Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1

Abstract

TopBP1 is a scaffold protein that coordinates activation of the DNA-damage-checkpoint response by coupling binding of the 9-1-1 checkpoint clamp at sites of ssDNA, to activation of the ATR-ATRIP checkpoint kinase complex. We have now determined the crystal structure of the N-terminal region of human TopBP1, revealing an unexpected triple-BRCT domain structure. The arrangement of the BRCT domains differs significantly from previously described tandem BRCT domain structures, and presents two distinct sites for binding phosphopeptides in the second and third BRCT domains. We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2.

Figure 1.

Structure of the N-terminus of TopBP1. (A) Schematic representation of human TopBP1, highlighting the position of each BRCT domain. The boxed region indicates the amino acid boundaries of the TopBP1(1–290) expression construct used in this study. (B) The X-ray crystal structure of TopBP1(1–290) reveals three consecutive BRCT domains, designated as BRCT0, BRCT1 and BRCT2 (coloured grey, yellow and orange, respectively). Amino acid boundaries of each domain are indicated in parentheses. This and all other molecular graphics figures were produced with MacPyMOL unless otherwise stated. (C) Superimposition of the first BRCT domain in the consecutive TopBP1 BRCT pairs BRCT0:1 and BRCT1:2 onto the first-BRCT domain in the canonical-tandem BRCT structure (cyan) of Crb2 in a phospho-H2A.1 peptide complex (PDB: 2VXC), reveals significant differences in juxtaposition of the two domains in both TopBP1 pairs (left, middle). In contrast (right), both BRCT domains in the canonical pair from BRCA1 (pink), align with their equivalents in Crb2. The bound phospho-peptide from the Crb2–pH2A.1 complex is shown in stick representation. In order to improve the clarity of the figure, some interconnecting loops have been omitted. (D) Comparison of the TopBP1 BRCT0:1 and BRCT1:2 pairs, based on superimposition of the outlined domain from each, but with the pairs separated for clarity, reveals distinct and idiosyncratic domain juxtapositions for each, and the absence of any repeating poly BRCT superstructure.

Figure 2.

Phosphopeptide-binding sites in TopBP1 N-terminus. (A) Comparison of the phosphopeptide-binding site in a canonical tandem BRCT structure, Crb2, with the topologically equivalent sites in TopBP1 BRCT0, BRCT1 and BRCT2. The pattern of residues that provide the phosphate interaction in Crb2 and in other known phosphopeptide-binding BRCT domains, are present in BRCT1 and BRCT2 but not BRCT0 where the site is hydrophobic in nature. (B) Surface representation of TopBP1(1–290) structure coloured by domain as in Figure 1. The conserved putative phosphopeptide-binding patches conserved in BRCT1 and BRCT2 are highlighted in green. (C) Difference Fourier maps from crystals of TopBP1(1–290) grown in a buffer containing MgSO₄, show significant density (>2.5σ) in the putative phosphopeptide-binding sites of BRCT1 and BRCT2, corresponding to bound sulphate. This confirms that both sites are competent for interacting with the phosphate of a phosphopeptide.

Figure 3.

Rad9-tail phosphorylation and TopBP1 binding. (A) Pull-down assay of binding of His₆-TopBP1(1–290) to a GST-Rad9 tail construct. His₆-TopBP1(1–290) was efficiently co-precipitated only when the GST-Rad9 tail construct was incubated with the protein kinase CK2α in the presence of Mg-ATP, but not by GST alone, or a mutant in which the target phosphorylation site, Ser387 was changed to alanine. (B) Pull-down assay using a synthetic biotin-coupled peptide incorporating a phosphoserine at the equivalent of Ser387. His₆-TopBP1(1–290) was efficiently co-precipitated by the biotin-pS387 peptide, but not by a different biotinylated-phosphopeptide, nor by the biotin-pS387 peptide treated with phosphatase. Incubation of phosphatase-treated peptide with CK2α + MgATP restored binding.

Figure 4.

Specificity of phospho-peptide binding to TopBP1(1–290). (A) Binding affinity of TopBP1(1–290) for a fluorescein-labeled Rad9-derived peptide, incorporating pSer387, as determined by fluorescence polarization. The calculated Kd (shown in parenthesis) indicates a specific interaction. For calculation of Kd see ‘Materials and Methods’ section. (B) As (A) but with a labelled phosphopeptide derived from the S. pombe checkpoint mediator protein Crb2. No significant binding is observed, confirming the specificity of the Rad9 interaction. C) As (A), but with TopBP1(1–290) constructs harbouring mutations in the putative phosphopeptide-binding site in BRCT1. The affinity of the interaction is substantially reduced, indicating a significant contribution to the interaction by BRCT1. (D) As (C), but with TopBP1(1–290) constructs harboring mutations in the putative phosphopeptide-binding site in BRCT2. The affinity of the interaction is comparable to that for wild-type TopBP1(1–290), indicating that there is no significant contribution to the interaction by BRCT2. (E) As (C), but with TopBP1(1–290) constructs with mutations in both putative phosphopeptide-binding sites. The weak-residual binding present in BRCT1 mutants is effectively abolished in the double mutants. (F) As (A), but with phosphopeptides corresponding to five different phosphorylation sites that have been mapped within the Rad9 tail. Only the peptide incorporating pSer387 shows significant affinity.

Figure 5.

BRCT-domain classes in TopBP1 and other BRCT domain proteins. (A) Array of the linkers connecting consecutive BRCT domains in structurally characterized tandem BRCT proteins. The linker segments in the triple-BRCT structure of TopBP1(1–290) are substantially shorter, resulting in the novel non-canonical domain arrangement. (B) Assignment of BRCT domain classes in TopBP1. Domains 1, 2, 3, 5 and 7 show the characteristics of phosphopeptide binding, but only BRCT7:8 resemble the canonical tandem pair arrangement found in checkpoint mediators such as Crb2, BRCA1, etc.