Elucidating the RAD51-BRC repeats interaction by integrating computational and experimental approaches

The interaction between the RAD51 and BRCA2 proteins is central in the homologous recombination pathway and DNA repair; through a series of BRC repeats, BRCA2 recruits RAD51, regulating its polymerization and chaperoning it to the damaged DNA site.1 The binding of the BRC repeats increases the structural flexibility of RAD51’s N-terminal domain, hindering the structural characterization of this complex via experimental methods.2 To reconstruct the conformational ensemble of the RAD51-BRC complex in solution, we combined Molecular Dynamics (MD) simulations with Small Angle X-ray Scattering (SAXS) experimental data. We initially generated an AlphaFold model of the complex between RAD51 and the BRC4 repeat. The predicted SAXS spectrum for this structure showed significant disagreement with the experimental data. Thus, we used steered MD to guide the structure towards a spectrum compatible with the experimental one. Interestingly, by using a forward model that neglects the solvent contribution for spectra prediction, we were not able to fulfill this task. Specifically, the simulations resulted in the detachment of BRC4 repeat from RAD51, in disagreement with experimental knowledge. Conversely, when including the solvent contribution, the simulations led to a conformation of the RAD51-BRC4 complex in remarkable agreement with the experiments. SAXS experimental spectra are averaged over multiple conformations accessible to the system in solution. Thus, starting from the structure obtained via steered MD, our next step will be to carry out MD simulations coupled with the maximum entropy principle3 to obtain an ensemble of configurations of the system whose average reflects the experimental SAXS spectrum.