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, 45 (21), 12374-12387

Characterization of the APLF FHA-XRCC1 Phosphopeptide Interaction and Its Structural and Functional Implications

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Characterization of the APLF FHA-XRCC1 Phosphopeptide Interaction and Its Structural and Functional Implications

Kyungmin Kim et al. Nucleic Acids Res.

Abstract

Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHA-FBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr. These high pK values are characteristic of the polyanionic peptides typically produced by CK2 phosphorylation. Binding affinity is greatly enhanced by residues flanking the crystallographically-defined recognition motif, apparently as a consequence of non-specific electrostatic interactions, supporting the role of XRCC1 in nuclear cotransport of APLF. The FHA domain-dependent interaction of XRCC1 with APLF joins repair scaffolds that support single-strand break repair and non-homologous end joining (NHEJ). It is suggested that for double-strand DNA breaks that have initially formed a complex with PARP1 and its binding partner XRCC1, this interaction acts as a backup attempt to intercept the more error-prone alternative NHEJ repair pathway by recruiting Ku and associated NHEJ factors.

Figures

Figure 1.
Figure 1.
Crystal structures of the APLF FHA domain unliganded and bound to a phosphorylated XRCC1 peptide. (A) Cartoon representation of apo-APLF FHA domain (green) with the 10 β-strands numbered. (B) Stereo view showing an overlay of α-carbon traces of APLF FHA (green) with the FHA domains of PNKP (blue) and APTX (magenta). The APLF FHA structures in complex with (C) XRCC1pSpT-9 diphosphopeptide (protein, light gray; peptide, blue) and (D) XRCC1EpT-9 monophosphopeptide (tan, protein; pink, peptide) are represented. Simulated annealing Fo-Fc omit maps of each phosphopeptide (green mesh) contoured at 3.0 σ for the diphosphopeptide and 2.5 σ for the monophosphopeptide are displayed. The peptide residues are annotated in blue with underlined, italic residue names, and the APLF FHA residues important for binding are annotated in black. Hydrogen-bond interactions are also depicted (red dotted line). (E) The topologies of the β3-β4 and β5-β6 loops in the APLF FHA domain for the apo (green), the XRCC1pSpT-9 diphosphopeptide-complexed (light gray), or the XRCC1EpT-9 -monophosphopeptide-complexed (tan) structures are represented. Hydrogen-bonds that sustain a binding-ready conformation are indicated by black, red or cyan dashed lines, respectively.
Figure 2.
Figure 2.
31P-NMR titration data for diphosphorylated XRCC1 and XRCC4 peptides. (A) 1D 31P-NMR spectra of XRCC1pSpT-9 at the pH values indicated are shown (upper panel). 31P chemical shifts for pSer (△) and pThr (▴) were plotted as a function of pH, and fit to a Henderson-Hasselbalch equation as described in Methods (lower panel). (B) 1D 31P NMR spectra of XRCC4pSpT-12 at the pH values indicated for pSer (O) and pThr (•), are shown (upper panel), with pK determinations in the lower panel. Samples contained trimethylphosphate (TMP) as an internal shift standard, and 50 mM sodium acetate, 50 mM sodium cacodylate, and 1 mM EDTA buffer.
Figure 3.
Figure 3.
31P NMR titration data for diphosphorylated XRCC1 and XRCC4 peptides as a function of the APLF FHA domain concentration. 31P NMR spectra of samples containing (A) 0.5 mM of an 18-residue XRCC1 peptide phosphorylated on Ser518/Thr519 (XRCC1pSpT-18), and (B) 0.5 mM of an 12-residue XRCC4 peptide phosphorylated on Ser232/Thr233 (XRCC4pSpT-12) were titrated with the APLF FHA domain at the ratios indicated. The pSer resonance is indicated by a blue arrow, and the pThr resonance by a red dotted arrow. Titration studies were performed in 25 mM HEPES, 25 mM MES, 150 mM NaCl, 1 mM EDTA, pH 7.4 in the presence of a 0.25 mM TMP chemical shift reference. Resonances arising from impurities in the XRCC1pSpT-18 sample are indicated with an X.
Figure 4.
Figure 4.
Chemical shift perturbations vs. peptide length. (A) Amide chemical shift changes for 0.1 mM U-[15N]APLF FHA domain in the presence of 0.8 mM XRCC1pSpT-9 (blue bars) or XRCC1pSpT-18 (orange bars). (B) 1H,15N-HSQC spectra of U-[15N]APLF FHA domain as a function of XRCC1pSpT-9 (left panel) or XRCC1pSpT-18 (right panel). Peptide concentrations (in μM): 0 (red), 10 (orange), 20 (yellow), 50 (green), 100 (blue), 200 (purple), 400 (cyan) and 800 (pink). Titration studies were performed in a NMR buffer containing 25 mM HEPES, 25 mM MES, 150 mM NaCl, 1 mM EDTA, pH 7.4.
Figure 5.
Figure 5.
Simulation of amide exchange data. Amide 1H NMR shifts obtained in the study shown in Figure 3B were simulated as described in Materials and Methods. The total shift differences between uncomplexed and fully complexed resonances were taken directly from the spectra. Color coding of the spectra and simulations as a function of peptide concentration (μM) is: 0 (black); 10 (orange); 20 (yellow); 50 (green); 100 (blue); 200 (purple); 400 (cyan); 800 (red).
Figure 6.
Figure 6.
Apparent Kd values as a function of pH. pH-dependent Kd values were measured by fluorescence polarization (FP) using N-terminal FITC-fluorophore labeled (A) XRCC1pSpT-18, (B) XRCC1pSpT-24 or (C) XRCC1EpT-18 (pH 7.4: •, ─; pH 7.0: □, ···; pH 6.5: *, —; pH 6.0: △, -·-, pH 5.5: ж, -··-). (D) The titration curves of the three phosphopeptides obtained at pH 7.4 are compared. The FP values were normalized to 1.0 to facilitate comparison of the results. Data were fit to a single-site binding equation as described in Materials and Methods. Studies were performed in 25 mM HEPES, 25 mM MES, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, and 0.05% Tween 20.
Figure 7.
Figure 7.
XRCC1 at the nexus of three different repair pathways. Schematic illustrating NHEJ backup recruitment by APLF. In addition to the standard abbreviations, KuBM—Ku binding motif; FBM—FHA domain binding motif. NHEJ proteins (blue), alt-NHEJ proteins (orange), proteins involved in multiple pathways (gray).

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