Characterisation of the BRCT Domains of the Breast Cancer Susceptibility Gene Product BRCA1 C.M.S. Ekblad, H.R. Wilkinson, J.W.H. Schymkowitz, F. Rousseau S.M.V. Freund and L.S. Itzhaki* MRC Centre for Protein Engineering, University Chemical Laboratory Lensfield Road Cambridge CB2 1EW, UK The breast cancer susceptibility gene product BRCA1 is a tumour sup- pressor but the biochemical and biological functions that underlie its role in carcinogenesis remain to be determined. Here, we characterise the solution properties of the highly conserved C terminus of BRCA1, con- sisting of a tandem repeat of the BRCT domain (BRCT-tan), that plays a critical role in BRCA1-mediated tumour suppression. The overall free energy of unfolding of BRCT-tan is high (14.2 kcal mol 21 at 20 8C in water) but unfolding occurs via an aggregation-prone, partly folded inter- mediate. A representative set of cancer-associated sequence variants was constructed and the effects on protein stability were measured. All of the mutations were highly destabilising and they would be expected to cause loss of function for this reason. Over half could not be purified in a soluble form, indicating that these residues are critical for maintaining structural integrity. The remaining mutants exhibited much greater aggre- gation propensities than the wild-type, which is most likely a consequence of their reduced thermodynamic stability relative to the partly folded intermediate. The mutations characterised here are located at different sites in the BRCT-tan structure that do not explain fully their effects on the protein’s stability. Thus, the results indicate an important role for biophysical studies in assessing the significance of sequence variants and in determining how they cause disease. q 2002 Elsevier Science Ltd. All rights reserved Keywords: BRCA1; BRCT; mutation; protein stability *Corresponding author Introduction The breast cancer susceptibility gene BRCA1 was identified in 1994. 1 Germline mutations in BRCA1 are found in almost half of families that display strong heritable susceptibility to breast cancer and in over 80% of families with multiple cases of both breast and ovarian cancer. The wild-type allele of BRCA1 is typically lost or inactivated in the tumours that arise in these families, suggesting that BRCA1 normally functions as a tumour suppressor. 2,3 BRCA1 encodes a predicted protein of 1863 amino acid residues. It contains at its N terminus a single RING finger domain. A globular domain has been identified by sequence analysis at the C terminus with similarities to regions of a number of other proteins that are involved mainly in cell-cycle checkpoint functions responsive to DNA damage. 4,5 These include 53BP1, a protein that binds the p53 tumour suppressor, and the proteins RAD4 and RAD9 involved in DNA repair. The domain appears to be the result of an internal duplication, each of the tandem domains being designated as a BRCT domain (for BRCA1 C termi- nus). The domain is not limited to the C termini of proteins, nor is it always found as multiple copies. Cancer-associated missense mutations are found in the BRCT repeat of BRCA1 and truncations within the repeat predispose to cancer. 6,7 Notably, a mutation Y1853term, which results in a truncation of only 11 amino acid residues, is found in a family predisposed to develop very early onset breast cancer. 8 Furthermore, growth suppression by BRCA1, which can be localised to the BRCT domains, is diminished or abolished by disease- associated mutations in that region. 9,10 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved Present address: J. W. H. Schmkowitz and F. Rousseau, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. E-mail address of the corresponding author: lsi@mrc-lmb.cam.ac.uk Abbreviations used: CD, circular dichroism; GdmCl, guanidinium chloride. doi:10.1016/S0022-2836(02)00478-3 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 320, 431–442