-resolution crystal structure of AfAlkA, which, together with the characterization of several site-directed mutants, forms a molecular rationalization for the newly discovered base excision activity. Comparison of the crystal structure of AfAlkA with the structure of AlkA from E. coli (EcAlkA) (Labahn et al, 1996; Yamagata et al, 1996; Hollis et al, 2000) reveals intriguing differences and conservations between the two enzymes. Results Excision of m1A, m3C and εA from DNA by AfAlkA To investigate whether the AfAlkA protein is able to excise m1A, m3C and εA from DNA, oligonucleotide substrates containing one of these base lesions inserted at a defined position (Figure 1A) were incubated with enzyme at 70°C (a temperature previously determined to be optimal for the excision of m3A by AfAlkA; Birkeland et al, 2002) for an increasing period of time. To avoid interference from less well-defined factors such as possible product inhibition (released base; AP site) and enzyme inactivation, activity was measured under single-turnover conditions employing enzyme 2–3 orders of magnitude in excess of substrate, as previously have been preferred for analysis of human MPG (hMPG) excision rate (Abner et al, 2001). The presence of repairable base lesions was confirmed by incubating the m1A- and m3C-containing DNA with EcAlkB (Trewick et al, 2002) and the εA-containing DNA with hMPG (Abner et al, 2001; Speina et al, 2003). The results showed that AfAlkA exhibits significant activity for the excision of all base lesions examined from DNA (Figure 1B). No excision of m1A or m3C was observed by incubating EcAlkA with substrate at 37°C under similar experimental conditions (Supplementary Figure 1). Figure 1.Excision of m1A, m3C and εA from DNA by AfAlkA. (A) The oligonucleotides containing m1A, εA or m3C at a specific position utilized as substrates. The size of the incision products following glycosylase excision of the specified base lesion and base-catalyzed phosphodiester bond cleavage of the resulting abasic site by alkali treatment is indicated. (B) Cleavage of 5′32P-labeled 49-nucleotides (nt) DNA into repair product (23 and 25 nt) is shown, for typical experiments. Incubation with EcAlkB (4.2 pmol) at 37°C was included as a positive control (in the case of m1A, 10 fmol DNA; m3C, 4.2 fmol DNA), where the DpnII cleavage site corresponds to 22 nt (Ringvoll et al, 2006) (not indicated). For excision of εA, incubation of 10 fmol DNA with a 26 kDa truncated hMPG protein (O'Connor, 1993) was included as a positive control. (C) Single-turnover kinetics for excision of m1A, m3C and εA, where 10 nM of DNA was incubated with the indicated concentrations of AfAlkA at 70°C for increasing time periods. Incubation with MNU-treated DNA (see Materials and methods) was performed under conditions where virtually only m3A is enzymatically excised (Birkeland et al, 2002), using 5000 d.p.m. of methylated DNA bases (∼1 nM of m3A). Each value represents the average of three independent measurements. Download figure Download PowerPoint To determine kinetic parameters for the excision of m1A, m3C and εA from DNA by AfAlkA, a single-turnover kinetic analysis based on certain considerations (presented in Supplementary data) was performed, where the reaction rate v for excision of a single base on a single DNA strand is described by the following equation: where [DNA]tot is the concentration of active substrate/DNA, [E]tot is the total concentration of the enzyme, k2 is the turnover number and KD=(k−1+k2)/k1. KD is analogous to the Michaelis constant KM describing the steady state (or rapid equilibrium) between substrate and the enzyme-substrate complex (see Supplementary data). The reaction rate v was determined by the slopes of the linear regression curves presented in Figure 1C. Dividing the rate by the active substrate concentration [DNA]tot leads to a first-order rate constant k, which describes the overall accumulation of product P during the excision: The amount of cleavable DNA (i.e. the active substrate concentration [DNA]tot) was determined by fitting the experimental [P] – time plots presented in Figure 1D to equation (2)), where the concentrations approached 5.3±0.1 nM for m1A, 6.4±0.3 nM for m3C, 8.2±0.2 nM for εA and 0.63±0.02 nM for m3A. The rate constant k is dependent on the total enzyme concentration as shown in equation (3) (see Supplementary data for derivation): Figure 2.