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Structure of human apurinic/apyrimidinic endonuclease 1 with the essential Mg2+ cofactor.

Manvilla BA, Pozharski E, Toth EA, Drohat AC - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: The structure reveals ideal octahedral coordination of Mg2+ via two carboxylate groups and four water molecules.One residue that coordinates Mg2+ directly and two that bind inner-sphere water molecules are strictly conserved in the DNase I superfamily.This structure, together with a recent structure of the enzyme-product complex, inform on the stoichiometry and the role of Mg2+ in APE1-catalyzed reactions.

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Affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, USA.

ABSTRACT
Apurinic/apyrimidinic endonuclease 1 (APE1) mediates the repair of abasic sites and other DNA lesions and is essential for base-excision repair and strand-break repair pathways. APE1 hydrolyzes the phosphodiester bond at abasic sites, producing 5'-deoxyribose phosphate and the 3'-OH primer needed for repair synthesis. It also has additional repair activities, including the removal of 3'-blocking groups. APE1 is a powerful enzyme that absolutely requires Mg2+, but the stoichiometry and catalytic function of the divalent cation remain unresolved for APE1 and for other enzymes in the DNase I superfamily. Previously reported structures of DNA-free APE1 contained either Sm3+ or Pb2+ in the active site. However, these are poor surrogates for Mg2+ because Sm3+ is not a cofactor and Pb2+ inhibits APE1, and their coordination geometry is expected to differ from that of Mg2+. A crystal structure of human APE1 was solved at 1.92 Å resolution with a single Mg2+ ion in the active site. The structure reveals ideal octahedral coordination of Mg2+ via two carboxylate groups and four water molecules. One residue that coordinates Mg2+ directly and two that bind inner-sphere water molecules are strictly conserved in the DNase I superfamily. This structure, together with a recent structure of the enzyme-product complex, inform on the stoichiometry and the role of Mg2+ in APE1-catalyzed reactions.

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Alignment of the new Mg2+-bound APE1 structure with DNA-bound structures. (a) Recently determined structure of the APE1 enzyme–product (EP) complex (green; PDB entry 4iem; Tsutakawa et al., 2013 ▶) aligned with the structure of Mg2+-bound APE1 (cyan) reported here. DNA from the EP complex (orange) contains a 3′-OH and a 5′-deoxyribose phosphate (dRP). The Mg2+ ion from the EP complex is colored green and its coordination is indicated by black dotted lines. The Mg2+ ion from the new DNA-free structure is colored cyan and its coordination is indicated by cyan dotted lines, with coordinating water molecules shown as red stars. Hydrogen-bond interactions (yellow dashes) are shown for the EP complex only. The approximate locations of the A and B sites are noted (gray symbols). (b) Structure of the APE1 enzyme–substrate (ES) complex (green; PDB entry 1dew; Mol et al., 2000 ▶) aligned with the new structure of Mg2+-bound APE1 (cyan). The DNA in the ES complex (orange) contains an intact abasic site and Mg2+ was omitted from the ES complex to halt P—O bond cleavage. The green sphere indicates the position of the Mg2+ ion in the EP complex (also aligned with DNA-free APE1). Mg2+ in the DNA-free structure is shown in cyan; its coordination is indicated by cyan dotted lines, with coordinating waters shown as red stars. Hydrogen-bond interactions (yellow dashes) are shown for the ES complex. The potential nucleophilic water from our DNA-free structure is shown as a red star, with cyan dashes indicating hydrogen bonds to Asp210 and Asn212. This water molecule was also observed in previous structures of APE1 with Sm3+ or a single Pb2+ ion (Fig. 1 ▶). The approximate locations of the A and B sites are noted (gray symbols).
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fig2: Alignment of the new Mg2+-bound APE1 structure with DNA-bound structures. (a) Recently determined structure of the APE1 enzyme–product (EP) complex (green; PDB entry 4iem; Tsutakawa et al., 2013 ▶) aligned with the structure of Mg2+-bound APE1 (cyan) reported here. DNA from the EP complex (orange) contains a 3′-OH and a 5′-deoxyribose phosphate (dRP). The Mg2+ ion from the EP complex is colored green and its coordination is indicated by black dotted lines. The Mg2+ ion from the new DNA-free structure is colored cyan and its coordination is indicated by cyan dotted lines, with coordinating water molecules shown as red stars. Hydrogen-bond interactions (yellow dashes) are shown for the EP complex only. The approximate locations of the A and B sites are noted (gray symbols). (b) Structure of the APE1 enzyme–substrate (ES) complex (green; PDB entry 1dew; Mol et al., 2000 ▶) aligned with the new structure of Mg2+-bound APE1 (cyan). The DNA in the ES complex (orange) contains an intact abasic site and Mg2+ was omitted from the ES complex to halt P—O bond cleavage. The green sphere indicates the position of the Mg2+ ion in the EP complex (also aligned with DNA-free APE1). Mg2+ in the DNA-free structure is shown in cyan; its coordination is indicated by cyan dotted lines, with coordinating waters shown as red stars. Hydrogen-bond interactions (yellow dashes) are shown for the ES complex. The potential nucleophilic water from our DNA-free structure is shown as a red star, with cyan dashes indicating hydrogen bonds to Asp210 and Asn212. This water molecule was also observed in previous structures of APE1 with Sm3+ or a single Pb2+ ion (Fig. 1 ▶). The approximate locations of the A and B sites are noted (gray symbols).

