Homo sapien BRCT Domain of
BRCA1
Brian Steginsky '06 and Alexis Marino '09
Contents:
I. Introduction
Genomic mutations and DNA damage are suspected to be correlated with cancer. Mutations arise as a result of replication errors and cellular exposure to mutagens. Alkylating- and oxidizing- reagents, as well as UV- and gamma- rays, are capable of inducing mutations into the genome. Fortunately, organisms invoke many cellular mechanisms to repair damaged DNA. Without DNA damage “check points,” DNA repair can not occur and these mutations are passed to daughter cells.
BRCA1 is a tumor suppressing gene that encodes a protein involved in the repair of double strand DNA breaks (DSB). BRCA1 is believed to serve as a mediator for the assembly of DNA repair complexes (Clappterton et al., 2004). BRCA1 interacts with various DNA damage proteins through its BRCT domain (BRCA1 C-termini domain). Mutations within the BRCT domain inhibit BRCA1 from interacting with partner proteins. As a result, damaged DNA escapes the checkpoints during the G2/M phase of the cell cycle. BRCT must bind BACH1, a member of the DEAH helicase family, in order to be operable and participate in DSB repair. Very little is known about the exact nature of these interactions, but the BRCA1/BACH1 complex appears to be an integral component to the DNA damage checkpoint during the G2 phase of the cell cycle.
II. General Structure
The BRCT domain consists of two repeats, the N-terminal BRCT and the C-terminal BRCT. The two repeats are extremely similar in structure and lie in a head-to-tail arrangement (Williams et al., 2001). The N-terminal BRCT domain is composed of 112 residues, whereas the C-terminal domain is slightly smaller, and is composed of only 102 residues.
Both BRCT repeats are composed of a 4-stranded ß-sheet
and 3-α-helices.
There is a ß hair-pin and
α-helical region
, connecting the two BRCT repeats. The
α-1 helix and α-3 helix of the C-terminal domain are packed
closely together, through hydrophobic interactions, with the
α-2 helix of the N-terminal domain.
The cleft formed between the three α-helices of the N- and C-terminal
BRCT repeats is highly conserved and extensively involved in phosphopeptide
binding.
III. BRCT-BACH1 Interface
Interactions between BRCT and BACH1 are essential for tumor suppression activity. Missense mutations within the BRCT or BACH1 binding domains are correlated to increased breast and ovarian cancer rates in women. The BRCT domain has a high affinity for the phosphorylated BACH1 peptide. Breast and ovarian cancer rates drastically increase in the absence of phosphorylation, which can be attributed to the fact that the BRCA1/BACH1 complex is not formed.
The BRCT tandem interacts with Ser988-Lys995 of the BACH1 phosphopeptide. Two amino acid residues of the BACH1 protein, phosphorylated Ser990 (pSer990) and Phe993, interact extensively with the hydrophobic cleft of the BRCT tandem (Shi et al., 2004). The pSer990 forms a hydrogen bond with the amide backbone of the Gly1656
residue of N-terminal BRCT repeat (2004) . The side chains of
Lys1702 and Ser1655, also of the N-terminal BRCT repeat, participate in hydrogen bonding with the pSer990. The BRCT tandem undergoes a slight conformational change upon phosphopeptide binding; as a result, Ser1655 and Gly1656 are shifted into an optimized position to engage in hydrogen bonding with the BACH1 domain.
The backbone atoms of Thr1700 and
Leu1701
form a hydrophobic cleft with the side chains of Phe1704,
Asn1774,
Met1775, and Leu1839
. Thr1700,
Leu1701, and Phe1704
are part of the N-terminal repeat; whereas Asn1774,
Met1775, Leu1839
are part of the C-terminal repeat. The aromatic side chain of Phe993 inserts
into the hydrophobic cleft, engaging in van der Waals interactions with
the BRCT domain.
IV. Mutations
BRCT residues involved in phosphopeptide binding, either by stabilizing or forming the phosphopeptide binding surface, are highly conserved in BRCA1 across species (Clapperton et al., 2004). Mutations associated with these residues are strongly correlated with increased cancer rates. Mutations can disrupt the entire BRCT domain, leading to the unfolding and collapse of the hydrophobic cleft. Another scenario invokes the idea that some mutations do not result in the collapse of the hydrophobic cleft, but rather interfere with ligand binding (2004).
