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

Abstract

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 repeats were stronger than any of the natural repeats, suggesting balancing of relative properties in BRC repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.44 kCal/mol compared to the strongest natural repeat, BRC4. Crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended -hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear foci after ionizing radiation.

Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

“…BRCA2 has many predicted disordered regions, which complicate understanding the functional organization and possible dynamic rearrangement of the reported structured domains. Several crystal structures of small fragments of BRCA2 are available: C-terminal BRCA2-DSS1 complex ( Yang et al, 2002 ) (PDB ID: 1IYJ), N-terminal BRCA2-PALB2 ( Oliver et al, 2009 ) (PDB ID: 3EU7), Brc4 BRCA2-RAD51 ( Pellegrini et al, 2002 ) (PDB ID: 1N0W), BRCA2 phosphopeptide-Plk1 ( Ehlén et al, 2020 ) (PDB ID: 6GY2), Brc8-2 BRCA2-RadA ( Lindenburg et al, 2020 ) (PDB ID: 6HQU), and a BRCA2 peptide residing in exon 12 with the C-terminal armadillo-type domain of HSF2BP ( Ghouil et al, 2020 ). Of these, the largest crystallized segment of BRCA2 is the DBD (736 amino acids), which encompasses the helical domain, tower domain, and the three oligonucleotide/oligosaccharide binding (OB) folds.…”