Structural basis to stabilize the domain motion of BARD1-ARD BRCT by CstF50

BRCA1 associated ring domain protein 1(BARD1) is a tumor suppressor protein having a wide role in cellular processes like cell-cycle checkpoint, DNA damage repair and maintenance of genomic integrity. Germ-line mutation Gln 564 His discovered in linker region of BARD1 leads to loss of binding to Cleavage stimulating factor (CstF50), which in turn instigates the premature mRNA transcript formation and apoptosis. We have studied the dynamics of ARD domain present in the BARD1 wild-type and mutant protein in association with CstF50 using biophysical, biochemical and molecular dynamics simulations. It has been observed that the ARD domain is relatively more flexible than the BRCT domain of BARD1. Further relative orientations of both the ARD and BRCT domains varies due to the highly flexible nature of the connecting linker region present between the domains. It has been observed that mutant ARD domain is more dynamic in nature compared to wild-type protein. Molecular docking studies between BARD1 Gln 564 His mutant and CstF50 shows the loss of interactions. Furthermore, domain motion of ARD present in BARD1 was stabilized when complexed with CstF50.

was performed to explore the effect of mutation on the secondary structure of BARD1 ARD-BRCT. The CD spectra for wild-type and mutant protein shows that secondary structure of the protein is majorly stabilized by α-helices because of prominent ellipticity minima at λ = 208 nm and 222 nm (Fig. 1A). However, the CD spectra do not show change in the secondary structure of mutant protein which indicates that mutation has no effect on the secondary structure of the wild-type BARD1 ARD-BRCT protein. However, the absence of characteristic random coils and β-sheets are due to the dominant contribution of CD signal from the α-helix portion of the structure. Additionally, this masking of random coil and β-sheets can also be due to the disulfide chromophores present in ARD-BRCT region which strongly contributes to the CD signal in the range of λ = 215-235 nm 27,28 . CD spectroscopy was further performed at near-UV range to investigate the effect of mutation on the tertiary structure of the protein. Near-UV spectra of wild-type and mutant proteins show peaks at λ max = 295 nm and 285 nm respectively, which indicates the hydrophobic microenvironments of intrinsic fluorophores substantiating the compact 3D structure of the proteins 29 (Fig. 1B). Furthermore, limited proteolysis profile of wild-type and mutant protein indicates that ARD and BRCT domain of BARD1 are two compact and independently folding domains ( Figure 3C and D Supplementary). Thermal stability of BARD1 ARD-BRCT. Thermal denaturation was performed to delineate the effect of mutation on the thermal stability of the BARD1 ARD-BRCT wild-type. Thermal induced denaturation was monitored from 25 °C-65 °C using CD spectroscopy. The loss in secondary structure was observed at 65 °C for both the wild-type and mutant proteins ( Figure 4A and B Supplementary). The unfolded fraction was calculated by using change in millidegree ellipticity at λ = 222 nm, which suggest that wild-type and mutant protein unfold via a two state pathway in a co-operative manner. The T m calculated for wild-type and mutant were 45.6 ± 0.60 °C and 45.2 ± 0.42 °C respectively, which further suggest that mutation does not drastically affect the thermal stability of the BARD1 ARD-BRCT wild-type (Fig. 1C). Chemical denaturation induced by GuHcl was monitored using fluorescence spectroscopy. Fluorescence emission maximum at λ max = 340 nm for wild-type and at λ max = 341 nm for mutant indicate that proteins are in folded conformation. However, at 1.8 M GuHcl concentration significant blue-shifts in the emission maxima was observed for wild-type at λ max = 335 nm and mutant at λ = 336 nm respectively. Wild-type and mutant completely unfold at 6 M GuHcl as indicated by the emission maximum at λ max = 348 nm. These observations suggest that wild-type and mutant unfold via an intermediate molten globule formation ( Fig. 1D and E), and ( Figure 4C and D Supplementary). Furthermore, to investigate the molecular interactions between CstF50 and BARD1, we have expressed His-tagged CstF50 in bacterial system that expressed mostly in inclusion bodies ( Figure 1C and D Supplementary). Hence 6XHis tagged fused CstF50 protein was extracted using 8 M urea, and then further refolded in the buffer D (50 mM Tris, pH 6.2, 500 mM NaCl, 0.1% triton, 10% glycerol pH 7.2, 2 mM EDTA, 700 mM arginine, 100 mM KCl, 50 mM MgCl 2 , 100 mM CaCl 2 and 5 mM DTT) containing Arginine. Refolding pattern of CstF50 protein was further confirmed by fluorescence spectroscopy. The fluorescence spectroscopy profile for denatured CstF50 in 8 M urea shows an emission maximum at λ max = 347 nm, however, folded protein shows a blue-shifted emission maximum at λ max = 337 nm, which indicates complete burial of tryptophans in the hydrophobic core of the protein (Fig. 1F). Furthermore, good quality CD spectra could not be achieved because arginine hydrochloride present in buffer D interfered with the measurement 30,31 . An attempt to dialyze out arginine hydrochloride resulted in precipitation of CstF50 protein.
