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

Laurens H Lindenburg1, Teodors Pantelejevs1, Fabrice Gielen1,2

  • 1Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.

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Shuffling protein modules in BRCA2’s BRC repeats created stronger RAD51 binders than natural ones. This protein engineering approach reveals new insights into DNA repair mechanisms and protein evolution.

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Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Genetics

Background:

  • Protein sequence modules facilitate evolutionary leaps in function.
  • BRCA2 protein contains eight BRC repeats, each with two RAD51-interacting modules.
  • Understanding these interactions is crucial for DNA repair and cancer research.

Purpose of the Study:

  • To investigate the role of RAD51-interacting modules within BRCA2 BRC repeats.
  • To determine if shuffling these modules can enhance RAD51 binding affinity.
  • To explore the functional implications of engineered BRCA2 variants.

Main Methods:

  • Created 64 chimeric BRC repeats by shuffling RAD51-interacting modules.
  • Measured binding affinities of natural and chimeric repeats to RAD51.
  • Determined the crystal structure of a high-affinity RAD51:BRC8-2 complex.
  • Assessed the function of BRC8-2 in human cells following ionizing radiation.

Main Results:

  • Certain shuffled module combinations exhibited stronger RAD51 binding than natural repeats.
  • The contribution of individual modules to affinity was poorly correlated with natural repeat affinities.
  • The strongest chimera, BRC8-2, showed a significant binding improvement (-2.4 kCal/mol) over the best natural repeat (BRC4).
  • Crystal structure revealed an improved interface fit and an extended β-hairpin in BRC8-2.

Conclusions:

  • Protein module shuffling is a viable strategy to engineer enhanced protein-protein interactions.
  • BRC8-2 demonstrates functional activity in human cells, inhibiting RAD51 foci formation.
  • This study provides novel insights into BRCA2-RAD51 interaction dynamics and potential therapeutic strategies.