Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by 22.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended beta-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.

Automated summary

The study focused on the role of RAD51-interacting modules within BRCA2, creating chimeric repeats to measure binding to RAD51. Surprisingly, certain shuffled module combinations showed stronger binding than natural repeats, with weak correlation to affinities of natural repeats. The strongest chimera, BRC8-2, showed improved binding to RAD51 and functioned in preventing the formation of nuclear RAD51 foci in human cells after ionizing radiation.