Dissecting the RAD51-BRC4 Interaction Landscape through Integrative Molecular Simulations and Experimental Biophysics

The interaction between the RAD51 and BRCA2 proteins is central for homologous recombination, a crucial pathway ensuring high-fidelity DNA repair. Recruitment of RAD51 involves eight highly conserved regions on BRCA2, named BRC repeats. The interaction between the fourth BRC repeat (BRC4) and the RAD51 C-terminal domain has been structurally characterized, while the complex of full-length RAD51 with the peptide remains elusive. This gap limits our understanding of the cytosolic RAD51 recruitment driven by the BRCA2 BRC-repeats, which is one of the first crucial steps in homologous recombination. Here, we report an integrative experimental and in silico approach to reconstruct the conformational ensemble in solution for full-length RAD51 in complex with BRC4. We combined AlphaFold2, Cross-linking Mass Spectrometry, and Small-Angle X-ray Scattering data with Molecular Dynamics simulations. Our results show that the full-length RAD51-BRC4 complex is a mixture of compact and elongated conformations and allow the identification of key residues at the interface between RAD51 N-terminus and BRC4, mediating complex conformational dynamics. Our evidence provides robust atomic-level insights into the RAD51-BRC4 interaction, shedding light on the molecular features that govern the recognition between these two proteins, while unveiling novel hotspots for developing novel anti-cancer agents.