Characterization of Mg2+ Binding to the DNA Repair Protein Apurinic/Apyrimidic Endonuclease 1 via Solid-State 25Mg NMR Spectroscopy
A. S. Lipton, R. W. Heck, S. Primak, D. R. McNeill, D. M. Wilson, III, and P. D. Ellis
Apurinic/apyrimidinic endonuclease 1 (APE1), a member of the divalent cation-dependent phosphoesterase superfamily of proteins that retain the conserved four-layered α/β-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 25Mg NMR spectroscopy and various mutants of APE1, we demonstrate that Mg2+ binds to APE1 and a functional APE1−substrate DNA complex with an overall stoichiometry of one Mg2+ 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 Mg2+ site is disordered. We discuss the probable reasons for the disorder at the Mg2+ binding site. The most likely source of this disorder is arrangement of the protein−ligands about the Mg2+ (cis and trans isomers). The existence of these isomers reinforces the notion of the plasticity of the metal binding site within APE1.