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Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity

Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity
Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity
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.

1553-734X
e1002096
Kowalczykowski, Stephen Charles
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Cole, Daniel J.
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Rajendra, Eason
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Roberts-Thomson, Meredith
70ae4c90-104c-41be-b317-d252cad1514a
Hardwick, Bryn
48937654-879e-483c-9ed8-bcb787735299
McKenzie, Grahame J.
5deec50a-154d-4401-9ad0-9849a8041c64
Payne, Mike C.
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Venkitaraman, Ashok R.
fe199119-1c25-4f0e-8275-0e07305c5384
Skylaris, Chris-Kriton
8f593d13-3ace-4558-ba08-04e48211af61
Kowalczykowski, Stephen Charles
da79918e-25e8-4c45-bd69-6cdf3547bd03
Cole, Daniel J.
cb208a53-13ef-4871-b59b-6373abaadc39
Rajendra, Eason
26c9a243-ec16-42b1-bca6-9884fd21fe25
Roberts-Thomson, Meredith
70ae4c90-104c-41be-b317-d252cad1514a
Hardwick, Bryn
48937654-879e-483c-9ed8-bcb787735299
McKenzie, Grahame J.
5deec50a-154d-4401-9ad0-9849a8041c64
Payne, Mike C.
abb730ea-f683-4bec-a7e0-766f0a180a05
Venkitaraman, Ashok R.
fe199119-1c25-4f0e-8275-0e07305c5384
Skylaris, Chris-Kriton
8f593d13-3ace-4558-ba08-04e48211af61

Kowalczykowski, Stephen Charles, Cole, Daniel J., Rajendra, Eason, Roberts-Thomson, Meredith, Hardwick, Bryn, McKenzie, Grahame J., Payne, Mike C., Venkitaraman, Ashok R. and Skylaris, Chris-Kriton (2011) Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity. PLoS Computational Biology, 7 (7), e1002096. (doi:10.1371/journal.pcbi.1002096).

Record type: Article

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.

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Published date: 14 July 2011
Organisations: Chemistry, Computational Systems Chemistry

Identifiers

Local EPrints ID: 336977
URI: http://eprints.soton.ac.uk/id/eprint/336977
ISSN: 1553-734X
PURE UUID: 8d42db72-e323-466a-8a93-ac160739c7eb
ORCID for Chris-Kriton Skylaris: ORCID iD orcid.org/0000-0003-0258-3433

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Date deposited: 12 Apr 2012 14:14
Last modified: 15 Mar 2024 03:26

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Contributors

Author: Stephen Charles Kowalczykowski
Author: Daniel J. Cole
Author: Eason Rajendra
Author: Meredith Roberts-Thomson
Author: Bryn Hardwick
Author: Grahame J. McKenzie
Author: Mike C. Payne
Author: Ashok R. Venkitaraman

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