Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction.
RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model's depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51(E42A), RAD51(E59A), RAD51(E237A), RAD51(E59A/E237A) and RAD51(E42A/E59A/E237A) maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction.
Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA, Center for the Physics of Living Cells, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA, Department of Biochemistry, Carver College of Medicine, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA and Division of Medicinal and Natural Products Chemistry, Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA.
Shyamal
S
Subramanyam
William T
WT
Jones
Maria
M
Spies
M Ashley
MA
Spies
D024682:BRCA2 Protein / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D040681:Structural Homology, Protein
D024682:BRCA2 Protein; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; C495151:RAD51 protein, S cerevisiae; C495153:RAD51 protein, human; D051135:Rad51 Recombinase