Characterization of Mg2+ binding to the DNA repair protein apurinic/apyrimidic endonuclease 1 via solid-state 25Mg NMR spectroscopy - PubMed (original) (raw)

. 2008 Jul 23;130(29):9332-41.

doi: 10.1021/ja0776881. Epub 2008 Jun 25.

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Characterization of Mg2+ binding to the DNA repair protein apurinic/apyrimidic endonuclease 1 via solid-state 25Mg NMR spectroscopy

A S Lipton et al. J Am Chem Soc. 2008.

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.

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Figures

Figure 1

Figure 1

All of the spectra were simulated at 9.4 T. The spectrum in (a) is a finite pulse simulation of a 25Mg NMR spikelet lineshape with a quadrupole coupling constant, Cq of 2.5 MHz with an asymmetry parameter, ηq of 1. Superimposed on the spikelet spectrum is an ideal pulse simulation of a static powder spectrum utilizing the same values of Cq and ηq. The chemical shift of these spectra was arbitrarily set to zero. The horizontal arrows indicate that under ideal conditions the height of the trays on either low or high frequency side of the lineshape is equal. The vertical arrow indicates the position of the shoulder for an eta equals 1 lineshape. The spectrum in (b) depicts a finite pulse simulation with Cq of 1.7 MHz and an isotropic chemical shift of 12.8 ppm to higher shielding. The spectrum in (c) depicts the sum of the spectra in (a) and (b) such that (b) is 60% of (a).

Figure 2

Figure 2

Data at 11.75T showing the binding of Mg2+ to WT APE1 in the absence of damaged DNA where (a) is the 25Mg NMR spectrum of 6X Mg2+ bound to APE1, (b) is the spectrum of 6X Mg2+ bound to ED in the absence of damaged DNA, (c) is an oversubtracted difference spectrum, and (d) is the correct difference spectrum. The corresponding fit to the difference spikelet spectrum in (d) shown in (e) utilized the parameters summarized in Table 1.

Figure 3

Figure 3

Summary of the results of HPLC analysis on a sample composed of 6 equivalents of Mg2+, APE1, and damaged DNA. Sample A was extracted before the start of the NMR experiment. Samples B and C were extracted after the NMR experiment. Sample C was incubated at 25 °C for 1 hour prior to DNA extraction. The indicators on the chromatograms denote retention volumes the various peaks maxima.

Figure 4

Figure 4

(a) The difference spectrum obtained at 11.75T that results from the binding of Mg2+ to APE1 in the presence of damaged DNA, and (b) its corresponding simulation from the extracted parameters listed in Table 1.

Figure 5

Figure 5

The difference spectrum obtained at 9.4T resulting from incubating the active APE1 damaged DNA complex for 1 hour at 25 °C. See text for the details.

Figure 6

Figure 6

All of the data were acquired at 9.4T. The top spectrum (a) denotes the 25Mg NMR spectrum of the E96Q damaged DNA complex of APE1. The middle spectrum (b) denotes the 25Mg NMR spectrum of ED damaged DNA complex. The lower spectrum (c) denotes the difference between the spectra in panels (a) and (b).

Figure 7

Figure 7

All of the data were acquired at 9.4T. The spectra result from collapsing the spikelet echo train into a single half echo and subsequent Fourier transformation. The top trace is the 25Mg NMR powder spectrum of APE1 at pH 8.0; the middle spectrum is the 25Mg NMR spectrum of APE1 at pH 6.5. The bottom trace is the difference spectrum formed by subtracting the pH 6.5 spectrum from the pH 8 spectrum.

Scheme 1

Scheme 1

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