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. 2022 Apr 14;50(8):4732–4754. doi: 10.1093/nar/gkac237

Figure 7.

Figure 7.

Non-conventional SUMO2-binding to XRCC4 coiled-coil overlaps with LIG4 and IFFO1 binding. (A) Intensity losses in the 1H–15N BEST-TROSY spectra of XRCC41–180 after addition of increasing concentrations of SUMO2 monomers (mSUMO2). Error bars represent 1 standard deviation from the plotted value, as calculated from the noise levels in the TROSY spectra using the standard error propagation formula. Grey bars indicate residues with too low intensity for reliable measurement (arbitrarily set to 1); lack of bars represents unassigned residues. Red and blue bars indicate assigned residues in XRCC41–180 and levels of intensity ratio loss of more or less than 30%, respectively. (B) 1H–15N BEST-TROSY spectra of XRCC41–180 (red) showing key residues affected after addition of increasing concentrations of mSUMO2 (blue). (C) GST-4×SUMO2 pulldowns of GFP-XRCC4 WT, SIM101, SIM163 or double SIM101/SIM163 (D) mutants ectopically expressed in HEK293T XRCC4 KO cells. Pulldowns were repeated twice with similar results. S2: SUMO2. (D) Intensity losses in the 1H–15N HSQC spectra of mSUMO2 after addition of 0.33 molar equivalents of XRCC4FL. Colour gradient for mSUMO2 structure (PDB 2N1W) from red (most affected by binding) to blue (unaffected by binding). (E) Electrostatic surface potential of SUMO2 using APBS (78). Dashed line highlights key residues implicated in binding to XRCC4FL according to (D). Electrostatic potential values are multiples of kT/e, kb: Boltzmann's constant, T: temperature (300 K); e: charge of an electron, conversion factor: 25.85 mV. (F) HADDOCK models of 2×SUMO2 (top) and 3×SUMO2 (bottom, based on PDB 2D07) interactions with XRCC4 (PDB 1IK9) head and coiled-coil domains. (G) Binding regions of SUMO2 (residues 163-EKCVSA-168; top), LIG4 (residues 173–19528; middle), and IFFO1 (residues 162–19630; bottom; PDB 6ABO30) on XRCC4 (PDB 1IK9). (H) GST pulldowns of GST and GST-4×SUMO2 with whole cell extracts obtained from HEK293T XRCC4 KO cells ectopically expressing GFP-XRCC4. (I) GFP-XRCC4 expressed in HEK293T cells co-precipitates a substantial fraction of total LIG4. Pulldowns were repeated twice with similar results. Inputs were 4% of the total.