Abstract
The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the analysis of protein-protein interactions for chemical biology and molecular therapeutics.
Highlights
The human breast cancer suppressor protein BRCA2 controls the functions of the RAD51 recombinase, an enzyme conserved in all kingdoms of life, which carries out the strand exchange reaction central to homologous DNA recombination (HDR) [1]
Due to lack of experimental structural information regarding the binding of seven of the eight BRC repeats to RAD51, it is unknown how subtle sequence variations in the repeats translate to measurable variations in their binding affinity
We have used a range of computational methods, firstly based on classical force fields, and secondly based on first principles quantum mechanical techniques whose computational cost scales linearly with the number of atoms, allowing us to perform calculations on the entire protein complex
Summary
The human breast cancer suppressor protein BRCA2 controls the functions of the RAD51 recombinase, an enzyme conserved in all kingdoms of life, which carries out the strand exchange reaction central to homologous DNA recombination (HDR) [1]. This essential cellular pathway is responsible for the error-free repair of DNA double strand breaks and is central to the maintenance of genome integrity and the prevention of diseases such as cancer [2]. The interaction between the BRC repeats of human BRCA2 and RAD51 has been characterised predominantly through structural and biochemical approaches and regulates many of RAD51’s activities including RAD51 oligomerisation, and its ordered assembly on single-stranded or double-stranded DNA substrates to control the stepwise events of the strand exchange reaction [5,6]
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