Cuneo MJ et al. (2011), The structural basis for partitioning of the XR...

XB-ART-53495

Nucleic Acids Res 2011 Sep 01;3917:7816-27. doi: 10.1093/nar/gkr419.

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The structural basis for partitioning of the XRCC1/DNA ligase III-α BRCT-mediated dimer complexes.

Cuneo MJ , Gabel SA , Krahn JM , Ricker MA , London RE .

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The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-α. For efficient ligation, ligase III-α is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-α BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.

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Species referenced: Xenopus
Genes referenced: xrcc1

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	Figure 1. Structure of the X1BRCTb homodimer. (A) Ribbon diagram of the X1BRCTb homodimer. The monomers are colored in cyan and blue. (B) Close-up view of the hydrogen bonding network between X1BRCTb monomers. (C) Close-up view of the homodimer interface illustrating the hydrophobic contacts. Amino acid side-chains that are found to be part of the interface are represented as stick models. Labels are shown only for hydrophobic residues.
	Figure 2. Structure of homodimeric L3BRCT. Two conformationally distinct homodimers were identified in the crystal structure and are shown in (A) and (B). The two structures differ by 13Â° in the relative orientation of the L3BRCT monomers. (C) Close-up view of the interface amino acids and hydrogen bonding network between the L3BRCTmonomers from the homodimer in (A). (D) Close-up view of the interface amino acids and hydrogen-bonding network between the L3BRCTmonomers from the homodimer in (B). Hydrogen bonds between monomers are represented as black dashed lines and residues involved in hydrogen bonds are labeled.
	Figure 3. Structure of X1BRCTb/L3BRCT tetramer. (A) Overall structure of the tetrameric X1BRCTb/L3BRCT complex (L3BRCT monomers are represented as red and magenta ribbon models; X1BRCTb monomers are represented as blue and cyan ribbon models). The 2-fold non-crystallographic axis in the z-plane of the page is represented as a black circle. (B) Close-up view of the X1BRCTb/L3BRCT heterodimer interface (amino acids found at the interface are shown in stick representation. (C) X1BRCTb/L3BRCT heterodimer from view (B) rotated 180Â° about the y-axis. The hydrophobic cleft on L3BRCT is shown in a grey surface representation. (D) Close-up view of the homodimer interface from the X1BRCTb/L3BRCT heterodimer (amino acids found at the interface are shown in stick representation, L596 is shown as a gray surface and water molecules are shown as red spheres).
	Figure 4. Gel-filtration of X1BRCTb complexed with L3BRCT. X1BRCTb/L3BRCT in 150âmM NaCl (black dashed line) and 1âM NaCl (gray solid line), and X1BRCTb Leu596Arg/L3BRCT in 150âmM NaCl (gray dashed line). The straight line (black) is a fit of the gel filtration standards (black squares) used to estimate the molecular mass of the complexes.
	Figure 5. Small-angle X-ray scattering of double mutant heterodimer. (A) SAXS intensity data of X1BRCTb(Y574R,L596R)/L3BRCT heterodimer. Black line is the raw scattering data with associated errors and the red line is the fit to the X-ray crystal structure of the X1BRCTb/L3BRCT dimer from the tetrameric crystal structure. Insets are the Guinier fit and the pair-wise distribution function. (B) Ab initio SAXS envelope model superimposed with the X-ray crystal structure of the X1BRCTb/L3BRCT dimer from the tetrameric crystal structure. X1BRCTb is shown in a cyan ribbon representation and L3BRCT is shown in a magenta ribbon representation.
	Figure 6. Conservation of X1BRCTb/L3BRCT protein interface residues. (A) Amino acid composition of the X1BRCTb and L3BRCT homodimer interface. Amino acids composing the protein interfaces are highlighted in red and those involved in hydrogen-bonding interactions are underlined. (B) X1BRCTb hydrogen-bonding network superimposed with the conserved residues from L3BRCT. X1BRCTb monomers are colored in blue tones; side-chains involved in forming the interface and homodimer hydrogen bonds are colored the same, numbered and shown as stick representations. Side-chains from the L3BRCT homodimer are also shown as stick representations that are colored orange and magenta. (C) L3BRCT hydrogen bonding network superimposed with the conserved residues from X1BRCTb [colored as in (B)]. Residues circled in (B) and (C) are circled in (A).
	Figure 7. Structure of the X1BRCTb(Y574R)-extended/L3BRCT complex. (A) Overall structure of the tetrameric X1BRCTb(Y574R)-extended/L3BRCT complex (L3BRCT monomers are represented as red and magenta ribbon models; X1BRCTb(Y574R)-extended monomers are represented as blue and cyan ribbon models). (B) Close-up view of the X1BRCTb-extended/L3BRCT heterodimer interface (amino acids found at the interface are shown in stick representation and amino acids composing the L3BRCT peptide-binding site are shown as a pale green surface; non-interacting surfaces of the ligase are colored in magenta). (C) Circular dichroism temperature melts of X1BRCTb (green), X1BRCTb(Y574) extended (blue), X1BRCTb/L3BRCT (black), X1BRCTb(Y574R)-extended/L3BRCT (red). Inset is a schematic representing the constructs used for the temperature melts (X1, X1BRCTb; L3, L3BRCT).

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