Thermal denaturation of the BRCT tandem repeat region of human tumour suppressor gene product BRCA1

Reduced stability of the tandem BRCT domains of human BReast CAncer 1 (BRCA1) due to missense mutations may be critical for loss of function in DNA repair and damage-induced checkpoint control. In the present thermal denaturation study of the BRCA1 BRCT region, high-precision differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy provide evidence for the existence of a denatured state that is structurally very similar to the native. Consistency between theoretical structure-based estimates of the enthalpy (DeltaH) and heat capacity change (DeltaCp) and the calorimetric results is obtained when considering partial thermal unfolding contained in the region of the conserved hydrophobic pocket formed at the interface of the two BRCT repeats. The structural integrity of this region has been shown to be crucial for the interaction of BRCA1 with phosphorylated peptides. In addition, cancer-causing missense mutations located at the inter-BRCT-repeat interface have been linked to the destabilization of the tandem BRCT structure.     

Crystal structure of the BARD1 BRCT domains

The interaction of the breast tumor suppressor BRCA1 with the protein BARD1 results in the formation of a heterodimeric complex that has ubiquitin ligase activity and plays central roles in cell cycle checkpoint control and DNA repair. Both BRCA1 and BARD1 possess a pair of tandem BRCT domains that interact in a phosphorylation-dependent manner with target proteins. We determined the crystal structure of the human BARD1 BRCT repeats (residues 568-777) at 1.9 A resolution. The composition and structure of the BARD1 phosphoserine-binding pocket P1 are strikingly similar to those of the BRCA1 and MDC1 BRCT domains, suggesting a similar mode of interaction with the phosphate group of the ligand. By contrast, the BARD1 BRCT selectivity pocket P2 exhibits distinct structural features, including two prominent histidine residues, His685 and His686, which may be important for ligand binding. The protonation state of these histidines has a marked effect on the calculated electrostatic potential in the vicinity of P2, raising the possibility that ligand recognition may be regulated by changes in pH. Importantly, the BARD1 BRCT structure provides insights into the mechanisms by which the cancer-associated missense mutations C645R, V695L, and S761N may adversely affect the structure and function of BARD1.     

Characterisation of the BRCT domains of the breast cancer susceptibility gene product BRCA1

The breast cancer susceptibility gene product BRCA1 is a tumour suppressor but the biochemical and biological functions that underlie its role in carcinogenesis remain to be determined. Here, we characterise the solution properties of the highly conserved C terminus of BRCA1, consisting of a tandem repeat of the BRCT domain (BRCT-tan), that plays a critical role in BRCA1-mediated tumour suppression. The overall free energy of unfolding of BRCT-tan is high (14.2 kcal mol(-1) at 20 degrees C in water) but unfolding occurs via an aggregation-prone, partly folded intermediate. A representative set of cancer-associated sequence variants was constructed and the effects on protein stability were measured. All of the mutations were highly destabilising and they would be expected to cause loss of function for this reason. Over half could not be purified in a soluble form, indicating that these residues are critical for maintaining structural integrity. The remaining mutants exhibited much greater aggregation propensities than the wild-type, which is most likely a consequence of their reduced thermodynamic stability relative to the partly folded intermediate. The mutations characterised here are located at different sites in the BRCT-tan structure that do not explain fully their effects on the protein's stability. Thus, the results indicate an important role for biophysical studies in assessing the significance of sequence variants and in determining how they cause disease.     

Crystal structure and interactions of the PAS repeat region of the Drosophila clock protein PERIOD

PERIOD proteins are central components of the Drosophila and mammalian circadian clock. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interactions with other clock components. Here we present the crystal structure of a Drosophila PERIOD (dPER) fragment comprising two tandemly organized PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) and two additional C-terminal alpha helices (alphaE and alphaF). Our analysis reveals a noncrystallographic dPER dimer mediated by intermolecular interactions of PAS-A with PAS-B and helix alphaF. We show that alphaF is essential for dPER homodimerization and that the PAS-A-alphaF interaction plays a crucial role in dPER clock function, as it is affected by the 29 hr long-period perL mutation.     

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.     

Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1

The breast cancer susceptibility gene, BRCA1, encodes a large nuclear phosphoprotein, the major isoform of which is 1863 amino acids in size. Structure-function studies have been largely restricted to the only two domains identified by homology searches: the RING (really interesting new gene) and BRCT (BRCA1 C-terminus) domains. However, we have recently reported the identification of a large central soluble region of BRCA1 (residues 230-534) that binds specifically to four-way junction DNA, a property that potentially facilitates its role in the repair of DNA lesions by homologous recombination. We have now used a combination of limited proteolysis and extension cloning to identify more accurately the DNA-binding region of BRCA1. Limited trypsinolysis of BRCA1-(230-534) resulted in the production of a soluble domain identified as residues 230-339. However, after cloning, expression and purification of this region, studies revealed that it was unable to bind to four-way junctions, suggesting that the DNA-binding activity, in part, resides within residues 340-534. A series of fragments extending from residue 340 were produced, and each was tested for its ability to bind to four-way junction DNA in gel retardation assays. In these experiments, residues 340-554 of BRCA1 were identified as the minimal DNA-binding region. We then went on to characterize the conformation of this region using CD spectroscopy and analytical centrifugation.     

Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1

The BRCT repeats in BRCA1 are essential for its tumor suppressor activity and interact with phosphorylated protein targets containing the sequence pSer-X-X-Phe, where X indicates any residue. The structure of the tandem BRCA1 BRCT repeats bound to an optimized phosphopeptide reveals that the N-terminal repeat harbors a conserved BRCT phosphoserine-binding pocket, while the interface between the repeats forms a hydrophobic groove that recognizes the phenylalanine. Crystallographic and biochemical data suggest that the structural integrity of both binding sites is essential for peptide recognition. The diminished peptide-binding capacity observed for cancer-associated BRCA1-BRCT variants may explain the enhanced cancer risks associated with these mutations.     

Role for the BRCA1 C-terminal repeats (BRCT) protein 53BP1 in maintaining genomic stability

p53-binding protein-1 (53BP1) is phosphorylated in response to DNA damage and rapidly relocalizes to presumptive sites of DNA damage along with Mre11 and the phosphorylated histone 2A variant, gamma-H2AX. 53BP1 associates with the BRCA1 tumor suppressor, and knock-down experiments with small interfering RNA have revealed a role for the protein in the checkpoint response to DNA damage. By generating mice defective in m53BP1 (m53BP1(tr/tr)), we have created an animal model to further explore its biochemical and genetic roles in vivo. We find that m53BP1(tr/tr) animals are growth-retarded and show various immune deficiencies including a specific reduction in thymus size and T cell count. Consistent with a role in responding to DNA damage, we find that m53BP1(tr/tr) mice are sensitive to ionizing radiation (gamma-IR), and cells from these animals exhibit chromosomal abnormalities consistent with defects in DNA repair. Thus, 53BP1 is a critical element in the DNA damage response and plays an integral role in maintaining genomic stability.     

Phosphopeptide binding specificities of BRCA1 COOH-terminal (BRCT) domains

Protein phosphorylation by protein kinases may generate docking sites for other proteins. It thus allows the assembly of signaling complexes in response to kinase activation. Several protein domains that bind phosphoserine or phosphothreonine residues have been identified, including the 14-3-3, PIN1, FHA, KIX, WD-40 domain, and polo box (Yaffe, M. B., and Elia, A. E. (2001) Curr. Opin. Cell Biol. 13, 131-138; Elia, A. E., Cantley, L. C., and Yaffe, M. B. (2003) Science 299, 1228-1231). The BRCA1 COOH-terminal (BRCT) domains are protein modules found in many proteins that regulate DNA damage responses (Koonin, E. V., Altschul, S. F., and Bork, P. (1996) Nat. Genet. 13, 266-268). Whether BRCT domains can mediate phosphorylation-dependent interactions has not been systematically investigated. We report here that the BRCT domains also recognize phosphopeptides. Oriented peptide library analysis indicated that the BRCT domains from BRCA1, MDC1, BARD1, and DNA Ligase IV preferred distinct phosphoserine-containing peptides. In addition, the interaction between BRCA1 and the BRCT binding motif of BACH1 was required for BACH1 checkpoint activity. Furthermore, BRCT domains of the yeast DNA repair protein Rad9 could bind phosphopeptides, suggesting that the BRCT domains represent a class of ancient phosphopeptide-binding modules. Potential targets of BRCT domains were identified through data base search. Structural analysis of BRCA1 BRCT repeats also predicted conserved residues that may form the phosphopeptide-binding pocket. Thus, the BRCT repeats are a new family of phosphopeptide-binding domains in DNA damage responses.     

