Conformational changes upon XLF-XRCC4 complexation initiate XRCC4 site-to-site interaction. A, superposition of XLF(1–224) and XRCC4 structures with the XLF(1–224)-XRCC4(1–140) complex. The stalk regions of XLF (orange) or XRCC4 (light blue) superimposed with the stalks in the XLF-XRCC4 complex (magenta and blue). The angle between the head domains and stalks in the complex is larger than in free XLF or free XRCC4. B, close up view of the XRCC4(1–140)-XRCC4(1–140) parallel binding site (blue, orange) highlighting conformational changes and interface symmetry upon complexation. XRCC4 (PDB code 1fu1, transparent gray) have been superimposed on the complexed XRCC4 structure to show conformational changes at the parallel interface β1, β2-β4, and β6,β7-α1 sheets. Interaction residues are highlighted. XLF mutations residues R57G and C123R found in patients are highlighted.

Overall arrangement of XRCC4-XLF filaments in solution. P(r) functions of XLF(1–224)-XRCC4(1–140) (A) and XLF^FL-XRCC4(1–140) (B) for dilution series (0.4, 2.3, 2.4, and 2.9 mg/ml as indicated). The P(r) functions are normalized to unity at their maxima. Four distinct distances at 85, 160, 240, and 320 Å are indicated, together with the XLF(1–224)-XRCC4(1–140) filament as shown in the crystal structure (Fig. 1A). C, P(r) of XLF(1–224) and XRCC4(1–140) (pink and blue) in comparison with a mixed sample observed 10–600 s after extensive mixing (from orange to green). The P(r) functions are not normalized. D, P(r) of XLF^FL and XRCC4(1–140) (cyan and blue) in comparison with a P(r) observed 10–600 s after extensive mixing (from orange to green). E, normalized intensity of the P(r) at r = 85 Å observed in time-dependent SAXS measurements: C, XLF(1–224)-XRCC4(1–140) (red); D, XLF^FL-XRCC4(1–140) (cyan); XLF(1–224) ^(L115D)-XRCC4(1–140) (magenta) and XLF(1–224)^(R64E,L65D) (blue). F, experimental scattering profiles of the collected SEC peak fractions at ∼0.4 mg/ml or XLF^FL-XRCC4(1–140) (cyan), XLF(1–224)-XRCC4(1–140) (red), and XLF^FL (pink) and XLF(1–224) (green). The theoretical scattering from the final MES model matching the experimental data (χ² = 1.9, XLF^FL-XRCC4(1–140) (cyan); χ² = 1.9, XLF(1–224)-XRCC4(1–140) (red); χ² = 1.3, XLF^FL; χ² = 1.3 XLF(1–224)). G, MES atomic model of XLF^FL-XRCC4(1–140) and XLF(1–224)-XRCC4(1–140) along with respective percentages. Bottom, plots show residual for the fit of the MES-atomistic models with corresponding χ² values.

Stabilization of XLF-XRCC4 filaments through DNA bridging. A, 20 top-ranked 20-bp DNA docking placements are shown as green helices and the 99 top-ranked placements as axis lines colored by helix direction. The protein surface is colored by electrostatic potential. B, atomistic model of XLF^FL-XRCC4(1–140)-40-bp DNA built based on the crystal structure of XLF(1–224)-XRCC4(1–140), the HDX, and DNA docking. C, P(r) functions of XLF(1–224)-XRCC4(1–140)-40-bp DNA assemblies calculated for samples with molar ratios of XLF(1–224)-XRCC4(1–140): 40-bp DNA of 1:0, 3:1, 2:1, 1:1, and 0:0.3 (magenta, light blue, blue, dark-blue, and gray). In the presence of 40-bp DNA, the P(r) function shows distinct distances at r = 50 and 100 Å, indicating new order in the filaments. Elongation in the P(r) indicates stabilization of the filaments. D, P(r) functions of XLF^FL-XRCC4(1–140)-40-bp DNA assemblies for samples with high protein concentration (∼3.0 mg/ml) and molar ratio of 40-bp DNA 2:1 (magenta and green) show changes and indicate new order in the filaments after DNA binding.