CstF50 stabilizes the ARD domain of BARD1: In-silico approach. To understand the function associated to protein-protein interactions for BARD1-CstF50 complex, BARD1 ARD-BRCT region and CstF50 were modeled using Robetta server 32,33 . This Model of BARD1 ARD-BRCT and CstF50 were further validated by MolProbity server 34 (Table 1 and 2 Supplementary). The 3-D structure of BARD1 ARD-BRCT which comprises 4, N-terminal ankyrin repeats containing α-helices, a linker, two BRCT repeats containing 3-α helices and three βstrands ( Fig. 2A). BARD1 ARD-BRCT Gln 564 His mutation was generated using SPDB viewer 35 . The BARD1 ARD-BRCT and Gln 564 His mutant structures were superimposed for direct comparison between wild-type and Mutant models (Fig. 2B). Modeled structure of CstF50 shows N-terminal helical region and C-terminal 7 WD-40 repeat comprised of beta sheets that fold into a propeller like structure (Fig. 2C).
Normal Mode Analysis (NMA) was performed on the BARD1 ARD-BRCT wild-type and Gln 564 His mutant structures to investigate the effect of mutation on protein dynamics. First few modes known as a low frequency modes describe the large domain motion within the protein 36 . Hence, to understand the structural dynamics between ARD and BRCT domain, we have selected the 1 st lowest frequency mode for BARD1 ARD-BRCT wild-type and Gln 564 His mutant protein structures ( Fig. 3A-D). A large inter-domain motion was observed between the ARD and BRCT domain of BARD1. The inter-domain motion is often observed in multidomain proteins having hinge bending 37 . The residues at ARD and BRCT domain show high positive correlated motion at the terminal regions. Interestingly, the linker region residues show high anti-correlated motion for the ARD and BRCT domain residues in wild-type and mutant protein structures. The region of hinge bending and local flexibility were investigated using deformation analysis ( Fig. 3E and F). The linker region was found to possess highest flexibility in both wild-type and mutant (thickness and color intensity) which shows that the linker between inter-domain region is responsible for large dynamics between ARD domain and BRCT domains.

BARD1-ARD domain motion and CstF50 complex.
To understand the function associated to protein-protein interactions of BARD1-CstF50 complex, and inter-domain motion between ARD and BRCT domains, the model structure of BARD1 ARD-BRCT, BARD1 Gln 564 His mutant, CstF50 and BARD1 ARD-BRCT-CstF50 complex were simulated for 100 ns under periodic boundary conditions. The RMSD (Root Mean Square Deviation) profile for BARD1 shows that mutant protein structure exhibits higher RMSD than the wild-type (Fig. 4A). During the first 5 ns of simulation, RMSD values of wild-type and mutant have increased up to a value of 5 Å. However, after 10 ns, RMSD of wild-type decreases to 3.5 Å and mutant increases up to 6.5 Å. At 16 ns, RMSD value for wild-type show sharp increase and reaches up to 8 Å. Hence, the mutant structure RMSD shows only a gradual rise to reach the level of wild-type RMSD (Fig. 4A). Beyond 16 ns, the decrease in the RMSD up to 4 Å for wild-type and mutant structure is indicative of structural stabilization. However, the RMSD of mutant protein structure remained high as compared to wild-type. After 35 ns of simulation, wild-type and mutant structures are stabilized for next 50 ns. Differences in RMSD were again observed at 98 ns and 96 ns of simulation for mutant and wild-type structures respectively. To investigate change in the RMSD, 0 ns input structures were superimposed onto the structure of 16 ns and 98 ns time frame for wild-type and mutant respectively. The two superimposed structures ( Fig. 5A and B) show large ARD domain movement with respect to BRCT domain for wild-type and mutant protein structures. Superimposed structure of wild-type with input structure shows that ARD domain rotates about 70° relative to BRCT domain contributing to the RMSD value of 8.55 Å. However, the mutant shows that rotation of ARD domain by 84.4° leading to a RMSD of 9.27 Å with respect to the structure of 0 ns ( Fig. 5A and B). The RMSD attained during the entire simulation by ARD domain