乳腺癌易感蛋白BRCA1的BRCT1结构域染色质伸展活性的定位

乳腺癌易感基因BRCA1（Breast cancer susceptibility gene 1）在乳腺癌的发生、发展中起重要作用。BRCA1 C末端含有2个BRCT结构域（BRCT1和BRCT2）,许多乳腺癌易感突变发生在BRCA1的BRCT结构域中。利用染色质结构检测技术表明,BRCT结构域具有染色质伸展活性。本文利用缺失突变技术构建了6种BRCT1结构域（1642-1736 aa）缺失突变体并将BRCT1结构域中与染色质伸展相关的重要区域定位到1691-1721之间的氨基酸残基;用丙氨酸扫描技术构建了10种BRCT1结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到1707-1711之间的IAGGK。利用定位的重要区域进行Blast分析,结果找到一新型同源蛋白质。BRCT1结构域的定位有助于预测BRCT1结构域突变后发生乳腺癌的风险,也为进一步研究BRCT1结构域的功能机制提供了有用的材料。     

Modification of BRCA1-associated breast cancer risk by the polymorphic androgen-receptor CAG repeat

Compared with the general population, women who have inherited a germline mutation in the BRCA1 gene have a greatly increased risk of developing breast cancer. However, there is also substantial interindividual variability in the occurrence of breast cancer among BRCA1 mutation carriers. We hypothesize that other genes, particularly those involved in endocrine signaling, may modify the BRCA1-associated age-specific breast cancer risk. We studied the effect of the CAG repeat-length polymorphism found in exon 1 of the androgen-receptor (AR) gene (AR-CAG). AR alleles containing longer CAG repeat lengths are associated with a decreased ability to activate androgen-responsive genes. Using a sample of women who inherited germline BRCA1 mutations, we compared AR-CAG repeat length in 165 women with and 139 women without breast cancer. We found that women were at significantly increased risk of breast cancer if they carried at least one AR allele with >/=28 CAG repeats. Women who carried an AR-CAG allele of >/=28, >/=29, or >/=30 repeats were given a diagnosis 0.8, 1.8, or 6.3 years earlier than women who did not carry at least one such allele. All 11 women in our sample who carried at least one AR-CAG allele with >/=29 repeats had breast cancer. Our results support the hypothesis that age at breast cancer diagnosis is earlier among BRCA1 mutation carriers who carry very long AR-CAG repeats. These results suggest that pathways involving androgen signaling may affect the risk of BRCA1-associated breast cancer.     

Primary structure-based function characterization of BRCT domain replicates in BRCA1

BRCA1 is a large protein that exhibits a multiplicity of functions in its apparent role in DNA repair. Certain mutations of BRCA1 are known to have exceptionally high penetrance with respect to familial breast and ovarian cancers. The structures of the N-terminus and C-terminus of the protein have been determined. The C-terminus unit consists of two alpha-beta-alpha domains designated BRCT. We predicated two homologous BRCT regions in the BRCA1 internal region, and subsequently produced and purified these protein domains. Both recombinant domains show significant self-association capabilities as well as a preferential tendency to interact with each other. These results suggest a possible regulatory mechanism for BRCA1 function. We have demonstrated p53-binding activity by an additional region, and confirmed previous results showing that two regions of BRCA1 protein bind p53 in vitro. Based on sequence analysis, we predict five p53-binding sites. Our comparison of binding by wild-type and mutant domains indicates the sequence specificity of BRCA1-p53 interaction.     

The BRCA1 RING and BRCT domains cooperate in targeting BRCA1 to ionizing radiation-induced nuclear foci

BRCA1 accumulates in nuclear foci during S-phase and reassembles into DNA repair-associated foci after DNA damage, reflecting its role in genome maintenance. BRCA1 comprises a RING domain at the N terminus and a BRCT domain at the C terminus, through which it associates with DNA repair proteins. The key sequences that target BRCA1 to DNA damage-induced foci have not been identified. Here, we mapped the BRCA1 foci-targeting domains of yellow fluorescence protein (YFP)-tagged BRCA1 in MCF-7 breast cancer cells exposed to ionizing radiation (IR). Cancer mutations specific to the BRCT domain, but not the RING domain, abolished BRCA1 recruitment to IR-induced foci. The YFP-BRCT domain itself, however, localized poorly at IR-induced foci, and the RING domain and other sequences were negative. We discovered that only when the RING and BRCT domains were combined was foci targeting restored to levels observed for wild-type BRCA1. The RING-BRCT fusion co-localized at foci with the MDC1 DNA damage response factor and inhibited entry of endogenous BRCA1 into nuclear foci. Our results explain why exon 11-deficient BRCA1 splice variants are targeted to IR-induced foci even though they are incapable of repairing DNA damage. We propose that both RING and BRCT domains together target BRCA1 to large focal assemblies at DNA double-stranded breaks.     

Crystal structure of human 53BP1 BRCT domains bound to p53 tumour suppressor

The BRCT (BRCA1 C-terminus) is an evolutionary conserved protein-protein interacting module found as single, tandem or multiple repeats in a diverse range of proteins known to play roles in the DNA-damage response. The BRCT domains of 53BP1 bind to the tumour suppressor p53. To investigate the nature of this interaction, we have determined the crystal structure of the 53BP1 BRCT tandem repeat in complex with the DNA-binding domain of p53. The structure of the 53BP1-p53 complex shows that the BRCT tandem repeats pack together through a conserved interface that also involves the inter-domain linker. A comparison of the structure of the BRCT region of 53BP1 with the BRCA1 BRCT tandem repeat reveals that the interdomain interface and linker regions are remarkably well conserved. 53BP1 binds to p53 through contacts with the N-terminal BRCT repeat and the inter-BRCT linker. The p53 residues involved in this binding are mutated in cancer and are also important for DNA binding. We propose that BRCT domains bind to cellular target proteins through a conserved structural element termed the 'BRCT recognition motif'.     

