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. 2000 Feb 15;19(4):758-66.
doi: 10.1093/emboj/19.4.758.

DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA

T Hollis  1 , Y Ichikawa, T Ellenberger
Affiliations

DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA

T Hollis et al. EMBO J. .

Abstract

The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin-helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner.

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Figures

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Fig. 1. Unbiased Fo – Fc difference electron density for the DNA. This electron density map was calculated with phases from the protein model after two molecules of the AlkA dimer were positioned in the asymmetric unit by molecular replacement and rigid body refinement. The resulting electron density for the bound DNA in the region of the active site clearly shows the flipped out 1–azaribose. The protein is shown in green and the fitted DNA is shown in yellow. Figures 1, 2, 4 and 5 were created with the program SETOR (Evans, 1990).
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Fig. 2. (A) AlkA-induced distortion of DNA. A 66° bend in the DNA results from the insertion of Leu125 and loops αD–αE and αG–αH (shown in green) into the minor groove. The DNA is anchored to the protein by the interactions with the HhH motif (shown in red). The local helical axis of the DNA is shown by a red line. (B) Schematic diagram of the AlkA–DNA contacts. The 1–azaribose abasic nucleotide is in an extrahelical conformation with N1′ of the sugar positioned 3.2 Å from the carboxylate oxygen of Asp238 (inset). Except for Lys170, all hydrogen-bonding contacts are made with the 1–azaribose-containing strand of the DNA. The HhH motif acts to anchor the DNA to the protein by providing several hydrogen bonds as well as a metal-mediated interaction. Having relatively few polar interactions with the DNA, AlkA also relies on van der Waals interactions for binding energy (see Figure 3). Residues with main chain atoms contacting the DNA are indicated by the prefix ‘mc’.
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Fig. 2. (A) AlkA-induced distortion of DNA. A 66° bend in the DNA results from the insertion of Leu125 and loops αD–αE and αG–αH (shown in green) into the minor groove. The DNA is anchored to the protein by the interactions with the HhH motif (shown in red). The local helical axis of the DNA is shown by a red line. (B) Schematic diagram of the AlkA–DNA contacts. The 1–azaribose abasic nucleotide is in an extrahelical conformation with N1′ of the sugar positioned 3.2 Å from the carboxylate oxygen of Asp238 (inset). Except for Lys170, all hydrogen-bonding contacts are made with the 1–azaribose-containing strand of the DNA. The HhH motif acts to anchor the DNA to the protein by providing several hydrogen bonds as well as a metal-mediated interaction. Having relatively few polar interactions with the DNA, AlkA also relies on van der Waals interactions for binding energy (see Figure 3). Residues with main chain atoms contacting the DNA are indicated by the prefix ‘mc’.
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Fig. 3. (A) The binding surface of AlkA colored according to its electrostatic potential (blue, positively charged; red, negatively charged) shows a relatively uncharged surface contacting the DNA. The intercalating Leu125 is indicated by an asterisk, and the catalytic Asp238 is indicated by the small red patch above the asterisk and adjacent to Leu125. (B) A rotated view of the AlkA–DNA complex showing that the abasic 1–azaribose has been rotated out of the DNA and into the aromatic active site cleft of the enzyme. Leu125 has been inserted into the gap created by the flipped out nucleotide. The minor groove has been widened substantially by the protein, contributing to the significant distortion of the DNA. Figures 1 and 6 were generated with the program GRASP (Nicholls et al., 1993).
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Fig. 4. The HhH motif serves to anchor the DNA to the protein through hydrogen bonds with Thr219 and the main chain amides of residues 216 and 214. The HhH motif is positioned adjacent to the active site (cf. Figure 2B) but it does not participate directly in base flipping or in catalysis. The hairpin turn of the HhH ligates a metal that contacts the DNA phosphate backbone and serves to organize the hairpin for additional hydrogen-bonding interactions with the DNA. The sodium ion modeled here (light blue) is coordinated by the main chain carbonyl oxygens of residues 215, 212 and 210, the phosphate oxygen of the DNA and a water molecule. One remaining potential site of metal coordination is unoccupied.
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Fig. 5. A 3–methyladenine substrate modeled in AlkA's active site. 3–methyladenine (pink) was superimposed on the 1–azaribose moiety in the crystal structure of the AlkA–DNA complex. In the resulting model, the 3–methyladenine base stacks face-to-face against Trp272 and makes edge-on contacts with Tyr222. The open architecture of AlkA's substrate-binding pocket would accommodate many types of modified bases. Trp218 is located behind the ribose of the flipped out nucleotide, leaving no room for a water nucleophile (cf. Figure 6).
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Fig. 6. Mechanistic implications of the AlkA active site. The closely interacting van der Waals surfaces of the protein (green) and the DNA (yellow) leave no room between the deoxyribose of a flipped out nucleotide and Trp218 to position a water molecule (red) for an attack on the back of the glycosylic bond. This is strong evidence against a direct displacement (SN2) mechanism of glycosylic bond cleavage (see text for details).
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Fig. 7. Structure-based sequence alignment of HhH base excision glycosylases. The sequences of AlkA, MutY and Endo III are aligned based on the best superposition of Cα atoms in the crystal structures, as determined by the program DALI (Holm and Sander, 1993). The secondary structure of AlkA is shown above the sequence. Members of the HhH superfamily not only share a similar fold, but also have a common surface chemistry in the region contacting DNA in the AlkA–DNA complex (cf. Figure 3). The areas shaded green are the residues in AlkA that are within van der Waals contact distance of the minor groove of the DNA. The residues of the HhH motif that make contact with the DNA are shaded pink and the catalytic aspartate is shaded blue. Residues lining the putative base-binding pocket are shaded orange (cf. Figure 5). It is likely that members of the HhH glycosylase superfamily all bind to DNA and expose a substrate base in a similar fashion.
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