of wild-type mutant shows that ARD domain is dynamic in nature comparative to the BRCT domain, which is stable during entire simulation (Fig. 4C). The RMSD change for ARD domain in mutant protein shows gradual increase but wild-type ARD domain is stable. Furthermore, 10 superimposed structures extracted for wild-type and mutant protein at different time points of trajectory show highly dynamic behavior of ARD domain, whereas the BRCT domain is stable in nature. The ARD domain in mutant shows conformational changes compared to wild-type, signifying that ARD domain in mutant protein is more dynamic than in wild-type ( Fig. 4F and G). However, RMSD profile for CstF50 structure indicates that CstF50 is compact protein and does not show any changes in the model structure (Fig. 4B).   Furthermore, changes in the radius of gyration (R gyr ) in a time dependent manner during the course of simulation determines the protein structure compactness 38 . During the early phase of simulation wild-type and mutant did not show any sharp changes in the R gyr , but steep rise in the R gyr was observed at 16 ns in wild-type and at 98 ns in mutant structure of BARD1. In mutant structure, R gyr change is significantly higher than that of wild-type, substantiating the inference that mutation has made the mutant protein dynamic than the wild-type structure (Fig. 4E).Change in R gyr is largely due to change in the position of ARD domain relative to the BRCT domain of BARD1. However, R gyr profile for CstF50 structure indicates that CstF50 has overall compact structure and does not show any changes in the conformation (Fig. 4D).
Comparative residual RMSF (Root Mean Square Fluctuation) values attained by wild-type and mutant suggest that the N-terminal ARD domain of BARD1 is highly flexible. The linker region (550-568)amino acids in BARD1 ARD-BRCT shows highest RMSF values in wild-type and Gln 564 His mutant structures (Fig. 5C). Hence, to explore the residual flexibility of BARD1 ARD-BRCT, RMSF structures showing maximum fluctuation from the trajectory were extracted (Fig. 5D and E). The RMSF structures for wild-type and mutant indicate that ARD domain is highly flexible, and high value of RMSF is observed for the residues in N-terminal, linker and loop regions of second BRCT repeat (Fig. 5C,E and F). However, in the CstF50 structure, RMSF indicated two flexible regions, first at the connecting loops between N-terminal and WD-40 domain, and second at the loop region between WD1 and WD2 repeat which also corroborates with the residual RMSF plotted for the CstF50 (Fig. 5D and G).
Furthermore, domain movement of the protein structures have been studied in relation to possible involvement with protein function 39 . To probe the collective motion during the simulation, the principal component analysis (PCA) was performed over the BARD1-ARD-BRCT, Gln 564 His mutant, CstF50 and BARD1-ARD-BRCT-CstF50 complex trajectories. The Eigenvalues and eigenvectors were calculated after the diagonalization of the covariance matrix. The area of trace of covariance matrix calculated for BARD1-ARD-BRCT, Gln 564 His mutant and CstF50 were 487.990 nm 2 , 714.963 nm 2 and 540.810 nm 2 respectively, which indicates that mutant shows more dynamic tertiary structural conformations than the wild-type protein. Hence, the trajectories for domain motion of BARD1 ARD-BRCT wild-type, Gln 564 His mutant and CstF50 were projected on the first three eigenvectors. Projection on eigenvector 2 and 1 for wild-type and mutant protein indicates that mutant protein is more dynamic in nature than the wild-type. Trajectory Projection on eigenvectors combination 3 and 1, 2 and 1 and 3 and 2 for wild-type (Fig. 6A,B and C) and mutant ( Figure 6D,E and F) indicates that both the structures can attain three major tertiary structure conformations whereas mutant structure shows higher periodic jump. Further, projection of eigenvectors 3 and 1, 2 and 1 for CstF50 shows that two major groups of tertiary structural conformation can be attained by the CstF50. The large transitions and periodic fluctuation between these conformers throughout the trajectory have been observed (Fig. 6G,H

and I).