Impact of BRCA1 BRCT domain missense substitutions on phosphopeptide recognition

The BRCA1 BRCT domain binds pSer-x-x-Phe motifs in partner proteins to regulate the cellular response to DNA damage. Approximately 120 distinct missense variants have been identified in the BRCA1 BRCT through breast cancer screening, and several of these have been linked to an increased cancer risk. Here we probe the structures and peptide-binding activities of variants that affect the BRCA1 BRCT phosphopeptide-binding groove. The results obtained from the G1656D and T1700A variants illustrate the role of Ser1655 in pSer recognition. Mutations at Arg1699 (R1699W and R1699Q) significantly reduce peptide binding through loss of contacts to the main chain of the Phe(+3) residue and, in the case of R1699W, to a destabilization of the BRCT fold. The R1835P and E1836K variants do not dramatically reduce peptide binding, in spite of the fact that these mutations significantly alter the structure of the walls of the Phe(+3) pocket.     

Structural basis of BACH1 phosphopeptide recognition by BRCA1 tandem BRCT domains

BRCT tandem domains, found in many proteins involved in DNA damage checkpoint and DNA repair pathways, were recently shown to be phosphopeptide binding motifs. Using solution nuclear magnetic resonance (NMR) spectroscopy and mutational analysis, we have characterized the interaction of BRCA1-BRCT domains with a phosphoserine-containing peptide derived from the DNA repair helicase BACH1. We show that a phenylalanine in the +3 position from the phosphoserine of BACH1 is bound to a conserved hydrophobic pocket formed between the two BRCT domains and that recognition of the phosphate group is mediated by lysine and serine side chains from the amino-terminal BRCT domain. Mutations that prevent phosphopeptide binding abolish BRCA1 function in DNA damage-induced checkpoint control. Our NMR data also reveal a dynamic interaction between BRCA1-BRCT and BACH1, where the bound phosphopeptide exists as an equilibrium of two conformations and where BRCA1-BRCT undergoes a transition to a more rigid conformation upon peptide binding.     

Crystal structure of a consensus-designed ankyrin repeat protein: implications for stability

Consensus-designed ankyrin repeat (AR) proteins are thermodynamically very stable. The structural analysis of the designed AR protein E3_5 revealed that this stability is due to a regular fold with highly conserved structural motifs and H-bonding networks. However, the designed AR protein E3_19 exhibits a significantly lower stability than E3_5 (9.6 vs. 14.8 kcal/mol), despite 88% sequence identity. To investigate the structural correlations of this stability difference between E3_5 and E3_19, we determined the crystal structure of E3_19 at 1.9 A resolution. E3_19 as well has a regular AR domain fold with the characteristic H-bonding patterns. All structural features of the E3_5 and E3_19 molecules appear to be virtually identical (RMSD(Calpha) approximately 0.7 A). However, clear differences are observed in the surface charge distribution of the two AR proteins. E3_19 features clusters of charged residues and more exposed hydrophobic residues than E3_5. The atomic coordinates of E3_19 have been deposited in the Protein Data Bank. PDB ID: 2BKG.     

乳腺癌易感蛋白BRCA1的BRCT2结构域染色质伸展活性的定位

40 %～ 5 0 %的遗传性乳腺癌和至少 80 %的既有乳腺癌又有卵巢癌家族史的患者是由BRCA1突变引起的 .BRCA1C末端含有 2个BRCT结构域 (BRCT1和BRCT2 ) ,它们与BRCA1的重要功能密切相关 .许多乳腺癌易感突变发生在BRCA1的BRCT结构域中 .利用染色质结构检测技术表明 ,BRCT结构域具有染色质伸展活性 .利用缺失突变技术构建了 6种BRCT2结构域 (175 6～ 185 2位氨基酸残基 )缺失突变体并将BRCT2结构域中与染色质伸展相关的重要区域定位到 175 6～ 180 8之间的氨基酸残基 ;用丙氨酸扫描技术构建了 6种BRCT2结构域丙氨酸扫描突变体并将重要氨基酸残基序列定位到 1784～ 1788之间的VQLCG .BRCT2结构域的定位有助于预测BRCT2结构域突变后发生乳腺癌的风险 ,也为进一步研究BRCT2结构域的功能机制提供了有用的材料 .