Structural implications of amyloidogenic rare variants Ser282Leu and Gln356Arg identified in h-BRCA1.

Preliminary studies have shown BRCA1 (170-1600) residues to be intrinsically disordered with unknown structural details. However, thousands of clinically reported variants have been identified in this central region of BRCA1. Therefore, we aimed to characterize h-BRCA1(260-553) to assess the structural basis for pathogenicity of two rare missense variants Ser282Leu, Gln356Arg identified from the Indian and Russian populations respectively. Small-angle X-ray scattering analysis revealed WT scores Rg -32 Å, Dmax -93 Å, and Rflex-51% which are partially disordered, whereas Ser282Leu variant displayed a higher degree of disorderedness and Gln356Arg was observed to be aggregated. WT protein also possesses an inherent propensity to undergo a disorder-to-order transition in the presence of cruciform DNA and 2,2,2-Trifluoroethanol (TFE). An increased alpha-helical pattern was observed with increasing concentration of TFE for the Gln356Arg mutant whereas Ser282Leu mutant showed significant differences only at the highest TFE concentration. Furthermore, higher thermal shift was observed for WT-DNA complex compared to the Gln356Arg and Ser282Leu protein-DNA complex. Moreover, mature amyloid-like fibrils were observed with 30 µM thioflavin T (ThT) at 37°C for Ser282Leu and Gln356Arg proteins while the WT protein exists in a protofibril state as observed by TEM. Gln356Arg formed higher-order aggregates with amyloidogenesis over time as monitored by ThT fluorescence. In addition, computational analyses confirmed larger conformational fluctuations for Ser282Leu and Gln356Arg mutants than for the WT. The global structural alterations caused by these variants provide a mechanistic approach for further classification of the variants of uncertain clinical significance in BRCA1 into amyloidogenic variants which may have a significant role in disease pathogenesis.

Tetrameric ZBRK1 DNA binding domain has affinity towards cognate DNA in absence of zinc ions.

Zinc finger transcription regulatory proteins play crucial roles in cell-cycle regulation, DNA damage response and tumor genesis. Human ZBRK1 is a zinc-finger transcription repressor protein, which recognizes double helical DNA containing consensus sequences of 5'GGGXXXCAGXXXTTT3'. In the present study, we have purified recombinant DNA binding domain of ZBRK1, and studied binding with zinc ions and DNA, using biophysical techniques. The elution profile of the purified protein suggests that this ZBRK1 forms a homotetramer in solution. Dissociation and pull down assays also suggest that this domain forms a higher order oligomer. The ZBRK1-DNA binding domain acquires higher stability in the presence of zinc ions and DNA. The secondary structure of the ZBRK1-DNA complex is found to be significantly altered from the standard B-DNA conformation.

Structural basis to characterise transactivation domain of BRCA1.

Familial inheritance of breast and ovarian cancer is attributed to mutations discovered in functional domains of BRCA1 gene. BRCA1 is a multifunctional protein responsible for maintaining the genomic integrity and has transcriptional regulatory function encoded in its C-terminal region. The different amino-terminal e extensions to BRCA1 BRCT domain are responsible for transcription activation. However, only BRCA1 BRCT (1649-1859) amino acids have been explored for its structural characteristics. Noting the importance of extended region to the N-terminus of BRCT different regions of BRCA1 which demonstrates maximum transactivation activity has been explored for their structure and functional activity. Secondary and tertiary structural analysis revealed a limited alpha-helical content with well-folded tertiary structure. In silico tools were used to corroborate the in vitro results. Amino acids composition and sequence analysis display a propensity for intrinsic disorder and coiled-coil formation in BRCA1 (1396-1863) (BRCA1-TAD). The results presented in this paper suggest the extreme flexibility in coiled-coil motif might be an important requirement in the establishment of protein-protein interaction networks for BRCA1.

Functional assessment of intrinsic disorder central domains of BRCA1.

The most studied function of BRCA1 is that of tumor suppression through its role in DNA repair and transcription regulation. Germline mutations discovered in a larger cohort of patients, abrogate BRCA1 interactions with reported cellular partners, and are responsible for breast and ovarian cancer. The different functional regions of BRCA1 interact with nearly 30 different cellular partners. Thus, it becomes clinically significant to understand the detailed protein-protein interactions associated with functional regions of BRCA1. Different overlapping central domains of BRCA1 have been characterized using in silico, in vitro and biophysical approaches. To our conclusions, it has been observed that central domains of BRCA1 are intrinsically disordered and has large hydrodynamic radius with random coil like structures.

Structural and functional implication of RAP80 ΔGlu81 mutation.

Receptor Associated Protein 80 (RAP80) is a member of RAP80-BRCA1-CCDC98 complex family and helps in its recruitment to the DNA damage site for effective homologous recombination repair. It encompasses two tandem UIMs (UIM1 and UIM2) motif at its N-terminus, which interact with K-63 linked polyubiquitin chain(s) on H2AX and thereby assemble the RAP80-BRCA1 complex at the damage site. Nevertheless, how RAP80 helps in the structural integrity of BRCA1 complex is still elusive. Considering the role of RAP80 in the recruitment of BRCA1 complex at the DNA damage site, we attempted to explore the molecular mechanism associated with RAP80 and mutation that causes chromosomal aberrations due to its loss of function. There is a significant loss in structural characteristics of RAP80 ΔE81, which impairs its binding affinity with the polyubiquitin chain. This leads to the defective recruitment of RAP80 and BRCA1 complex at the DNA damage site. The results presented here are very useful in understanding the cause of various repair defects (chromosomal aberration) that arise due to this mutation. Comparative study of wild type and ΔE81 could be helpful in designing the small molecules that can potentially compensate the deleterious effect(s) of ΔE81 and hence useful for therapeutic application.