There have been over 80 cancer-derived mutations associated with the BRCT tandem, but only a handful have actually been confirmed to directly increase breast and ovarian cancer rates (2004). The BRCT-BACH1 binding interactions provide an excellent platform to study cancer-related mutations of the BRCT domain. As mentioned in the previous section, BRCA1 must bind with BACH1 in order to successfully participate in DSB repairs. Mutations, either by sterically hindering ligand binding or disrupting the global structure of the BRCT domain, are directly correlated with the ability of BRCT to bind BACH1 and BRCA1-BACH1 to participate in DSB repair.
Arg1699 and Met1775 are buried in the hydrophobic cleft. R1699Q/W and M1775R are the two most studied mutations of the BRCT domain, because of their importance associated with the BACH1-BRCT interface.
The Arg1699 side chain donates two H-bonds to the backbone of Phe993 of the BACH1 domain.
R1699Q results in a loss of phospho-specificity and the mutant binds to both phosphorylated and nonphosphorylated peptides.
In comparison, R1699W completely diminishes the ability of BRCT to bind BACH1, which is likely due to the fact that the W1699 side chain contributes less hydrogen bonding than the wildtype and R1699Q mutant .
Met1775 stacks against Phe993 of the BACH1 domain.
The van der Waals stacking interactions between M1775 and Phe993 are lost in the M1775R mutation (Shi et al., 2004). The guanidine of the arginine side (M1775R) chain protrudes into the BRCT cleft, resulting in steric hinderence that prevents Phe993 of BACH1 from inserting into the hydrophobic cleft.
Met1652 and Ala1708
lie deep within the surface of the BRCT domain.
Mutations associated with Met1652 and Ala1708disrupt the folding and global structure of the BRCT domain, as a result, BRCT is unable to bind BACH1.
Numerous other missense mutations have been studied: D1692Y, C1697R, S1715R, G1738E, P1749R, and Y1853X. All of these residues are located at the phosphopeptide-binding interface of the BRCT domain and result in a reduced binding affinity for BACH1.
V. Conclusion
BRCA1 interacts with several DNA damage repair proteins during the DNA damage checkpoint and initiates homologous recombination to repair DSBs. BRCA1, along with many other DNA damage repair proteins, appear to be involved in a cascade of phosphorylation signaling. The BRCT domain of BRCA1 has a high affinity for phosphopeptides, which appears to be a commonly recurrent theme of DNA damage repair proteins such as XRCC1, 53BP1, and MDC1. Mutations in the BRCT domain lead to a decreased affinity for phosphorylated proteins, usually as a result of the cleft unfolding or steric hinderence. As a result, damaged DNA is able to elude BRCA1 during the G2/M phase check point and damaged DNA is replicated.
Recent studies have directly linked a handful of BRCT mutations with increased breast and ovarian cancer rates. The BACH1-BRCT interface provides an excellent medium to study the effects of cancer causing mutations. The discovery of BRCA1 has led to several advancements in the search to cure hereditary breast and ovarian cancer. However, after twelve years of extensive research, the exact function and mechanism of BRCA1 is still unclear. Perhaps a more lucid understanding BRCA1, its interactions with various proteins, and precise role in DNA repair will lead to the cure for breast and ovarian cancer. Future studies associated with BRCA1 are aimed at resolving these unidentified interactions.
VI. References
Clapperton, Julie A., Isaac A Manke, Drew M. Lowery, Timmy Ho, Lesley R. Haire, Michael B. Yaffe, and Stephen J. Smerdon. 2004. Structure and mechanism of BRCA1 BRCT domain recognition of phosphorylated BACH1 with implications for cancer. Nature Structural and Molecular Biology 11:512-518.
Glover, Mark J.N., R. Scott Williams, and Megan S. Lee. 2004. Interactions between BRCT repeats and phosphoproteins: tangled up in two. TRENDS in Biochemical Sciences 29: 579-585.
Schiozaki, Eric N., Lichuan Gu, Nieng Yan, and Yigong Shi. 2004. Structure of the BRCT repeats of BRCA1 bound to a BACH1 phosphopeptide: Implications for signaling. Molecular Cell
14:405-412.
Williams, R. Scott, Ruth Green and J.N. Mark Glover. 2001. Crystal structure of the BRCT repeat region from the breast cancer-associated protein BRCA1. Nature Structural Biology 8: 838-842.
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