Insights from the crystal structure of the sixth BRCT domain of topoisomerase IIbeta binding protein 1

Abstract

Topoisomerase IIbeta binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy-terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP-ribose) polymerase 1 (PARP-1), where the latter interaction is responsible for the poly(ADP-ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 A. The crystal structure reveals a degenerate phospho-peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho-peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho-peptide binding domain. We further provide insight into poly(ADP-ribose) binding and sites of potential modification by PARP-1.

Figure 1

Conserved BRCT fold of TopBP1 BRCT6. (A) Ribbon model of TopBP1 BRCT6 in stereoview. Secondary structure elements are labeled. (B) Superposition of TopBP1 BRCT6 (blue) with the N-terminal BRCT domains of BRCA1 (pink) and MDC1 (yellow) in stereoview.

Figure 2

Comparison of TopBP1 BRCT6 to phospho-peptide binding tandem BRCT repeats. (A) Sequence alignment of TopBP1 BRCT6 with N-terminal BRCT domains of BRCA1, MDC1, and BARD1. Residues are shaded based on levels of conservation. Secondary structure elements are plotted above the sequences. Conserved residues involved in pSer binding are shown as purple squares. Conserved residues involved in BRCT repeat packing are shown as green circles. (B) Structural alignment of the pSer binding pocket of BRCA1 (pink) with TopBP1 BRCT6 (blue). The BACH1 phospho-peptide is shown in red. Hydrogen bonding is designated as dotted lines. Hydrogen bonding residues in BRCA1 (below) aligned with TopBP1 (above) are displayed. (C) Structural alignment of the α₂ helix of BRCA1 (pink) and TopBP1 BRCT6 (blue) packed against the C-terminal BRCT of BRCA1 (represented as electrostatic surface). Equivalent residues of TopBP1 BRCT6 (above) and BRCA1 (below) are shown.

Figure 3

Mapping of putative PAR binding and PARP-1 modification residues in TopBP1 BRCT6. Consensus PAR-binding motif residues are shown in orange. Putative PARP-1 auto-modification Glu residues common in TopBP1 BRCT6 are indicated in red.