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 rep...

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Autores principales:	Daniel J Cole, Eeson Rajendra, Meredith Roberts-Thomson, Bryn Hardwick, Grahame J McKenzie, Mike C Payne, Ashok R Venkitaraman, Chris-Kriton Skylaris
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/d7e5bba212ce4a52b4caf3a858b0a352
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spelling	oai:doaj.org-article:d7e5bba212ce4a52b4caf3a858b0a3522021-11-18T05:50:25ZInterrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.1553-734X1553-735810.1371/journal.pcbi.1002096https://doaj.org/article/d7e5bba212ce4a52b4caf3a858b0a3522011-07-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21789034/?tool=EBIhttps://doaj.org/toc/1553-734Xhttps://doaj.org/toc/1553-7358The 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.Daniel J ColeEeson RajendraMeredith Roberts-ThomsonBryn HardwickGrahame J McKenzieMike C PayneAshok R VenkitaramanChris-Kriton SkylarisPublic Library of Science (PLoS)articleBiology (General)QH301-705.5ENPLoS Computational Biology, Vol 7, Iss 7, p e1002096 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biology (General) QH301-705.5
spellingShingle	Biology (General) QH301-705.5 Daniel J Cole Eeson Rajendra Meredith Roberts-Thomson Bryn Hardwick Grahame J McKenzie Mike C Payne Ashok R Venkitaraman Chris-Kriton Skylaris Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.
description	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.
format	article
author	Daniel J Cole Eeson Rajendra Meredith Roberts-Thomson Bryn Hardwick Grahame J McKenzie Mike C Payne Ashok R Venkitaraman Chris-Kriton Skylaris
author_facet	Daniel J Cole Eeson Rajendra Meredith Roberts-Thomson Bryn Hardwick Grahame J McKenzie Mike C Payne Ashok R Venkitaraman Chris-Kriton Skylaris
author_sort	Daniel J Cole
title	Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.
title_short	Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.
title_full	Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.
title_fullStr	Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.
title_full_unstemmed	Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.
title_sort	interrogation of the protein-protein interactions between human brca2 brc repeats and rad51 reveals atomistic determinants of affinity.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/d7e5bba212ce4a52b4caf3a858b0a352
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Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.

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