Mentions: One of the two residues that directly coordinate the Mg2+ ion, Glu96, is strictly conserved in the DNase I superfamily, while the other, Asp70, seems to be restricted to mammalian APE1 (Gorman et al., 1997 ▶; Castillo-Acosta et al., 2009 ▶). We note that Glu96 also directly coordinates the single Mg2+ ion that was observed in a recently reported enzyme–product (EP) complex of APE1, while the Asp70 carboxylate binds a water molecule that in turn coordinates Mg2+ (Fig. 2 ▶a; Tsutakawa et al., 2013 ▶). Previous studies found that the E96Q mutation causes a 2300-fold loss in AP endonuclease activity (Erzberger & Wilson, 1999 ▶), consistent with an important role of Glu96 in coordinating the Mg2+ cofactor. An important role of Asp70 is indicated by the 26-fold loss in endonuclease activity of the D70A or D70R mutations (Erzberger & Wilson, 1999 ▶). Moreover, D70A and E96Q variants exhibit diminished endonuclease activity relative to native APE1 under conditions of limiting Mg2+ (Nguyen et al., 2000 ▶; Castillo-Acosta et al., 2009 ▶).


Structure of human apurinic/apyrimidinic endonuclease 1 with the essential Mg2+ cofactor.

Manvilla BA, Pozharski E, Toth EA, Drohat AC - Acta Crystallogr. D Biol. Crystallogr. (2013)

Alignment of the new Mg2+-bound APE1 structure with DNA-bound structures. (a) Recently determined structure of the APE1 enzyme–product (EP) complex (green; PDB entry 4iem; Tsutakawa et al., 2013 ▶) aligned with the structure of Mg2+-bound APE1 (cyan) reported here. DNA from the EP complex (orange) contains a 3′-OH and a 5′-deoxyribose phosphate (dRP). The Mg2+ ion from the EP complex is colored green and its coordination is indicated by black dotted lines. The Mg2+ ion from the new DNA-free structure is colored cyan and its coordination is indicated by cyan dotted lines, with coordinating water molecules shown as red stars. Hydrogen-bond interactions (yellow dashes) are shown for the EP complex only. The approximate locations of the A and B sites are noted (gray symbols). (b) Structure of the APE1 enzyme–substrate (ES) complex (green; PDB entry 1dew; Mol et al., 2000 ▶) aligned with the new structure of Mg2+-bound APE1 (cyan). The DNA in the ES complex (orange) contains an intact abasic site and Mg2+ was omitted from the ES complex to halt P—O bond cleavage. The green sphere indicates the position of the Mg2+ ion in the EP complex (also aligned with DNA-free APE1). Mg2+ in the DNA-free structure is shown in cyan; its coordination is indicated by cyan dotted lines, with coordinating waters shown as red stars. Hydrogen-bond interactions (yellow dashes) are shown for the ES complex. The potential nucleophilic water from our DNA-free structure is shown as a red star, with cyan dashes indicating hydrogen bonds to Asp210 and Asn212. This water molecule was also observed in previous structures of APE1 with Sm3+ or a single Pb2+ ion (Fig. 1 ▶). The approximate locations of the A and B sites are noted (gray symbols).
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Related In: Results  -  Collection