(D) Plots of product formation as a function of time ([P]–time plots, together with a curve fit of equation (2) to the experimental data, to determine the active substrate concentration [DNA]tot (see Results)). (E) Calculated k values (determined from the slopes of the curves presented in C) as a function of [E]tot, together with a curve fit of equation (3) to the experimental data (see Results). Download figure Download PowerPoint Experimental results are in good agreement with equation (3). k values determined as function of [E]tot are presented in Figure 1E, together with a curve fit of equation (3) to the experimental data. From this, k2 and KD for the different substrates were estimated (Table I). Table 1. Single-turnover kinetic parameters of AfAlkAa Parameter Entity Substrate m1A m3C εA m3A Wild type k2 min−1 0.0109±0.0002 0.022±0.004 0.12±0.02 3.9±0.8 KD nM 530±40 900±500 700±200 230±60 Gln128Ala k2 min−1 0.011±0.001 0.058±0.004 KD nM 1300±500 170±50 Phe133Ala k2 min−1 0.008±0.002 0.037±0.001 KD nM 7000±4000 220±30 Phe282Ala k2 min−1 0.0084±0.0009 0.046±0.004 KD nM 4000±1000 160±50 Phe133Ala/Phe282Ala k2 min−1 ≈5 × 10−7 KD 0.008±0.001 KD nM ≫10 000 400±200 Arg286Ala k2 min−1 0.027±0.003 0.033±0.001 KD nM 4000±1000 280±30 Asp240Ala k2 min−1 Not detectable Not detectable a Because KD values depend on the binding kinetics between DNA and the enzyme, a precise interpretation of the physical nature of KD is difficult to make. See Supplementary data section for a more detailed discussion. The results show that AfAlkA excises m3C (k2=0.022±0.004 min−1) from DNA about twice as fast as m1A (k2=0.0109±0.0002 min−1). The rate for εA (k2=0.12±0.02 min−1) is further 5–10 times higher (Figure 1E). The results also indicate m3A as the principal substrate for AfAlkA, although the nature of the DNA in this case makes a direct comparison to the other three damaged bases inaccurate. Overall structure and comparison with EcAlkA The crystal structure of AfAlkA was determined using the single-wavelength anomalous dispersion (SAD) method on a mercury-soaked crystal. Redundant diffraction data to 1.8 Å resolution, collected on the absorption peak of the mercury LIII edge were sufficient to solve the structure. The native structure was thereafter determined by molecular replacement using data collected to 1.9 Å resolution on a native crystal. Of the 295 amino-acid residues, 289 from each of the two protein monomers in the asymmetric unit were ordered and included in the refinement. In addition, 2–3 residues in the C-terminus were observed to interact with a symmetry molecule and were modeled with reduced occupancy. This interaction is most likely an artifact of the crystal packing. Crystallographic data are presented in Supplementary Table I. The two monomers in the asymmetric unit overlay with a root mean square deviation (r.m.s.d.) of 0.24 Å for all Cα atoms. Similar to the helix–hairpin–helix (HhH) protein EcAlkA (Labahn et al, 1996), AfAlkA has a relatively compact globular structure, with overall dimensions of about 35 Å by 40 Å by 50 Å. AfAlkA is a three-domain protein, where the N-terminal domain, comprising residues 1–81, consists of a five-stranded antiparallel β-sheet (βA–βE) with two α-helices adjacent to the β-sheet (αA–αB; Figure 2A and B). Two helices (αC–αD), comprising residues 82–109, are close to the C-terminal domain. The central domain is mainly helical and consists of residues 110–236 in a total of eight α-helices (αE–αL). Two short β-strands (βF–βG; residues 148–161) protrude from the middle domain, contacting strands βC–βE in the N-terminal domain. A short linker region connects the middle domain with the C-terminal domain, which comprises residues 237–289, forming the three last α-helices in the protein (αM–αO). A study on EcAlkA in complex with DNA containing a modified abasic nucleotide, 1-azaribose (Hollis et al, 2000), established the HhH motif to be important for DNA binding. In AfAlkA, this motif is located in the C-terminal part of the central domain, comprising residues 205–229 in α-helices αK–αL (colored green in Figure 2A and B). Figure 3.Structure of AfAlkA. β-Strands are shown in blue and α-helices in red (except for the two α-helices forming the HhH motif, αK–αL, which are shown in green). (A) Stereo representation of the overall ribbon structure. (B) Primary and secondary structures of AfAlkA (above) aligned with EcAlkA (below). Identical amino-acid residues are boxed in blue, whereas structurally conserved residues are highlighted