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

DOI: 10.1073/pnas.2017708118 DOI Help

Authors: Laurens H. Lindenburg (University of Cambridge) , Teodors Pantelejevs (University of Cambridge) , Fabrice Gielen (University of Cambridge; University of Exeter) , Pedro Zuazua-Villar (The Institute of Cancer Research) , Maren Butz (University of Cambridge) , Eric Rees (University of Cambridge) , Clemens F. Kaminski (University of Cambridge) , Jessica A. Downs (The Institute of Cancer Research) , Marko Hyvonen (University of Cambridge) , Florian Hollfelder (University of Cambridge)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences , VOL 118

State: Published (Approved)
Published: November 2021
Diamond Proposal Number(s): 18548

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 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 −2.4 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 β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.

Journal Keywords: protein evolution; modular protein engineering; synthetic biology; RAD51; BRC repeats

Diamond Keywords: Breast Cancer

Subject Areas: Biology and Bio-materials

Instruments: I03-Macromolecular Crystallography

Added On: 17/11/2021 11:20

Discipline Tags:

Life Sciences & Biotech Health & Wellbeing Cancer Non-Communicable Diseases Structural biology

Technical Tags:

Diffraction Macromolecular Crystallography (MX)