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The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex - PubMed

  • ️Thu Jan 01 2015

The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex

Maryam Faridounnia et al. J Biol Chem. 2015.

Abstract

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation.

Keywords: COFS; DNA repair; ERCC1; XPF; biophysical characterization; nuclear magnetic resonance (NMR); nucleotide excision repair; protein structure; surface plasmon resonance (SPR).

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.
FIGURE 1.

DNA binding and stability of the wild type and F231L ERCC1-XPF (HhH)2 heterodimers. A, SPR with different concentrations of bubble10 DNA binding to the wild type ERCC1-XPF (HhH)2 heterodimer. B, same as A but for the mutant F231L ERCC1-XPF (HhH)2 complex. C, DNA binding curves of wild type (□) and mutant (▴) ERCC1-XPF based upon the SPR data from A and B. The figure displays the ratio R60[ERCC1-XPF]free/R60[ERCC1-XPF]total as a function of the concentration of bubble10 DNA. D, thermal shift assay for wild type (□, black line) and mutant (▴, gray line) ERCC1-XPF. The stability of ERCC1-XPF (HhH)2 is measured using the Sypro Orange Thermofluor assay (40). Note that the concentration of mutant ERCC1-XPF and thus also XPF homodimer was slightly higher than that of wild type, causing an apparent higher transition temperature for XPF homodimer at ∼75 °C. E, thermal stability of wild type (□) and mutant (▴) ERCC1-XPF measured by CD spectroscopy. F, dissociation of ERCC1-XPF measured by SPR at 150 m

m

NaCl and 12 °C. The graphs show the dissociation of XPF from immobilized wild type ERCC1 (data in black, fit in blue) and immobilized F231L ERCC1 (data in gray, fit in red). RU, arbitrary response units.

FIGURE 2.
FIGURE 2.

Comparison of NMR spectra for wild type and F231L ERCC1-XPF (HhH)2 heterodimers. A, overlay of 1H,15N HSQC for wild type (black) and F231L mutant (red) ERCC1-XPF (HhH)2 heterodimer. B, selected regions from a three-dimensional NOESY-1H,13C HSQC spectrum comparing the wild type and F231L ERCC1-XPF (HhH)2 heterodimers.

FIGURE 3.
FIGURE 3.

1H,15N HSQC chemical shift and signal intensity analysis of wild type and F231L ERCC1-XPF (HhH)2 heterodimers. A, chemical shift differences for amide protons: ΔHN(δWT-δF231L) (in ppm). Unassigned residues are marked with an asterisk, red for the mutation site. B, variation in 1H,15N HSQC cross-peak intensities. The intensities are depicted in black for the wild type and in blue for the F231L mutant. Values are normalized to the highest signal intensity (Lys905). The red horizontal line is the average of the signal intensity for residues in secondary structure elements of the wild type ERCC1-XPF (HhH)2 heterodimer. C, projection of chemical shift differences on the structure of the wild type ERCC1-XPF (HhH)2 heterodimer, representing residues with maximum perturbation as red broad ribbons, residues with medium perturbation as yellow broad ribbons, and residues with small to no difference in green. The F231L mutation site is represented as gray spheres.

FIGURE 4.
FIGURE 4.

Proton-deuterium exchange of wild type and F231L ERCC1-XPF (HhH)2 heterodimers. Shown are initial 1H,15N HSQCs after the dead time of the experiment for wild type (A) and mutant (B) ERCC1-XPF heterodimers. Shown is the time course of the H/D exchange behavior of Leu253 (C) and Val859 (D). Signal intensities for the wild type system are shown in blue, and signal intensities for the mutant are shown in red.

FIGURE 5.
FIGURE 5.

Secondary structure elements of wild type and F231L ERCC1-XPF (HhH)2 heterodimers. Shown are chemical shift-derived secondary structure elements for wild type (WT) and mutant (F231L) ERCC1 predicted by TALOS+ and secondary structure elements detected in the lowest energy structure after (re)calculation of the NMR ensembles. Top, ERCC1; bottom, XPF. Unassigned amide protons are shown in blue in the sequence. The N-terminal extension assigned in mutant ERCC1, the His6 tag, and N-terminal methionines of the construct are shown in gray.

FIGURE 6.
FIGURE 6.

Solution structures of wild type and F231L ERCC1-XPF (HhH)2 heterodimers. A, superimposed NMR ensembles of F231L and recalculated wild type ERCC1-XPF (HhH)2. The backbone of ERCC1 is colored blue (cyan, wild type; blue, mutant); the backbone of XPF is colored green (lemon yellow, wild type; dark green, mutant). The mutated residue Leu231 is shown in red. Shown are a surface depiction and side chain organization for phenylalanines Phe231, Phe889, and Phe894 of wild type ERCC1-XPF (HhH)2 (B) and for residues Leu231, Phe889, and Phe894 of F231L ERCC1-XPF (HhH)2 (C). Backbones, Phe889, and Phe894 are colored as in A; the side chain of Phe231/Leu231 is colored in red, and the labels in gray depict residues surrounding Phe231/Leu231.

FIGURE 7.
FIGURE 7.

Side chain orientations in wild type and F231L ERCC1-XPF (HhH)2 heterodimer. Side chains of residues showing the largest differences between the NMR ensembles are depicted for the recalculated complex of wild type ERCC1 (HhH)2 (top) and XPF (bottom) (A) and the calculated complex of F231L mutant ERCC1 (HhH)2 (top) and XPF (bottom) (B). Backbone coloring is as in Fig. 6A. Amino acid side chains are shown in yellow, with polar hydrogen in gray, oxygen in red, and nitrogen in blue. The monomers of ERCC1 and XPF are separated for a better view.

FIGURE 8.
FIGURE 8.

Interaction interface of ERCC1 and XPF and the comparison of Phe293 and Phe894 cavities at the interface of wild type and F231L mutant ERCC1-XPF. A, schematic representation of the three major interaction regions: anchor Phe293/pocket 1, anchor Phe894/pocket 2, and the hydrophobic core (helices α and γ) surrounding Phe840. ERCC1 is shown in blue, and XPF is shown in green. Side chains of important phenylalanines are shown. Numbers of residues involved in intermolecular interactions are indicated. Residue 231 is labeled in red. The Phe293 cavity in the wild type (B) and mutant (C) ERCC1-XPF (HhH)2 complex is shown. The Phe894 cavity in the wild type (D) and mutant (E) ERCC1-XPF (HhH)2 complex is shown. Side chains of the anchors Phe293 and Phe894 are shown as sticks. The side chain of residue 231 (Phe or Leu) is shown in red.

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