In Silico and Structure-Based Assessment of Similar Variants Discovered in Tandem Repeats of BRCT Domains of BRCA1 and BARD1 To Characterize the Folding Pattern.

BRCA1 and BARD1 are important proteins in the homologous DNA damage repair pathways. Different genetic variants identified in these proteins have been clinically correlated with the occurrence of hereditary breast and ovarian cancer (HBOC). Variants of unknown significance (VUS) reported in the BRCT domains of BRCA1 and BARD1 substantiate the importance of BRCT domain-containing proteins for genomic integrity. To classify the pathogenicity of variants, in silico, structural and molecular dynamics (MD)-based approaches were explored. Different variants reported in the BRCT region were retrieved from cBioPortal, LOVD3, BRCA Exchange, and COSMIC databases to evaluate the pathogenicity. Multiple sequence alignment and superimposition of the structures of BRCA1 BRCT and BARD1 BRCT domains were performed to compare alterations in folding patterns. From 11 in silico predictions servers, variants reported to be pathogenic by 70% of the servers were considered for structural analysis. To our observations, four residue pairs of both the proteins were reported, harboring 11 variants, H1686Y, W1718L, P1749L, P1749S, and W1837L variants for BRCA1 BRCT and H606D, H606N, W635L, P657L, P657S, and W762F for BARD1 BRCT. MD simulations of the BRCT repeat regions of these variants and wild-type proteins were performed to evaluate the differences of folding patterns. Root mean square deviation (RMSD), R g, solvent-accessible surface area (SASA), and root mean square fluctuation (RMSF) of variants showed slight differences in the folding patterns from the wild-type proteins. Furthermore, principal components analysis of H1686Y, P1749S, and W1718L variants of BRCA1 showed less flexibility than the wild type, whereas that of H606D, W635L, and W762F of BARD1 showed more flexibility than the wild type. Normal mode analysis of the energy minima from the simulation trajectories revealed that most of the variants do not show much differences in the flexibility compared to the wild-type proteins, except for the discrete regions in the BRCT repeats, most prominently in the 1798-1801 amino acid region of BRCA1 and at the residue 744 in BARD1.

Conserved residues at the MAPKs binding interfaces that regulate transcriptional machinery.

Signaling through c-Raf downstream pathways is the crucial subject of extensive studies because over expressed or mutated genes in this pathway lead to a variety of human cancers. On the basis of cellular localization, this pathway has been sub-divided into two cascades. The first RAF1-MEK1-ERK2 cascade which remains in the cytosol, whereas the second MEK1-ERK2-RSKs transduces into the nucleus and regulates the transactivation function. But how a few amino acids critically regulate the transcriptional function remains unclear. In this paper, we have performed in silico studies to unravel how atomic complexities at the MEK1-ERK2-RSKs pathways intercedes different functional responses. The secondary structure of the ERK, RSKs have been modeled using Jpred3, PSI-PHRED, protein modeler, and Integrated sequence analyzer from Discovery Studio software. Peptides of RSKs isozymes (RSK1/2/3/4) were built and docked on ERK2 structure using ZDOCK module. The hydropathy index for the RSKs molecules was determined using the KYTE-DOOLITTLE plot. The simulations of complex molecules were carried out using a CHARMM force field. The protein-protein interactions (PPIs) in different cascade of MAP kinase (MAPK) have been shown to be similar to those predicted in vivo. PPIs elucidate that the amino acids located at the conserved domains of MAPK pathways are responsible for transactivation functions.

Role of MERIT40 in stabilization of BRCA1 complex: a protein-protein interaction study.

MERIT40 is a novel associate of the BRCA1-complex, thus play an essential role in DNA damage repair mechanism. It is the least implicit protein and its structural and functional aspects of regulating the stability of BRCA1-MERIT40 complex remain equivocal. Analysis of protein-protein interactions between BRCA1 and its cellular binding partners like ABRAXAS, RAP80 and MERIT40 would help to understand the role of protein complex integrity in DNA repair mechanism. The recombinant proteins were purified and their structural aspects were elucidated by spectroscopic methods. Interaction analysis was carried out to determine binding partners of MERIT40. MERIT40 showed interaction with bridging molecule, called ABRAXAS, thus generate a scaffold among various members which further stabilizes the entire complex. It acts as an adapter molecule by interacting with BRCA1-BRCT in non-phosphorylation dependent manner. The feature enlighten on structural and interaction profile of BRCA1-complex member to elucidate their role in complex stability and DNA repair process.

Preliminary crystallographic studies of BRCA1 BRCT-ABRAXAS complex.

The BRCA1 holoenzyme complex plays an important role in DNA damage repair. ABRAXAS is a newly discovered component of this complex and its C-terminal region directly binds to the BRCA1 BRCT domain. Single crystals of the BRCA1 BRCT-ABRAXAS complex grown by co-crystallization belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 187.18, c = 85.31 Å. Diffraction data were collected on the BM-14 beamline at the ESRF. Molecular-replacement calculations using Phaser led to three molecules in the asymmetric unit and a high solvent content of 76%.