Interrogation of the Protein-Protein Interactions between Human BRCA2 BRC Repeats and RAD51 Reveals Atomistic Determinants of Affinity

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.

Correlation between Selected XRCC2, XRCC3 and RAD51 Gene Polymorphisms and Primary Breast Cancer in Women in Pakistan

“…Different meta-analysis and studies on RAD51 have earlier shown an association of this polymorphism with an elevated breast cancer risk (Cole et al, 2011;Falvo et al, 2011;Gao et al, 2011;Hosseini et al, 2012). The RAD51 135G/C polymorphism located in the 5' untranslated region seems to be of functional relevance.…”

Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction

“…Both negative (22,55–57) and positive (19,58) effects have been reported. Our experimental conditions were more similar to those reported in (57,59), in that we used a synthesized peptide, whereas Carreira and colleagues used Glutathione S-transferase-fused BRC4. Similar to (22,55–57), we observed destabilization of the extended RAD51 nucleoprotein filament.…”

Electrostatic considerations affecting the calculated HOMO–LUMO gap in protein molecules

“…This allows an accurate QM description of systems of thousands of atoms [5], including entire proteins [14,29,30]. The ONETEP formalism is based on a reformulation of conventional Kohn-Sham DFT [1,2] in terms of the single-particle density matrix…”