Comparison of BRCT domains of BRCA1 and 53BP1: a biophysical analysis

Abstract

53BP1 interacts with the DNA-binding core domain of the tumor suppressor p53 and enhances p53-mediated transcriptional activation. The p53-binding region of 53BP1 maps to the C-terminal BRCT domains, which are homologous to those found in the breast cancer protein BRCA1 and in other proteins involved in DNA repair. Here we compare the thermodynamic behavior of the BRCT domains of 53BP1 and BRCA1 and examine their ability to interact with the p53 core domain. The free energies of unfolding are of similar magnitude, although slightly higher for 53BP1-BRCT, and both populate an aggregation-prone partly folded intermediate. Interaction studies performed in vitro by analytical size-exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry reveal that 53BP1-BRCT interacts with p53 with a K(d) in the low micromolar range. Despite their homology with 53BP1-BRCT domains, the BRCT domains of BRCA1 did not bind p53 with any detectable affinity. In summary, although other studies have indicated that the BRCT domains of both BRCA1 and 53BP1 interact with p53 core domain, the quantitative biophysical measurements performed here indicate that only 53BP1 can bind. Although both proteins may be involved in the same DNA repair pathways, our study indicates that a direct role in p53 function is unique to 53BP1.

Figure 1.

Schematic representation of the structures of the BRCT domains of BRCA1 and 53BP1. (A) BRCA1-BRCT with the linker region in black. (B) An overlay of the BRCT domains of BRCA1 (red) and 53BP1 (blue); the linker regions are shown in black and in turquoise, respectively. (C) 53BP1-BRCT (blue) bound to p53 core domain (yellow). The binding interface is concentrated on the L2 and L3 loops of p53, which interacts with the C terminus of the BRCT-N and the linker region of 53BP1. Interacting residues are shown in green (53BP1) and in red (p53).

Figure 2.

GdmCl-induced denaturation measured at 20°C for 53BP1-BRCT, together with that measured previously for BRCA1-BRCT (Ekblad et al. 2002) for comparison. The buffer used was 50 mM sodium phosphate (pH 8.0), 500 mM NaCl, and 5 mM DTT. The protein concentration was 0.6 μM, and the excitation wavelength was 280 nm. Data are plotted as the average emission wavelength (see Ekblad et al. 2002), and the dotted lines are the fits to a three-state model.

Figure 3.

Interaction experiments monitored by analytical gel filtration. (A) 53BP1-BRCT and p53-core. The squares and dotted lines represent, respectively, p53-core (elution volume, 16.1 mL) and 53BP1-BRCT (elution volume, 15.3 mL), and the solid line is a mixture of the two proteins (elution volume, 14.6 mL). The protein concentration is 75 μM of each protein, and the ratio in the mixture is 1 : 1. The shift to lower elution volume for the mixture of 53BP1-BRCT and p53 indicates binding. (B) BRCA1-BRCT and p53-core. The squares and the dotted lines represent, respectively, p53-core (elution volume, 16.1 mL) and BRCA1-BRCT (elution volume, 15.9 mL), and the solid line is a mixture of the two proteins (elution volume, 16.0 mL). Protein concentrations are as in A. No interaction is observed. The buffer was 50 mM Tris-HCl (pH 7.5), 75 mM NaCl, and 5 mM DTT, and the analysis was performed at room temperature.

Figure 4.

Calorimetric titration of 53BP1-BRCT into p53-core. The protein concentrations of 53BP1-BRCT and p53-core were 311.4 and 53.2 μM, respectively. The experiment was carried out at 8°C in 50 mM Tris-HCl buffer (pH 7.5), 75 mM NaCl, and 1 mM DTT. (Top) The exothermic heat pulse of each injection. (Bottom) The integrated heat data fit to single-site binding model. This gave n = 0.88 ± 0.01 and K_a = (1.6 ± 0.1) × 10⁵ M⁻¹ and thus a K_d of 6.2 ± 0.5 μM.

Figure 5.

Sedimentation equilibrium profiles of mixture of p53-core and BRCA1-BRCT (A) and 53BP1-BRCT (B). (Inset) The fit error distribution. Curves at 15,000 and 21,000 rpm for the same sector are shown.

Figure 6.

Proteolysis approach for mapping the p53-binding site on 53BP1-BRCT. (A) Digestion of 53BP1-BRCT with proteases clostripain, Lys-C, and Asp-N. Some precipitation occurred during the digestion: ‘s’ and ‘p’ refer to the soluble and the insoluble fractions, respectively, after centrifugation. Only the soluble fraction was used subsequently for binding to p53-core. Intact 53BP1-BRCT is shown in lane 7. Molecular weight markers (Mw) are shown. (B) Analytical gel filtration elution profile of clostripain-digested 53BP1-BRCT incubated with p53-core; collected fractions are numbered. (C) Concentrated fractions from the gel filtration run shown in B. Lane 1 indicates p53-core alone; lane 2, fraction 15; lane 3, fraction 16; and lane 4, fraction 17. The peptide fragment in lane 2 is highlighted by a circle. Fractions 15, 16, and 17 were analyzed by MALDI-TOF mass spectrometry. Comparison of the obtained masses with the masses of expected fragments from clostripain cleavage of 53BP1-BRCT identified the peptides NYLLPAGYSLEEQR (residues 1845–1858) and ILDWQPR (1859–1865) in fraction 15. A peptide corresponding to both these fragments (NYLLPAGYSLEEQRILDWQPR) was identified in fraction 17. A peptide that could correspond to residues 1812–1864 was identified in fraction 16. From the gel, it is clear that there is also some digestion of p53-core by residual clostripain. However, this species is likely to have only small truncations from the termini that should not disrupt the integrity of the structure or the ability to bind 53BP1-BRCT. The shift in elution volume for fraction 15 is larger than expected if the peptides observed by MALDI-TOF bind as monomers, indicating that peptide oligomerization has occurred.