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.     

Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast

Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain-mediated and damage-specific BRCT domain-phospho-H2A-dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest.     

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.     

Supramodular nature of GRIP1 revealed by the structure of its PDZ12 tandem in complex with the carboxyl tail of Fras1

The scaffold protein GRIP1 (glutamate receptor interacting protein 1) binds to and regulates both the trafficking and membrane organization of a large number of transmembrane proteins. Mutation of GRIP1 in mice displays essentially the same phenotype of the mutations of Fras1 or Frem2, which are the animal models of the human genetic disorder Fraser syndrome. However, the molecular basis governing the interaction between GRIP1 and Fras1/Frem2 is unknown. Here, we show that interaction between Fras1 and GRIP1 requires the first two PDZ domains (PDZ1 and PDZ2) to be connected in tandem, as the folding of PDZ1 strictly depends on the covalent attachment of PDZ2. The crystal structure of GRIP1 PDZ12 in complex with the Fras1 C-terminal peptide reveals that the PDZ12 tandem forms a supramodule in which only the peptide-binding groove of PDZ1 is bound with the Fras1 peptide. The GRIP1 PDZ12/Fras1 peptide complex not only provides a mechanistic explanation of the link between GRIP1 and the Fraser syndrome but may also serve as a foundation for searching for potential mutations in GRIP1 that could lead to the Fraser syndrome.     

Structure of the BRCT repeat domain of MDC1 and its specificity for the free COOH-terminal end of the gamma-H2AX histone tail

MDC1 (mediator of DNA damage checkpoint protein 1) regulates the recognition and repair of DNA double strand breaks in mammalian cells through its interactions with nuclear foci containing the COOH-terminally phosphorylated form of the histone variant, H2AX. Here we demonstrate that the tandem BRCT repeats of MDC1 directly bind to the phosphorylated tail of H2AX-Ser(P)-Gln-Glu-Tyr, in a manner that is critically dependent on the free carboxylate group of the COOH-terminal Tyr residue. We have determined the x-ray crystal structure of the MDC1 BRCT repeats at 1.45 Angstroms resolution. By a comparison with the structure of the BRCA1 BRCT bound to a phosphopeptide, we suggest that two arginine residues in MDC1, Arg(1932) and Arg(1933) may recognize the COOH terminus of the peptide as well as the penultimate Glu of H2AX, while Gln(2013) may provide additional specificity for the COOH-terminal Tyr.     

Homo-oligomerization is the essential function of the tandem BRCT domains in the checkpoint protein Crb2

BRCT (BRCA1 C terminus) domains are frequently found as a tandem repeat in proteins involved in DNA damage responses, such as Saccharomyces cerevisiae Rad9, human 53BP1 and BRCA1. Tandem BRCT domains mediate protein-protein and protein-DNA interactions. However, the functional significance of these interactions is largely unknown. Here we report the oligomerization of Schizosaccharomyces pombe checkpoint protein Crb2 through its tandem BRCT domains. Truncated Crb2 without BRCT domains is defective in DNA damage checkpoint signaling. However, addition of either of two heterologous dimerization motifs largely restores the functions of truncated Crb2 without BRCT domains. Replacement of Crb2 BRCT domains with a dimerization motif also renders cells resistant to the dominant negative effect of overexpressing Crb2 BRCT domains. These results demonstrate that the crucial function of the tandem BRCT domains is to oligomerize Crb2.     

Structure and mechanism of BRCA1 BRCT domain recognition of phosphorylated BACH1 with implications for cancer

Germline mutations in the BRCA1 tumor suppressor gene often result in a significant increase in susceptibility to breast and ovarian cancers. Although the molecular basis of their effects remains largely obscure, many mutations are known to target the highly conserved C-terminal BRCT repeats that function as a phosphoserine/phosphothreonine-binding module. We report the X-ray crystal structure at a resolution of 1.85 A of the BRCA1 tandem BRCT domains in complex with a phosphorylated peptide representing the minimal interacting region of the DEAH-box helicase BACH1. The structure reveals the determinants of this novel class of BRCA1 binding events. We show that a subset of disease-linked mutations act through specific disruption of phospho-dependent BRCA1 interactions rather than through gross structural perturbation of the tandem BRCT domains.     

