• BRCA1 BARD1 Heterodimer
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 BRCA1/BARD1 RING-domain heterodimer (PDB ID: 1JM7) from Homo Sapiens

     The BRAC1/BARD1 RING-domain heterodimer (PDB ID: 1JM7) from Homo sapiens is an antitumor protein for breast and ovarian cancers. When the protein is malformed, individuals are more susceptible to cancer, genomic integrity is compromised, and transcription is less regulated (1). Inherited germline mutations, loss of the functional wild-type allele, in the BRCA1 (and BRCA2) DNA repair genes are associated with approximately 20% of ovarian cancer occurrences (7, 8, 11). Somatic mutations of the gene and protein were more often a novel mutation, and if there was a germline mutation there was no somatic mutation (10). The type of BRCA mutation is not related to the stage of the cancer (10). The exact mechanism is not known for how the protein acts in the body but the mutation of the protein of the gene that encodes the protein is known to be involved.

The Dali server is used to analyze the three-dimensional structure of the protein, to find structurally similar proteins, and to discover functional clues (2). A Z-score greater than 2 indicates two proteins have significantly similar 3-D structure, the Z-score must be two or greater. The PSI-Blast server is used to analyze the primary structure and find proteins with similar amino acid sequences (3). An E-value of 0.05 or less is needed to qualify as structurally similar.

The BRAC1/BARD1 heterodimer has a molecular weight of 25932.32 Da and an isoelectric point of 8.49 (4). The heterodimer is composed of two subunits, and while homodimers can still form, the heterodimer is the preferential form of the protein (6). Subunit A contributes three alpha helices and three beta sheets to the overall structure, and subunit B contributes two alpha helices, one 3/10 alpha helix, and two beta sheets (5). Residues 104-112 of chain A and residues of 123-142 of chain B are missing from the crystallized structure due to disorder (5). The anti-parallel 4-helix bundle interface resulting from subunit association is formed by helices that flank the RING motifs (9). The carboxylate side chain of the Asp-40 residue brings the A subunit close by interacting with Lys-96 and Arg-99 of chain B (1). Dysfunction in the Arg-7 residue can lead to a rare type of cancer when the side chain interacts with other side chains or forms a salt bridge.

The RING portion of the protein is specific to binding the zinc ligand with the presence of conserved cysteine and histidine residues (1). Zinc is the only ligand that binds to the heterodimer. Overall there are four Zn²⁺ ligands, two per subunit. Ligand bonding is important to the function of the protein. Missense mutations in the BRCA1 gene usually target the residues involved in binding zinc, which often yield partial or complete loss of protein function when the second zinc binding site folds one the first binding site and the affinity for binding at the second site is reduced (9). Disulfide bonds in proteins often require metal ions to assist in the bond formation since the cell disfavors disulfide bonds with the more negative cell interior (as compared to the extracellular matrix) (9). The binding sites are created by a pattern of seven cysteine residues and one histidine residue in an interleaved fashion forms the two zinc-binding sites in each subunit (9). The mutations that affect the zinc-binding site, through altering Cys-39, Cys-61, or Cys-64, interrupt the protein formation but do not prevent heterodimer formation (9). These mutations can also affect E3 ubiquitin activity (9).

An important function in the protein is the association between the subunits, and many residues are important in this function. The structural domains for the RING structure are composed of residues 1-109 of BRCA1 and residues 26-119 of BARD1 (6). The RING finger motif corresponds to residues 24-64 for BRCA1 and residues 50-86 for BARD1. In general, the flanking regions of the RING motif assist in heterodimer formation (8). The amino acid side chains from subunit A of Glu-19, Leu-30, Leu-22, Ile-31, Lys-32, Val-83, Glu-84, Leu-87 are all involved in orienting the helix in the central RING motif (1). The residues in subunit B, Glu-45, Leu-48, and Ile-105, also help to orient the helix in central RING motif by connecting with Ile-56, and Leu-57. The biochemical function of this heterodimeric RING finger made with the N-terminus is not known (8).