Comparative residual displacement along the Eigenvectors. To investigate the residual fluctuation, first two eigenvector extracted from trajectories of BARD1 ARD-BRCT wild-type, Gln 564 His mutant and CstF50 were projected on the residues. The plot of projection on eigenvector 1 and 2 of residues for wild-type ( Figure 5A

Positive correlation motion of BARD1 ARD domain. Cross correlation for PCA of BARD1 wild-type,
Gln 564 His mutant protein, CstF50 and complex was plotted to investigate the correlated motion within the proteins. Cross-correlation of PCA indicates high positive correlation within the ARD domain. The residues within the ARD domain show strong negative correlation with the linker region which is in consistent with the NMA results (Fig. 7A,B). Comparatively, BARD1 wild-type shows higher positive correlation motion within the ARD domain and linker region residues as compared to the mutant protein. Furthermore, mutation has also affected the positive correlation within the BRCT repeat as wild-type shows higher positive correlation within the N-terminal BRCT and C-terminal repeat residues. CstF50 cross-correlation for PCA indicates that the N-terminal residues show little positive correlation within themselves as compared to the WD-40 repeat domain, which shows very high positive correlated motion within their domain (Fig. 7C). However, BARD1 ARD-BRCT-CstF50 complex cross-correlation for PCA indicates increased positive correlated motion within the N-terminal residues and WD-40 repeat for CstF50 in complex as compared to native condition. Furthermore, higher positive correlation is also observed in the ARD domain as well as BRCT domain in complex compared to the BARD1 ARD-BRCT alone (Fig. 7D).

Protein-Protein Interactions between BARD1 ARD-BRCT and CstF50.
The minimum free energy model structures of BARD1 ARD-BRCT, Gln 564 His mutant and CstF50 were selected for protein-protein docking studies ( Figure 6A,B and C Supplementary). It is well established that 7 th WD 40 (395-431) repeat domain of CstF50 is required to establish BARD1 complex 26 . The Gln at 564 to His mutation reduces the binding affinity of CstF50 to BARD1 17 . Therefore, we considered BARD1 Gln 564 as an important residue for interaction study with CstF50. In-silico docking was performed to understand the residual interactions between BARD1 ARD-BRCT, Gln 564 His mutant with CstF50. In the intermolecular interface of BARD1-CstF50 complex, glutamine 564 of BARD1 ARD-BRCT forms hydrogen bond with Asn 409 and Tyr 425 of CstF50, and also participates in  Figure 7A and B Supplementary). However, in the BARD1 Gln 564 His mutant-CstF50 complex, BARD1 His 564 lost hydrogen bonding interactions but forms non-covalent interactions with Gly 394 and Leu 395 of the CstF50 ( Figure 7C and D Supplementary). The BARD1 Thr 562 was found to be involved in the non-covalent interactions with CstF50 Glu 100. Loss of weak intermolecular interactions in case of mutant rationalizes the loss of binding affinity between mutant BARD1 and CstF50 (Figure 7D Supplementary).

Binding Interface of BARD1 ARD-BRCT and CstF50 complex. To determine the stability of BARD1
ARD-BRCT-CstF50 complex molecule we have simulated the structure for 100 ns to analyze the comparative RMSD, RMSF, R gyr and hydrogen bonding (Fig. 8). Comparative RMSD and R gyr indicate that binding of CstF50 to the BARD1 ARD-BRCT restricts the ARD domain fluctuation relative to the BRCT domain ( Fig. 8A and C). Hydrogen bonding analysis between the linker region and 7 th WD-40 domain was performed to analyze the domain stability of complex (Fig. 8B). It was found that BARD1 ARD-BRCT wild-type linker and 7 th WD-40 repeat form stable hydrogen bonds throughout the simulation, thus enabling formation of a stable complex (Fig. 8D). Further, binding energy and other energy component calculated for BARD1-CstF50 complex for entire simulation indicated that interaction between BARD1 and CstF50 is stable (Table 4 Supplementary). Interestingly, interactions between CstF50 and the BARD1 linker rigidifies the linker flexibility and thereby controls the architectural placement of the BARD1 C-terminal domain relative to the N-terminal ARD domain (Fig. 8E). This phenomenon thereby reduce the relative inter domain flexibility of the ankyrin repeats and BRCT domains. The short length of the linker and germ-line cancer-predisposing mutations suggest that the relative positioning of ARD and BRCT domains within BARD1 is critical in the binding to proteins and functionality of BARD1 (Fig. 8E and F).

