Biophysical evaluation to categorize pathogenicity of cancer-predisposing mutations identified in the BARD1 BRCT domain

The BRCT domain of BARD1 (BARD1 BRCT) is involved in many cellular processes such as DNA damage repair (DDR) and cell-cycle checkpoint regulation. BARD1 BRCT performs tumor suppressor function by recruiting BRCA1 at DNA damage site via interactions with other DNA damage repair (DDR) proteins. Considering the importance of the BRCT domain in genomic integrity, we decided to evaluate reported mutations of BARD1 BRCT Cys645Arg, Val695Leu, and Ser761Asn for their pathogenicity. To explore the effect of the mutation on the structure and function, BARD1 BRCT wild-type proteins and the mutant proteins were studied using different biochemical, biophysical and in silico techniques. Comparative fluorescence, circular dichroism (CD) spectroscopy and limited proteolysis studies demonstrate the wellfolded structural conformation of wild-type and mutant proteins. However, thermal and chemical denaturation studies revealed similarity in the folding pattern of BARD1 BRCT wild-type and Cys645Arg mutant proteins, whereas there was a significant loss in the thermodynamic stability of Val695Leu and Ser761Asn mutants. Molecular dynamics (MD) simulation studies on wild-type and mutant protein structures indicate the loss in structural integrity of mutants compared with the wild-type protein.