in gray. AfAlkA residues investigated by site-directed mutagenesis are indicated by an asterisk. The sequence alignment in (B) is based on a structural alignment of the crystal structures. The secondary structure elements of AfAlkA are indicated above the alignment, whereas those of EcAlkA are indicated below. Download figure Download PowerPoint Two metal ions with octahedral coordination were identified in electron density in each monomer of AfAlkA. These were modeled as sodium, according to mean bond distances of 2.43 Å (2.42 Å) to water ligands and 2.44 Å (2.46 Å) to main-chain carbonyl atoms (where numbers in parentheses are the expected distances), as well as its presence as the major cation (0.3 M) during purification (Harding, 2006). One sodium ion is located in proximity to the αA–βB loop of the N-terminal domain and is coordinated by the carbonyl oxygens of Leu21, Pro22, Leu24 and Asp28, the side-chain oxygen of Thr26 and a water molecule. This ion is most likely important only in stabilizing the folded state of the protein. The other sodium ion is located in proximity to the HhH region and will be described in more detail later. AfAlkA and EcAlkA share 20.5% amino-acid sequence identity, and similarity in secondary structure is indicated by their native crystal structures (Supplementary Figure 2) (Labahn et al, 1996; Yamagata et al, 1996), where 159 residues can be superimposed with an overall r.m.s.d. of 1.58 Å for Cα atoms. Highest structural similarity is within the central domain, where Gly112–Leu241 of AfAlkA overlaps with Leu107–Tyr239 of EcAlkA (Figure 2B). However, larger conformational differences are observed between the N- and C-terminal regions of the proteins (Supplementary Table II and Supplementary Figure 2), explaining why the crystal structure of AfAlkA could only be determined by experimental phasing methods. A three-dimensional structural homology search using DALI (Holm and Sander, 1993), with one monomer of AfAlkA as the search template, resulted in structural hits for several HhH proteins. As expected, EcAlkA showed the highest similarity with a Z-score of 28.2, followed by the bifunctional human 8-oxoguanine-DNA glycosylase/lyase (hOGG1) (Bruner et al, 2000) with a Z-score of 20.7. The sequence of hOGG1 comprises 345 amino acids sharing ∼17% identity with that of AfAlkA. The HhH motifs of the three proteins align particularly well and a more detailed comparison was therefore performed as described later. The DNA-binding region There are numerous amino-acid substitutions in the active site region and in the DNA-binding area of AfAlkA compared with EcAlkA (Figure 3A–D). On the other hand, the extensively conserved hairpin region of the HhH motif (Figure 2B) suggests a retained overall mode of enzyme–DNA binding. Superimposing the crystal structures of AfAlkA and EcAlkA, when the latter is in complex with a modified AP-site DNA (Hollis et al, 2000), specifies the DNA-contacting residues of AfAlkA (Figure 3A and B), showing a conserved mode of contacting and bending (>60°) DNA. A requirement for dsDNA is indicated, since both enzymes seem to possess more and stronger charged interactions with the complementary strand than with the damage-containing strand, along with amino-acid residues forming van der Waals interactions to the DNA minor groove. The number of positively charged residues putatively in contact with the phosphate backbone of the complementary strand appears to be slightly higher in AfAlkA (Lys138, Lys142, Arg176 and Arg182) compared with EcAlkA (Lys133, Arg137 and Lys170). In addition to the positively charged Arg245 and Lys216 expected to strengthen AfAlkA–DNA binding, the damage-containing strand appears to almost exclusively form polar interactions with the protein backbone and a sodium ion present in proximity to the hairpin loop of the HhH motif. This ion is coordinated by three main-chain protein contacts (the carbonyl oxygens of Thr213, Phe215 and Ile218) as well as three water molecules, and may mediate DNA phosphate backbone interactions, as suggested for a similarly coordinated sodium ion in EcAlkA. The latter is coordinated by the main-chain carbonyl oxygens of the structurally conserved Gln210, Phe212 and Ile215 (EcAlkA numbering) (Hollis et al, 2000). AfAlkA and EcAlkA have similar electrostatic surface potentials (Figure 4A and B), which also may relate to their overall DNA-binding strength. Figure 4.Crystal structures of (A) EcAlkA in complex