Variant of uncertain significance Arg866Cys enhances disorderedness of h-BRCA1 (759-1064) region.

ABSTRACT

High structural flexibility has been reported in the central region of BRCA1, which hinders the structural and functional evaluations of mutations identified in the domain. Additionally, the need to categorize variants of unknown significance (VUS) has increased due to the growth in the number of variants reported in clinical settings. Therefore, unraveling the disease-causing mechanism of VUS identified in different functional domains of BRCA1 is still challenging. The current study uses a multidisciplinary approach to assess the structural impact of BRCA1 Arg866Cys mutation discovered in the central domain of BRCA1. The structural alterations have been characterized using Circular-Dichroism spectroscopy, nano-DSF, and molecular-dynamics simulations. BRCA1 Arg866Cys mutant demonstrated more flexibility and lesser affinity to DNA than the wild-type protein. The BRCA1(759-1064) wild-type protein was shown to be a ßII-rich protein with an induced D-O transition in the presence of DNA and 2,2,2-Trifluoroethanol (TFE). The protein's alpha-helical composition did not significantly change in the presence of TFE, besides an increase in ß-turns and loops. Under Transmission Electron Microscopes (TEM), amyloid-like fibrils structure was detected for Arg866Cys mutant whereas the wild-type protein showed amorphous aggregates. An increased ThT fluorescence indicated ß-rich composition and aggregation-prone behaviour for BRCA1 wild-type protein, while the fluorescence intensity was significantly quenched in the Arg866Cys mutant. Furthermore, increased conformational flexibility in the Arg866Cys variant was observed by principal component analysis. This work aims to comprehend the inherently disordered region of BRCA1 as well as the impact of missense mutations on folding patterns and binding to DNA for functional aspects.