RAD51 conformational dynamics explored through advanced molecular simulations

RAD51, a recombinase involved in DNA repair, is a promising drug target in the anticancer strategy known as Synthetic Lethality, a paradigm that leads a cancer cell to death by inhibiting two pathways simultaneously. RAD51 is found in fibril-like homo-oligomers; such structure is disassembled by the eight conserved BRC repeats of BRCA2, which recruit RAD51 monomers. RAD51 is then chaperoned to the damaged DNA site, where it catalyzes strand exchange during homologous recombination. The inhibition of the interaction between RAD51 and BRCA2 has proven to be synthetically lethal in association with PARP inhibition. Many drug discovery endeavors have been carried out to hinder the interaction between RAD51 and BRCA2, however, the details of such interface remain elusive because of the two proteins’ high flexibility. To overcome this challenge, we combined experimental data from Small Angle X-ray Scattering and Crosslinking Mass Spectrometry with Molecular Dynamics simulations leveraging the Maximum Entropy principle to reconstruct the RAD51-BRC4 complex’s conformational ensemble. Our results indicate that such complex assumes both compact and elongated conformations, and their interchange is guided by long range interaction between polar residues. We then approached the issue from a wider perspective, considering all eight RAD51-BRC complexes. We applied both end-point and alchemical perturbation based free energy estimation methods to identify residues that are possibly responsible for the different affinity of the BRC repeats for RAD51. Lastly, we tackled the modelling of a RAD51 filament assembly process. Our tool of election, given the availability of the filament’s structure, was a Gō model, which allowed us to simulate the assembly in a coarse-grained setting, starting from RAD51 monomers.