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Show All Figures
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fig2: Alignment of the new Mg2+-bound APE1 structure with DNA-bound structures. (a) Recently determined structure of the APE1 enzyme–product (EP) complex (green; PDB entry 4iem; Tsutakawa et al., 2013 ▶) aligned with the structure of Mg2+-bound APE1 (cyan) reported here. DNA from the EP complex (orange) contains a 3′-OH and a 5′-deoxyribose phosphate (dRP). The Mg2+ ion from the EP complex is colored green and its coordination is indicated by black dotted lines. The Mg2+ ion from the new DNA-free structure is colored cyan and its coordination is indicated by cyan dotted lines, with coordinating water molecules shown as red stars. Hydrogen-bond interactions (yellow dashes) are shown for the EP complex only. The approximate locations of the A and B sites are noted (gray symbols). (b) Structure of the APE1 enzyme–substrate (ES) complex (green; PDB entry 1dew; Mol et al., 2000 ▶) aligned with the new structure of Mg2+-bound APE1 (cyan). The DNA in the ES complex (orange) contains an intact abasic site and Mg2+ was omitted from the ES complex to halt P—O bond cleavage. The green sphere indicates the position of the Mg2+ ion in the EP complex (also aligned with DNA-free APE1). Mg2+ in the DNA-free structure is shown in cyan; its coordination is indicated by cyan dotted lines, with coordinating waters shown as red stars. Hydrogen-bond interactions (yellow dashes) are shown for the ES complex. The potential nucleophilic water from our DNA-free structure is shown as a red star, with cyan dashes indicating hydrogen bonds to Asp210 and Asn212. This water molecule was also observed in previous structures of APE1 with Sm3+ or a single Pb2+ ion (Fig. 1 ▶). The approximate locations of the A and B sites are noted (gray symbols).
Mentions: One of the two residues that directly coordinate the Mg2+ ion, Glu96, is strictly conserved in the DNase I superfamily, while the other, Asp70, seems to be restricted to mammalian APE1 (Gorman et al., 1997 ▶; Castillo-Acosta et al., 2009 ▶). We note that Glu96 also directly coordinates the single Mg2+ ion that was observed in a recently reported enzyme–product (EP) complex of APE1, while the Asp70 carboxylate binds a water molecule that in turn coordinates Mg2+ (Fig. 2 ▶a; Tsutakawa et al., 2013 ▶). Previous studies found that the E96Q mutation causes a 2300-fold loss in AP endonuclease activity (Erzberger & Wilson, 1999 ▶), consistent with an important role of Glu96 in coordinating the Mg2+ cofactor. An important role of Asp70 is indicated by the 26-fold loss in endonuclease activity of the D70A or D70R mutations (Erzberger & Wilson, 1999 ▶). Moreover, D70A and E96Q variants exhibit diminished endonuclease activity relative to native APE1 under conditions of limiting Mg2+ (Nguyen et al., 2000 ▶; Castillo-Acosta et al., 2009 ▶).

Bottom Line: The structure reveals ideal octahedral coordination of Mg2+ via two carboxylate groups and four water molecules.One residue that coordinates Mg2+ directly and two that bind inner-sphere water molecules are strictly conserved in the DNase I superfamily.This structure, together with a recent structure of the enzyme-product complex, inform on the stoichiometry and the role of Mg2+ in APE1-catalyzed reactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, USA.

ABSTRACT
Apurinic/apyrimidinic endonuclease 1 (APE1) mediates the repair of abasic sites and other DNA lesions and is essential for base-excision repair and strand-break repair pathways. APE1 hydrolyzes the phosphodiester bond at abasic sites, producing 5'-deoxyribose phosphate and the 3'-OH primer needed for repair synthesis. It also has additional repair activities, including the removal of 3'-blocking groups. APE1 is a powerful enzyme that absolutely requires Mg2+, but the stoichiometry and catalytic function of the divalent cation remain unresolved for APE1 and for other enzymes in the DNase I superfamily. Previously reported structures of DNA-free APE1 contained either Sm3+ or Pb2+ in the active site. However, these are poor surrogates for Mg2+ because Sm3+ is not a cofactor and Pb2+ inhibits APE1, and their coordination geometry is expected to differ from that of Mg2+. A crystal structure of human APE1 was solved at 1.92 Å resolution with a single Mg2+ ion in the active site. The structure reveals ideal octahedral coordination of Mg2+ via two carboxylate groups and four water molecules. One residue that coordinates Mg2+ directly and two that bind inner-sphere water molecules are strictly conserved in the DNase I superfamily. This structure, together with a recent structure of the enzyme-product complex, inform on the stoichiometry and the role of Mg2+ in APE1-catalyzed reactions.

Show MeSH
Related in: MedlinePlus