Characterization of Mg²⁺ Binding to the DNA Repair Protein Apurinic/Apyrimidic Endonuclease 1 via Solid-State ²⁵Mg NMR Spectroscopy

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1), a member of the divalent cation-dependent phosphoesterase superfamily of proteins that retain the conserved four-layered alpha/beta-sandwich structural core, is an essential protein that functions as part of base excision repair to remove mutagenic and cytotoxic abasic sites from DNA. Using low-temperature solid-state (25)Mg NMR spectroscopy and various mutants of APE1, we demonstrate that Mg(2+) binds to APE1 and a functional APE1-substrate DNA complex with an overall stoichiometry of one Mg(2+) per mole of APE1 as predicted by the X-ray work of Tainer and co-workers (Mol, C. D.; Kuo, C. F.; Thayer, M. M.; Cunningham, R. P.; Tainer, J. A. Nature 1995, 374 , 381-386). However, the NMR spectra show that the single Mg(2+) site is disordered. We discuss the probable reasons for the disorder at the Mg(2+) binding site. The most likely source of this disorder is arrangement of the protein-ligands about the Mg(2+) (cis and trans isomers). The existence of these isomers reinforces the notion of the plasticity of the metal binding site within APE1.

Conserved Structural Chemistry for Incision Activity in Structurally Non-homologous Apurinic/Apyrimidinic Endonuclease APE1 and Endonuclease IV DNA Repair Enzymes

“…It is more than 3.5 Å from where the attacking water would be positioned. Moreover, evidence from NMR has suggested that His-309 in the APE1-DNA complex is protonated (75,88) and thus incapable of serving as a general base. Recently, MD studies suggested that there may be a second metal binding site B, where the Mg 2ϩ coordinates with Asp-210 and Asn-212, and that the one metal moves from site B to the experimentally observed metal site A during catalysis (44,69).…”

Human Apurinic/Apyrimidinic Endonuclease 1

“…Other important residues include N68, D70, Y171, N212, D283, and D308, which are generally conserved throughout the diverse members of the phosphoesterase superfamily. Still, further studies are needed to delineate the precise catalytic reaction mechanism, which is debated in several recent publications (123,148,152,210).…”

Magnesium incorporation into hydroxyapatite

“…Indeed, just like calcium, Mg 2+ is a closed shell diamagnetic cation, and magnesium-25, the NMR active isotope, is a spin-5/2 low-nucleus of low natural abundance (~10.0%), [38] with a relatively large quadrupole moment, meaning that the NMR signals can be very broad. Although techniques like 25 Mg solid state NMR [38,39,76,88] and Mg K-edge EXAFS and XANES [89][90][91] are being developed to help investigate the local structure around magnesium, they cannot be used routinely, because they require access to ultra-high field magnets or soft X-ray synchrotron sources. Furthermore, these experiments can be very time-consuming, especially for samples in which the Mg content is low.…”