Hypertonic saline enhances expression of phosphorylated histone H2AX after irradiation

Phosphorylation of histone H2AX at serine 139 occurs at sites surrounding DNA double-strand breaks, producing discrete spots called "foci" that are visible with a microscope after antibody staining. This modification is believed to create changes in chromatin structure and assemble various repair proteins at sites of DNA damage. To examine the role of chromatin structure, human SiHa cells were exposed to hypertonic salt solutions that are known to condense chromatin and sensitize cells to chromosome damage and killing by ionizing radiation. Postirradiation incubation in 0.5 M Na(+) increased gammaH2AX expression about fourfold as measured by flow cytometry and immunoblotting, and loss of gammaH2AX was inhibited in the presence of high salt. Focus size rather than the number of radiation-induced gammaH2AX foci was also increased about fourfold. When high-salt treatment was delayed for 1 h after irradiation, effects on focus size and retention were reduced. The increase in focus size was associated with a decrease in the rate of rejoining of double-strand breaks as measured using the neutral comet assay. We conclude that gammaH2AX expression after irradiation is sensitive to salt-induced changes in chromatin structure during focus formation, and that a large focus size may be an indication of a reduced ability to repair DNA damage.     

Structural basis of adhesion-molecule recognition by ERM proteins revealed by the crystal structure of the radixin-ICAM-2 complex

ERM (ezrin/radixin/moesin) proteins recognize the cytoplasmic domains of adhesion molecules in the formation of the membrane-associated cytoskeleton. Here we report the crystal structure of the radixin FERM (4.1 and ERM) domain complexed with the ICAM-2 cytoplasmic peptide. The non-polar region of the ICAM-2 peptide contains the RxxTYxVxxA sequence motif to form a beta-strand followed by a short 3(10)-helix. It binds the groove of the phosphotyrosine-binding (PTB)-like subdomain C mediated by a beta-beta association and several side-chain interactions. The binding mode of the ICAM-2 peptide to the FERM domain is distinct from that of the NPxY motif-containing peptide binding to the canonical PTB domain. Mutation analyses based on the crystal structure reveal the determinant elements of recognition and provide the first insights into the physical link between adhesion molecules and ERM proteins.     

Oligomeric structure of the MALT1 tandem Ig-like domains

Background

Mucosa-associated lymphoid tissue 1 (MALT1) plays an important role in the adaptive immune program. During TCR- or BCR-induced NF-κB activation, MALT1 serves to mediate the activation of the IKK (IκB kinase) complex, which subsequently regulates the activation of NF-κB. Aggregation of MALT1 is important for E3 ligase activation and NF-κB signaling.

Principal Findings

Unlike the isolated CARD or paracaspase domains, which behave as monomers, the tandem Ig-like domains of MALT1 exists as a mixture of dimer and tetramer in solution. High-resolution structures reveals a protein-protein interface that is stabilized by a buried surface area of 1256 Ų and contains numerous hydrogen and salt bonds. In conjunction with a second interface, these interactions may represent the basis of MALT1 oligomerization.

Conclusions

The crystal structure of the tandem Ig-like domains reveals the oligomerization potential of MALT1 and a potential intermediate in the activation of the adaptive inflammatory pathway.

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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.     

NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain

DNA double-strand breaks represent the most potentially serious damage to a genome; hence, many repair proteins are recruited to nuclear damage sites by as yet poorly characterized sensor mechanisms. Here, we show that NBS1, the gene product defective in Nijmegen breakage syndrome (NBS), physically interacts with histone, rather than damaged DNA, by direct binding to gamma-H2AX. We also demonstrate that NBS1 binding can occur in the absence of interaction with hMRE11 or BRCA1. Furthermore, this NBS1 physical interaction was reduced when anti-gamma-H2AX antibody was introduced into normal cells and was also delayed in AT cells, which lack the kinase activity for phosphorylation of H2AX. NBS1 has no DNA binding region but carries a combination of the fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT). We show that the FHA/BRCT domain of NBS1 is essential for this physical interaction, since NBS1 lacking this domain failed to bind to gamma-H2AX in cells, and a recombinant FHA/BRCT domain alone can bind to recombinant gamma-H2AX. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for relocalization of hMRE11/hRAD50 nuclease complex to the vicinity of DNA damage.     

MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks

Histone variant H2AX phosphorylation in response to DNA damage is the major signal for recruitment of DNA-damage-response proteins to regions of damaged chromatin. Loss of H2AX causes radiosensitivity, genome instability, and DNA double-strand-break repair defects, yet the mechanisms underlying these phenotypes remain obscure. Here, we demonstrate that mammalian MDC1/NFBD1 directly binds to phospho-H2AX (gammaH2AX) by specifically interacting with the phosphoepitope at the gammaH2AX carboxyl terminus. Moreover, through a combination of biochemical, cell-biological, and X-ray crystallographic approaches, we reveal the molecular details of the MDC1/NFBD1-gammaH2AX complex. These data provide compelling evidence that the MDC1/NFBD1 BRCT repeat domain is the major mediator of gammaH2AX recognition following DNA damage. We further show that MDC1/NFBD1-gammaH2AX complex formation regulates H2AX phosphorylation and is required for normal radioresistance and efficient accumulation of DNA-damage-response proteins on damaged chromatin. Thus, binding of MDC1/NFBD1 to gammaH2AX plays a central role in the mammalian response to DNA damage.     