with DNA containing the modified abasic nucleotide 1-azaribose (Hollis et al, 2000) and (B) AfAlkA, including an enlarged view of their respective active site regions (C, D). The damage-containing strand is shown in cyan and the complementary strand is shown in yellow. The HhH motif of EcAlkA and AfAlkA is colored dark blue, whereas the amino-acid residue replacing the flipped-out nucleotide is colored red in (A and B). In (D), an εA moiety has been modeled into the substrate-binding pocket of AfAlkA, with the molecular surface of the εA moiety shown in atom colors. Furthermore, the AfAlkA residue Arg286 is flexible and, as a consequence, was refined in two conformations, indicated in (D). Download figure Download PowerPoint Figure 5.Estimated electrostatic surface potential of (A) EcAlkA and (B) AfAlkA colored according to electrostatic potential at a contour level of ±7 kBT (blue, positively charged; red, negatively charged). The damage-containing strand is shown in light blue and the complementary strand in yellow. The DNA fragment from the EcAlkA–DNA complex structure has been modeled to fit to the AfAlkA DNA-binding region in (B). Download figure Download PowerPoint Amino-acid residues involved in damage detection and binding As indicated by sequence analysis (Birkeland et al, 2002), the crystal structures of AfAlkA (Figure 3B) and EcAlkA (Figure 3A) demonstrate several differences in the composition of the active site (Figure 3C and D). As EcAlkA and hOGG1 were found to have the highest structural similarity to AfAlkA, their crystal structures were utilized to identify conformational similarities and potential differences between the three proteins. Although superimposing the DNA backbone of the hOGG1-DNA (Bruner et al, 2000) and EcAlkA-DNA (Hollis et al, 2000) complexes results in a reasonably good match, the orientation of the two active site grooves indicates that the flipped-out damaged bases will be oriented in different ways. Furthermore, the aromatic residues in the substrate-binding pockets of EcAlkA and AfAlkA will direct rotation of the substrate base at roughly 90°, compared with its orientation in hOGG1. Aromatic residues are electron donors attracting the damaged, and in most cases electron-deficient, alkylated DNA base. Such π–π (or π–cation in the case of positively charged bases) interactions have been suggested to be important in the recognition and binding of alkylated base lesions (Eichman et al, 2003) (Figure 5A–F). In AfAlkA, base stacking appears to be more pronounced than in EcAlkA (Figure 3C and D), with Phe133 and Phe282 ideally positioned in the binding pocket to form interactions with the flipped-out substrate base (Figure 3D). The binding pocket of EcAlkA (Figure 3C) possesses less-optimized residues (Val128 and Trp272) in order to attract damaged bases (Figure 5C). Figure 6.Substrate-binding pockets of (A) AfAlkA with εA modeled into the substrate-binding pocket and (B) hMPG crystallized in complex with an εA-containing DNA fragment. The εA moiety is indicated with orange carbon atoms. The dotted line indicates the potential hydrogen bond between the damaged base and the protein. (C) Binding pocket of EcAlka crystallized in complex with DNA containing 1-azaribose. (D–F) AfAlkA binding pockets with m1A, m3C and m3A modeled, otherwise as above. The potential repulsion between Arg286 and the amine of the damaged base is indicated. Download figure Download PowerPoint Arg286 provides binding strength and productive orientation of substrate by forming a hydrogen bond specifically with the εA base (Figure 5A), where the N6 nitrogen of εA projects a lone pair in the direction of the NH2+ (Nη2) arginine side chain. This may also explain the efficiency of hypoxanthine, the deamination product of adenine, as a substrate for AfAlkA (Mansfield et al, 2003). In this case, an oxygen atom (O6) instead of a nitrogen atom (N6) provides the lone pair. The equivalent position (N6) of m1A is unable to accept a proton necessary to form a similar interaction with Arg286 (Figure 5D), which also is the case for the N4 atom of m3C being in a spatially overlapping position (Figure 5E). The m1A/m3A-N6 and m3C-N4 amine groups are protonated and potentially cause repulsion of the protonated side chain of Arg286 (Figure 5D–F). The substrate-binding pocket of AfAlkA indicates some selectivity against naturally occurring purine bases. While a
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