Conclusion
Multidisciplinary approaches were applied to characterize the domain motion and binding affinity between BARD1 ARD-BRCT, Gln 564 His mutant and CstF50 proteins. It has been observed that the BARD1 ARD-BRCT Gln 564 His mutant is not affecting the monomeric property, secondary structure, hydrodynamic radii and thermal stability of the wild-type protein. The limited proteolysis of wild-type and mutant confirms that ARD and BRCT domain are stable and independent folded domains of BARD1 connected by a short stretch of a linker region. These results from limited proteolysis are consistent with earlier NMR studies which conclude that in the absence of any interacting protein, the ARD and tandem BRCT region behave as an independent domain in solution 40,41 . The thermal unfolding study indicates that BARD1 ARD-BRCT wild-type and mutant unfold via a two state pathway. However, chemical unfolding of wild-type and mutant suggest that both proteins unfold via a molten globule intermediate.
The results from NMA and MD simulation substantiates well with the earlier reported short angle X-ray scattering (SAXS) findings. In solution ARD and BRCT domains sample a wide range of relative orientations with respect to one another 42 . The Small Angle X-ray Scattering (SAXS) for BARD1 ARD-BRCT domain shows two additional features in the p(r) function: a shoulder at r ∼40 Å and tailing-off of the at r > 90 Å 42 . SAXS for ARD-BARD1BRCT suggests that Dmax of the domain is around 90 Å 42 . The RMSD values calculated for wild-type and mutant are very close (87 Å) to the experimental values (r > 90 Å) 42 . The distance calculated for wild-type N-terminal ARD domain and C-terminal BRCT domain shows a maximum change of 40 Å, which shows dynamic behavior of the protein due to the flexible linker region. The NMA and deformation analysis indicate that the linker connecting ARD and BRCT domains makes the wild-type and mutant protein flexible. MD simulation studies show that ARD domain is highly dynamic than the BRCT domain due to flexible linker which acts as the connecting bridge between the ARD and BRCT domain of BARD1. Flexibility in linker segments of the BARD1 ARD-BRCT backbone allow corresponding domain motions to occur with minor structural perturbations. The RMSD, R gyr and RMSF profiles show that BARD1 Gln 564 His mutant protein structure is more flexible than the wild-type. The relative orientation of ARD domain is significantly affected due to the Gln 564 His mutation. In BARD1 ARD-BRCT domain, flexible linker has different orientation that allows free twist and rotation of the ARD domain through space which can facilitates the recruitment of CstF50. The mutation Gln 564 His is present in the disordered flexible linker region of BARD1 ARD-BARD1BRCT domain. Lack of specific secondary structure provides the accessibility of different orientations to the linker region. The increase in the relative flexibility may be due to twist in the linker region by histidine. The linker region acts as a binding region for two large proteins CstF50 and p53. The difference in hydrogen bond donor group in glutamine and histidine may be the other reason in the loss of BARD1-CstF50 interactions. The results from wild-type, mutant and CstF50 docking study indicates that glutamine 564 to histidine mutation shows loss in hydrogen bonding interactions between wild-type and CstF50. To our conclusion, BARD1 Gln at 564 position plays a pivotal role in the BARD1 ARD-BRCT-CstF50 complex formation. The study further shows CstF50 binding provides rigidity to the ARD domain of BARD1. Therefore, flexibility in the linker region is immensely required to adopt such unique orientation to accommodate CstF50 protein.

Material and Methods
All the chemicals used in this study were purchased form Sigma-Aldrich, unless otherwise specified. The buffers were prepared in double distilled, 0.44 µM filtered Milli-Q water (Millipore, USA). Filtered 0.44 µM (Millipore, USA) protein and buffer solutions were degassed prior to use.