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.     

Structure of C-terminal tandem BRCT repeats of Rtt107 protein reveals critical role in interaction with phosphorylated histone H2A during DNA damage repair

Rtt107 (regulator of Ty1 transposition 107; Esc4) is a DNA repair protein from Saccharomyces cerevisiae that can restore stalled replication forks following DNA damage. There are six BRCT (BRCA1 C-terminal) domains in Rtt107 that act as binding sites for other recruited proteins during DNA repair. Several Rtt107 binding partners have been identified, including Slx4, Rtt101, Rad55, and the Smc5/6 (structural maintenance of chromosome) protein complex. Rtt107 can reportedly be recruited to chromatin in the presence of Rtt101 and Rtt109 upon DNA damage, but the chromatin-binding site of Rtt107 has not been identified. Here, we report our investigation of the interaction between phosphorylated histone H2A (γH2A) and the C-terminal tandem BRCT repeats (BRCT(5)-BRCT(6)) of Rtt107. The crystal structures of BRCT(5)-BRCT(6) alone and in a complex with γH2A reveal the molecular basis of the Rtt107-γH2A interaction. We used in vitro mutagenesis and a fluorescence polarization assay to confirm the location of the Rtt107 motif that is crucial for this interaction. In addition, these assays indicated that this interaction requires the phosphorylation of H2A. An in vivo phenotypic analysis in yeast demonstrated the critical role of BRCT(5)-BRCT(6) and its interaction with γH2A during the DNA damage response. Our results shed new light on the molecular mechanism by which Rtt107 is recruited to chromatin in response to stalled DNA replication forks.     

Specific recognition of HIV TAR RNA by the dsRNA binding domains (dsRBD1-dsRBD2) of PKR

PKR (double-stranded RNA-dependent protein kinase) is an important component of host defense to virus infection. Binding of dsRNA to two dsRBDs (double-stranded RNA binding domains) of PKR modulates its own kinase activation. How structural features of natural target RNAs, such as bulges and loops, have an effect on the binding to two dsRBDs of PKR still remains unclear. By using ITC and NMR, we show here that both the bulge and loop of TAR RNA are necessary for the high affinity binding to dsRBD1-dsRBD2 of PKR with 1:1 stoichiometry. The binding site for the dsRBD1-dsRBD2 spans from upper bulge to lower stem of the TAR RNA, based on chemical shift mapping. The backbone resonances in the 40 kDa TAR.dsRBD1-dsRBD2 were assigned. NMR chemical shift perturbation data suggest that the beta1-beta2 loop of the dsRBD1 interacts with the TAR RNA, whereas that of the dsRBD2 is less involved in the TAR RNA recognition. In addition, the residues of the interdomain linker between the dsRBD1 and the dsRBD2 also show large chemical perturbations indicating that the linker is involved in the recognition of TAR RNA. The results presented here provide the biophysical and spectroscopic basis for high-resolution structural studies, and show how local RNA structural features modulate recognition by dsRBDs.     

The tandem BRCT domains of Ect2 are required for both negative and positive regulation of Ect2 in cytokinesis

Epithelial cell transforming protein 2 (Ect2) is a guanine nucleotide exchange factor (GEF) for Rho GTPases, molecular switches essential for the control of cytokinesis in mammalian cells. Aside from the canonical Dbl homology/pleckstrin homology cassette found in virtually all Dbl family members, Ect2 contains N-terminal tandem BRCT domains. In this study, we address the role of the Ect2 BRCT domains in the regulation of Ect2 activity and cytokinesis. First, we show that the depletion of endogenous Ect2 by small interfering RNA induces multinucleation, suggesting that Ect2 is required for cytokinesis. In addition, we provide evidence that Ect2 normally exists in an inactive conformation, which is at least partially due to an intramolecular interaction between the BRCT domains and the C-terminal domain of Ect2. This intramolecular interaction masks the catalytic domain responsible for guanine nucleotide exchange toward RhoA. Consistent with a role in regulating Ect2 GEF activity, overexpression of an N-terminal Ect2 containing the tandem BRCT domains, but not single BRCT domain or BRCT domain mutant, leads to a failure in cytokinesis. Surprisingly, although ectopically expressed wild-type Ect2 rescues the multinucleation resulting from the depletion of endogenous Ect2, expression of a BRCT mutant of Ect2 failed to restore proper cytokinesis in these cells. Taken together, the results of our study indicate that the tandem BRCT domains of Ect2 play dual roles in the regulation of Ect2. Whereas these domains negatively regulate Ect2 GEF activity in interphase cells, they are also required for the proper function of Ect2 during cytokinesis.     

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'.