High-Resolution Crystal Structures Reveal Plasticity in the Metal Binding Site of Apurinic/Apyrimidinic Endonuclease I

Abstract

Apurinic/apyrimidinic endonuclease I (APE1) is an essential base excision repair enzyme that catalyzes a Mg2+-dependent reaction in which the phosphodiester backbone is cleaved 5′ of an abasic site in duplex DNA. This reaction has been proposed to involve either one or two metal ions bound to the active site. In the present study, we report crystal structures of Mg2+, Mn2+, and apo-APE1 determined at 1.4, 2.2, and 1.65 Å, respectively, representing two of the highest resolution structures yet reported for APE1. In our structures, a single well-ordered Mn2+ ion was observed coordinated by D70 and E96; the Mg2+ site exhibited disorder modeled as two closely positioned sites coordinated by D70 and E96 or E96 alone. Direct metal binding analysis of wild-type, D70A, and E96A APE1, as assessed by differential scanning fluorimetry, indicated a role for D70 and E96 in binding of Mg2+ or Mn2+ to APE1. Consistent with the disorder exhibited by Mg2+ bound to the active site, two different conformations of E96 were observed coordinated to Mg2+. A third conformation for E96 in the apo structure is similar to that observed in the APE1–DNA–Mg2+ complex structure. Thus, binding of Mg2+ in three different positions within the active site of APE1 in these crystal structures corresponds directly with three different conformations of E96. Taken together, our results are consistent with the initial capture of metal by D70 and E96 and repositioning of Mg2+ facilitated by the structural plasticity of E96 in the active site.

Phosphodiester hydrolysis computed for cluster models of enzymatic active sites

“…The single magnesium ion is coordinated by one glutamate and one aspartate residue, one oxygen atom of the phosphate group and a water molecule. Motivated by several high resolution crystal structures of the active sites of endonuclease enzymes [31][32][33][34][35], the magnesium ion is positioned either on the "attack site", binding to O2P, and termed MgA, or on the "departure site", coordinated by O1P…”

“…Available high-resolution X-ray structures of many endonuclease enzymes in complex with target DNA or RNA or substrate analogues [31][32][33][34][35] and various biochemical studies have shown that metal cofactors are essential for the enzymatic phosphodiester cleavage [36][37][38][39]. The metal ions not only play a direct role in the chemical step but also influence the binding interaction of the enzyme with the DNA or RNA [40,41].…”

Effects of mono- and divalent metal ions on DNA binding and catalysis of human apurinic/apyrimidinic endonuclease 1

“…The Mg 2+ coordination geometry and the functions of metal ions in substrate binding, cleavage, and product release are actively discussed. [14][15][16][22][23][24][25][26] The first reported APE1 crystal structure contains one Sm 3+ ion in the active site. 14 The metal-binding site identified in this APE1 structure (PDB ID: 1BIX) is often referred to as the ''A-site''.…”

“…14,15 The latest structural studies on human APE1 showed that repositioning of Mg 2+ is facilitated by the structural plasticity of Glu-96 in the active site of the enzyme. 26 Structures of an enzyme-product (EP) complex and enzyme-substrate (ES) complex with a single Mg 2+ ion in the active site were determined recently. 19,27 In the EP complex, Mg 2+ is coordinated directly by Glu-96, the 3 0 -OH, a non-bridging O atom of the nascent 5 0 -phosphate, and by three water molecules, one of which is bound to Asp-70.…”

Small molecule activation of apurinic/apyrimidinic endonuclease 1 reduces DNA damage induced by cisplatin in cultured sensory neurons

“…As assessed by differential scanning fluorimetry, nicorandil does not bind directly to either wild-type or R177A enzymes in the presence or absence of Mg 2+ , which as previously reported stabilizes the wild-type enzyme [33]. R177A APE1 exhibited a Tm that was ∼4 °C lower than that of the wild-type APE1 but was stabilized by the addition of Mg 2+ as shown by a ∼2 °C increase in melting temperature.…”