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

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 0 -deoxyribose phosphate and the 3 0 -OH primer needed for repair synthesis. It also has additional repair activities, including the removal of 3 0 -blocking groups. APE1 is a powerful enzyme that absolutely requires Mg 2+ , but the stoichiometry and catalytic function of the divalent cation remain unresolved for APE1 and for other enzymes in the DNase I superfamily. 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 Mg 2+ in APE1-catalyzed reactions.

The role of Asn-212 in the catalytic mechanism of human endonuclease APE1: Stopped-flow kinetic study of incision activity on a natural AP site and a tetrahydrofuran analogue

“…6A). In the recently published crystal structure of free APE1 with the Mg 2+ cofactor Asp-70 was found in the first coordination sphere of the metal ion, whereas Asp-308 participated in the metal coordination through a water molecule [10]. Our results revealed that the side chain of Asp-308 was lying closer to Mg 2+ than Asp-70, making possible the immediate contact.…”

“…Previous crystallographic and computational studies suggested two alternative reaction mechanisms: one predicts the presence of two metal ions in the APE1 active site for catalysis [8], while the other model suggests that Mg 2+ moves from the B-site (Asp-210, Asn-212 and His-309) to the A-site (Asp-70 and Glu-96) during the catalytic cycle [9]. However, the recently published high resolution crystal structure of free APE1 with the essential Mg 2+ cofactor has confirmed the presence of a single metal ion in the A-site, while the B-site is most probably occupied by a water molecule [10]. The authors also suggest that the water molecule, coordinated by Asn-212 and Asp-210 side chains, serves as a nucleophile for phosphodiester bond hydrolysis.…”

“…When Asn-212 is substituted by Asp, the imidazole ring of His-309 is rotated around the C˛-Cˇbond, resulting in an increase in the distance between the N1 atom and the DNA target phosphate from 3.8Å to 9.4Å. As known from literature, His-309 and Glu-96 are essential catalytic residues responsible for correct location of abasic ribose in the APE1 active site [10,11]. The observed changes most likely give rise to a decrease in the N212D APE1 catalysis rate, but do not preclude completion of substrate cleavage.…”

“…A significant problem for MD simulation is the correct positioning of the metal ion in the APE1 structure. While editing the PDB file, we placed the Mg 2+ ion by taking into account the general principles underlying metal ion coordination in proteins and the recently published data [10,11,24]. The resulting structure was subjected to minimization in which the metal ion and amino acids were allowed to adopt an optimal conformation.…”

Structural insights into the ligand-binding hot spots of APEX1: an in silico analysis

“…During preparation of ligands, number of rotatable bonds was restraint up to six to avoid conformational explosion due to too many rotational degrees of freedom. The binding site of APEX1 receptor was already reported in the previous studies (Beernink et al, 2001;Manvilla et al, 2013). Autogrid was engaged to create grid maps around the active site of the protein.…”