Toward Classification of BRCA1 Missense Variants Using a Biophysical Approach

Pamela J.E Rowling,Rebecca Cook,Laura S Itzhaki

doi:10.1074/jbc.m109.088922

Abstract

Carriers of germ line mutations in breast cancer susceptibility gene BRCA1 have an increased risk of developing breast and ovarian cancers; missense mutations have, however, been difficult to assess for disease association. Here we have used a biophysical approach to classify these variants. We established an assay for measuring the thermodynamic stability of the BRCA1 BRCT domains and investigated the effects of 36 missense mutations. The mutations show a range of effects. Some do not change the stability, whereas others destabilize the protein by as much as 6 kcal mol−1; one-third of the mutants could not be expressed in soluble form in Escherichia coli, and we conclude that these destabilize the protein by an even greater amount. We tested several computer algorithms for their ability to predict the mutant effects and found that by grouping them into two classes (destabilizing by less than or more than 2.2 kcal mol−1), the algorithms could predict the stability changes. Importantly, with the exception of the few mutants located in the binding site, none showed a significant reduction in affinity for phosphorylated substrate. These results indicate that despite very large losses in stability, the integrity of the structure is not compromised by the mutations. Thus, the majority of mutations cause loss of function by reducing the proportion of BRCA1 molecules that are in the folded state and increasing the proportion of molecules that are unfolded. Consequently, small molecule stabilization of the structure could be a generally applicable preventative therapeutic strategy for rescuing many BRCA1 mutations.

Highlights

Carriers of germ line mutations in BRCA1 have an increased lifetime risk of developing breast and ovarian cancers, and mutations in the BRCA1 gene account for 80% of all familial breast and ovarian cancer cases [1, 2]
The reasons are: first, a number of studies have indicated that the BRCT domains are critical for tumor suppression; second, many mutations in BRCA1 are located in the BRCT domains; third, the structure of the BRCT repeat is known, and we can relate our experimental results to the location of the mutations in the structure; we can look at whether computer algorithms, which require a structure, are able to predict these effects
We expressed wild-type and mutant BRCA1 BRCT in E. coli. 13 of the 36 mutants were found to be expressed exclusively in the inclusion body fraction, even when the growth and expression conditions were varied by, for example, lowering the temperature or the concentration of isopropyl-␤-D-thiogalactoside used for induction of protein expression

Summary

Introduction

Carriers of germ line mutations in BRCA1 have an increased lifetime risk of developing breast and ovarian cancers, and mutations in the BRCA1 gene account for 80% of all familial breast and ovarian cancer cases [1, 2]. Despite the large changes in stability, all of the solubly expressed mutants (with the exception of those located in the binding site) were able to bind a phosphorylated peptide with near wild-type affinities (at the lower temperature), indicating that the mutations do not induce a misfolded conformation.

Results

Conclusion