Combined crystallographic and SAXS structures explain the synergy of Ku-XLF-XRCC4 interactions in ligating DSBs. A, molecular surface of the XLF-XRCC4-BRCT-DNA assembly (magenta-blue-cyan-green). The XRCC4-BRCT structure (PDB code 3ii6, cyan) (35, 36) was superimposed with XRCC4. B, half-turn of the superhelical channel (8 units of XLF-XRCC4 dimers) with 60-bp DNA (green). C, superhelical channel molecular surface. The parallel XLF-XRCC4 filaments are shown as seen in the crystal structure (building unit, dotted line). D, Ku-nucleated XLF-XRCC4 filament appears suitable to maintain DNA end alignment via its grooved DNA-binding surface. This model, which utilizes the filament groove, is distinguished by having Ku moved distal to the DSB, as shown with BRCT-bound LigIV (LIV, cyan) with its identified DNA-binding site (+) at the break. Two alternative testable models would keep Ku (orange) and DNA-PKcs (yellow) proximal to the DSB, allowing possible DNA-PK activation of partners and providing steric access to processing enzymes and LigIV with the DNA either anti-parallel (E) or parallel (F).

Crystal structural of XLF(1–224)-XRCC4(1–140). A, two orthogonal views of the biological unit of the XLF(1–224)-XRCC4(1–140) complex (magenta and blue, respectively), as seen in the crystal lattice. Parallel strands are in surface representation. B, view of the biological unit rotated by 90°. C, overall architecture of the XLF(1–224)-XRCC4(1–140) complex, colored magenta and blue. Critical secondary structure elements are indicated. Inset, close-up view of the parallel interaction of XLF(1–224)-XRCC4(1–140) in the biological unit. D, XLF(1–224)-XRCC4(1–140) interface highlighting the interaction of the XLF β6-β7 loop (Leu-115) with an XRCC4 cavity formed by β6-β7 (Leu-108 and Phe-106) and α1-α2 (Met-59, Met-61, and Lys-65). E, electron density of the 2F_o − F_c map for the XLF(1–224)-XRCC4(1–140) interaction region (green and blue) is shown contoured at 1.5σ. Crystallographic statistics are in supplemental Table S1. F, XLF(1–224)-XRCC4(1–140) interface highlighting the interaction of the XRCC4 β6-β7 loop (Arg-107) and α2 helix (Glu-55 and Asp-58) (blue) with XLF α2-α3 loop (Arg-64 and Leu-65, magenta).

HDX analysis of XRCC4-XLF complexation. A, superposition of the complexation-induced mass shifts on the XLF-XRCC4 model. Significant reductions (yellow) and increases (green) in deuteration upon complexation are indicated. B, the structures are rotated by 90°. See supplemental Fig. S3 for details.

HDX analysis of XRCC4-XLF-DNA complexation. Superposition of the complexation-induced mass shifts on the XLF(1–224) structure arising from DNA binding (A) and the XLF(1–224)-XRCC4(1–178) structure arising from DNA binding (B). Significant reductions (yellow) and increases (green) in deuteration upon complexation are indicated. For additional details see supplemental Fig. S3.

Overall arrangement of the Ku-XLF-XRCC4-DNA assembly. A, SEC of Ku-XLF^FL-XRCC4(1–140)-40-bp DNA (magenta) in comparison with Ku-40-bp DNA (blue) and XLF^FL-XRCC4(1–140) (green). The fraction collected for SAXS measurements is highlighted. B, SDS-PAGE analysis and agarose gel for DNA analysis of the peak fractions. C, P(r) of Ku-XLF^FL-XRCC4(1–140)-40-bp DNA (magenta) in comparison with Ku-40-bp DNA (blue), Ku (gray), and XLF^FL-XRCC4(1–140) (green). The P(r) functions are normalized to unity at their maxima. D, experimental scattering profiles of the collected Ku-XLF^FL-XRCC4(1–140)-40-bp DNA fraction (black) with its atomic model matching the experimental data (χ² = 1.8, magenta). The atomic model is shown in F. A Guinier plot with linear fit in the limit qR_G >1.6 is shown in the inset. E, five representative SAXS envelopes of Ku-XLF^FL-XRCC4(1–140)-40-bp DNA were calculated by DAMMIF (51). F, average SAXS envelope is superimposed on the atomic model of Ku-XLF^FL-XRCC4(1–140)-40-bp DNA.