Combining computational and experimental methods to characterize the RAD51-BRC repeats interaction

Given its implication in homologous recombination and DNA repair, the interaction between the RAD51 and BRCA2 proteins has gained relevance from a pharmaceutical standpoint. (Davies et al., 2001) However, binding of BRCA2’s BRC repeats increases the flexibility of RAD51’s N-terminal domain, hampering the experimental characterization of the full structure of this complex.(Pellegrini et al., 2002) To overcome this limitation, we combine experimental data from Small Angle X-ray Scattering (SAXS) with Molecular Dynamics (MD) simulations, aiming at reconstructing the conformational ensemble of the RAD51-BRC peptides complexes at atomistic resolution. As initial guess for our simulations, we used an AlphaFold-generated model of the full RAD51 in complex with the BRC4 peptide. The SAXS spectrum computed for this model displayed remarkable disagreement with the experimentally measured one. Therefore, we performed Steered MD simulations to guide the system towards a configuration compatible with the experimental data. Notably, we found that taking into account the solvent contribution(Ballabio et al., 2023) in the calculation of SAXS spectra proved necessary to sample realistic configurations of the complex, and to avoid detachment of the BRC4 peptide from RAD51. We then used enhanced sampling, namely Metadynamics, to generate a heterogeneous ensemble of configurations for subsequent reweighting via the Maximum Entropy(Cesari et al., 2018) principle. Through this strategy, we aim at identifying a conformational ensemble whose average spectrum is compatible with the experimental one.