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Protein Science (2004), 13:617-625. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Comparison of BRCT domains of BRCA1 and 53BP1: A biophysical analysis

Caroline M.S. Ekblad1,2,3, Assaf Friedler1,2, Dmitry Veprintsev1,2, Richard L. Weinberg1,2 and Laura S. Itzhaki1,2,3

1 MRC Centre for Protein Engineering, Cambridge CB2 2QH, UK
2 Department of Chemistry, Cambridge CB2 1EW, UK

(RECEIVED September 29, 2003; FINAL REVISION November 25, 2003; ACCEPTED November 28, 2003)



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 Kd 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.

Keywords: 53BP1; BRCA1; BRCT; p53

Abbreviations: AUC, analytical ultracentrifugation • 53BP1, p53 binding protein 1 • BRCT, BRCA1 C terminus • IPTG, Isopropylthiogalactoside • ITC, isothermal titration calorimetry • PMSF, Phenylmethylsulhponyl fluoride

Reprint requests to: Laura S. Itzhaki, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 2XZ, UK; e-mail: lsi10{at}cam.ac.uk.

3 Present address: Hutchison/MRC Research Centre, Cambridge CB2 2XZ, UK.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03461404.


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