Protein expression and purification. BARD1 ARD-BRCT (425-777) cloned into a modified pET-6H vector (generous gift by Dr. Richard Baer, Institute for Cancer Genetics, Columbia University, USA). BARD1 ARD-BRCT wild-type domain was further PCR amplified and cloned into pGEX-KT vector (Amersham) in such a way that TEV protease site was incorporated between the GST and BARD1 ARD-BRCT. Site directed mutagenesis was performed to engineer the Gln 564 His mutation in BARD1 ARD-BRCT, and further sequenced for confirmation. BARD1 ARD-BRCT wild-type and Gln 564 His mutant were expressed in Escherichia coli BL21 (DE3). The GST-BARD1 ARD-BRCT wild-type and Gln 564 His mutant fusion protein were purified using affinity chromatography (GST-sepharose 4B column) in 50 mM Borate buffer, pH 9.0, 300 mM NaCl(Buffer A). The affinity purified proteins were treated with TEV to remove the fusion tag. Molecular exclusion chromatography was further performed to achieve high purity and homogenous monomeric population of BARD1 ARD-BRCT wild-type and Gln 564 His mutant proteins. Superdex-75 column was used for the purification of the BARD1 ARD-BRCT wild-type protein. The Mutant protein was purified using the superdex-200 analytical column. Thermal and chemical denaturation. Thermal denaturation was monitored by circular dichroism spectroscopy. BARD1 ARD-BRCT wild-type and Gln 564 His mutant were allowed to unfold over the temperature ranges from 20 °C to 70 °C. Further fraction unfolded was calculated at each temperature by using millidegree ellipticity at λ 222 and data was fitted in to a two state unfolding pathway. BARD1 ARD-BRCT wild-type and mutant Gln 564 His were allowed to unfold by incubating 2 μM protein in different concentrations of GuHcl at 10 °C for 18 hrs. Chemical denaturation of BARD1 ARD-BRCT wild-type and Gln 564 His mutant were performed at 10 °C and the fluorescence from frequently used fluorophores was monitored using fluorescence spectrophotometer (Horiba, USA) at excitation wavelength of λ = 295 nm. BARD1 ARD-BRCT wild-type and Gln 564 His mutant at a concentration of 2 μM was mixed with the GuHcl. The concentration of GuHcl varied from 0 M to 6 M, while the protein concentration was fixed at 2 μM. Fluorescence emission spectra were recorded over the range of wavelength λ = 310-400 nm in order of increasing GuHcl concentration, and blank subtraction was done to increase signal to noise ratio.

Molecular dynamics simulation. The validated Model structures of BARD1 ARD-BRCT wild-type,
mutant Gln 564 His, CstF50 and BARD1-CstF50 complex were subjected to MD simulations using GROMACS 4.5.5 [45][46][47] software. Protein were solvated with explicit solvent Single Point Charge water, in a cubic box which left 2 Å space nearby the solute. The counter ions in the form of Na + and Cl − ions were added to make the system electrically neutral. Further energy minimization by the steepest descents method was used, and system was coupled to an external bath using Berendsen's method. The reference temperature for all simulation was fixed at 300 K. LINC method was used to constrain all bonds 48,49 . OPLS-AA force field in GROMACS was used and grid type neighbor searching was done, and long range electrostatics was controlled using PME 50,51 . During simulations, the respective energy minimized structures were subjected to a position restrained MD Simulation for 100 ns with a time step of 2 fs. The trajectories were saved at every 2 ps to form respective trajectories. GROMACS built in tools were used to calculate RMSD (Root Mean Square Deviation), RMSF (Root Mean Square Fluctuations) and R gyr (radius of gyration).
Normal mode, Deformation and fluctuation analysis. Normal mode, deformation and fluctuation analysis were performed to study the domain motion in BARD1 ARD-BRCT wild-type and mutant protein using R 3.2 bio3D package 52 . First five modes were calculated by building the hessian matrix, and further extracted to explain the relative fluctuation in ARD domain of wild-type and mutant protein.
Principal component analysis (PCA). PCA reduces the dimensionality of the MD trajectory data and provides a concise way to visualize, analyze and compare large-scale concerted motions observed over the course of the simulation. PCA was performed using GROMACS in-built tool g_covar, g_anaeig and covariance matrix was built after removing net translational or rotational motion of the system by fitting the coordinate data to a reference structure. Further, diagonalization of the symmetric 3 N × 3 N covariance matrix was performed via eigenvector decomposition method and an orthogonal set of eigenvectors which are also called as functional "modes" were calculated with eigenvalues. Molecular docking. BARD1 ARD-BRCT wild-type, Gln 564 His and CstF50 docking was performed using HADDOCK server 2.0 which uses data-driven method of docking, with support from an extensive variety of experimental data obtained from diverse biophysical and biochemical experimental method 53 . Binding energy calculation. In common term binding free of complex in solvent can be calculated by ∆G Binding = G complex −(G protein + G Ligand ). Free energy and other energy parameters of the complex were calculated by using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method in GROMACS 4.5.5 package. g_mmpbsa tool was used to integrate high-throughput molecular dynamics simulation with the estimation of free energy of interaction 54 .