Although the exact cellular mechanism for the BRCA1/BARD1-cell interaction is not known, its activity is associated with chromatin structures in the cell and RNA polymerase II (6). The amounts of BRCA1 and BARD1 reach a maximum during the S-phase of the cell cycle when they coalesce (8). The RING itself is a part of larger functional domains 6). The ubiquitin ligase activity of the RING finger is disrupted with mutations in the BRCA1 gene or protein formation, which also correlates to breast cancer formation with a loss of polyubiquitination function (8,9). In addition to this correlation to from the RING, the disruption of ubiquitin ligase activity in individuals with breast and ovarian cancer, suggests that additional function is associated with the areas of subunit association (8).

There is strong protection from proteolysis for regions outside the RING finger motif in the heterodimer that suggest that those regions are involved in heterodimer interaction (6). The protection of residues from proteolysis is supported by the susceptibility of Lys-55 in both the homodimer and heterodimer, which suggests it is on the surface of the protein in both forms (6). Trp-34 and Trp-91 flank the RING motif and are most likely buried in the dimer (6).

The site of the BRCA1 mutation is important when dealing with chemotherapy that uses platinum and PARP (7). For a patient with cancer, the loss of BRCA1 expression increases the sensitivity of cells to ionizing radiation, interstrand DNA crosslinking agents, and loss of high accuracy DSB repair (7). Mutations between BRCA1 and BRCA 2 cause different risks for breast and ovarian cancer, and correspond to different treatment, and BRCA1 has been show to increase risks for breast cancer over ovarian cancer (7). As the second or third line of treatment, platinum based treatments create a partial or complete response (reduction in tumor cells) 80% and 100% in BRCA1 and BRCA2 respectively (7). Taxanes are often added in the treatment with platinum to decrease resistance, and arrest the cell at the spindle assembly checkpoint via microtubule disassembly (7) which help to prevent the spread and growth of the cancer cells whose. Poly (ADP-ribose) polymerase-1 (PARP1) may be effective in cancers where homologous recombination function is compromised by somatic aberrations, and this also increases response to platinum drugs (10).

Another protein with similar structure is the Phosphotyr371-C-Cbl-Ubch5b complex (Chain A) (PDB ID: 4A49), a ligase protein from Homo sapiens (1). This molecule is also a heterodimer but it contains five beta sheets in subunit A, unlike BRCA1/BARD1 RING-domain heterodimer, which only has three beta sheets (1).  The Z score for the Phosphotyr371-C-Cbl-Ubch5b Complex is 2.5 and the E value is 0.002 (2,3). This protein is similar to the heterodimer protein in that it binds to zinc (Zn²⁺) but this protein also binds to potassium (K⁺) (1).  Both A subunits for these proteins have two zinc binding sites, but the B subunits differ in their metal binding because the B subunit of Phosphotyr371-C-Cbl-Ubch5b complex binds the potassium.  Phosphotyr371-C-Cbl-Ubch5b complex assists in receptor tyrosine kinase signal transduction (11). The ubiquitination activity of Phosphotyr371-C-Cbl-Ubch5b Complex suggests that the BRAC1/BARD1 RING-domain heterodimer also has this activity due to the similarity of their structures and functions. The tertiary structures of these two proteins differ in that the BRCA1 protein forms a RING-domain and the Phosphotyr371-C-Cbl-Ubch5b complex forms a globular shape (1). This difference in tertiary structure demonstrates the different overall functions of the proteins because the BRCA protein assists in tumor suppression and the Phosphotyr371-C-Cbl-Ubch5b complex protein assists in ligase activity, going between an active and inactive state (1, 12). This active and inactive state for Phosphotyr371-C-Cbl-Ubch5b complex is relies on the phosphorylation of 

BRAC1/BARD1 RING-domain heterodimer (PDB ID: 1JM7) is a antitumor protein, but when it is mutated, originating from the BRAC1/BARD1 gene, it causes an increase in susceptibility to breast and ovarian cancers. It is important to study BRAC1/BARD1 RING-domain heterodimer because understanding the mechanism of the protein activity in the tumor suppression could help doctors and researchers learn how to more accurately target chemotherapy or radiation for individuals, namely women, who now have cancer.