Nature Structural Biology
8, 822 - 824 (2001)
doi:10.1038/nsb1001-822
With the ends in sight: images from the BRCA1 tumor suppressorRichard BaerRaichard Baer is at the Institute of Cancer Genetics, College of Physicians & Surgeons, Columbia University, New York, New York 10032, USA. rb670@columbia.edu Structures of two regions of BRCA1 offer important insights into the molecular properties of this tumor suppressor.Since its isolation in 1994 (ref. 1), the BRCA1 tumor suppressor has been implicated in a remarkable range of cellular processes, including DNA repair, cell cycle checkpoint control, and RNA transcription (see refs 2, 3 for recent reviews). Nevertheless, the molecular mechanisms by which BRCA1 affects these processes remain obscure, and it is still unclear why inherited mutations in the BRCA1 gene predispose women to breast and ovarian cancers. The major product of BRCA1 is a nuclear protein of 1,863 residues that contains two recognizable amino acid motifs: a RING domain at the N-terminus and two tandem copies of the BRCT domain at the C-terminus (Fig. 1a)1. Unlike most tumor suppressors, BRCA1 is very poorly conserved. True orthologs of human BRCA1 have been found only in vertebrates, and the primary amino acid sequence of BRCA1 has drifted considerably with evolution, such that the mouse and human polypeptides share only  60% sequence identity. However, sequence conservation is particularly high in the terminal regions that encompass the RING and BRCT motifs. Moreover, the missense mutations seen in BRCA1-linked breast cancers usually affect residues within these same regions, implying that the RING or BRCT sequences are specifically involved in BRCA1-mediated tumor suppression. The physical structures of these motifs are now presented in two papers in this issue of Nature Structural Biology4,
5. The new data offer important insights into the molecular properties of BRCA1, and by uncovering novel modes of protein interaction, they also have broader implications for the large families of eukaryotic polypeptides harboring RING or BRCT motifs.
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The BRCA1−BARD1 heterodimer
In living cells, BRCA1 exists as a heterodimeric complex with the BRCA1-associated RING domain (BARD1) protein, which resembles BRCA1 in that it also contains an N-terminal RING domain and two C-terminal BRCT motifs (Fig. 1a)6,
7. The in vivo interaction between BRCA1 and BARD1 is mediated by sequences that encompass their respective RING domains and is disrupted by tumor-associated missense mutations in the RING motif of BRCA1 (ref. 6). Although homodimeric interactions between BRCA1 and BARD1 have been reported based on in vitro data8, only heteromeric complexes have been observed in vivo6, suggesting that the BRCA1−BARD1 heterodimer represents the physiologically relevant form of BRCA1.
The RING motif is quite common among eukaryotes: the genome of budding yeast specifies 35 different RING proteins, whereas the human genome potentially encodes more than 200. The motif itself consists of 50−60 amino acids, including four pairs of conserved metal binding residues — mostly cysteines and histidines — that coordinate two zinc ions9. Recent advances in the field of protein ubiquitination reveal that the RING domain is an essential component of many ubiquitin E3 ligases, a large group of enzymatic factors that catalyze the final steps of protein ubiquitination10. Thus, although RING proteins appear to be functionally diverse, they may share a common enzymatic property. Indeed, the RING sequences of both BRCA1 and BARD1 have been shown to ubiquitinate model substrates in vitro, and this activity is ablated by tumor-associated missense mutations in the BRCA1 motif11,
12,
13. Significantly, the E3 ligase activity of the BRCA1−BARD1 heterodimer is dramatically higher than those of the isolated RING domains from either BRCA1 or BARD1 (ref. 11). These important studies have uncovered the first catalytic function for the BRCA1 tumor suppressor, and they provide further evidence that the BRCA1−BARD1 complex is the natural mediator of BRCA1 action.
The N-terminal end
On page 833, Brzovic et al.4 describe the solution structure of a heterodimeric complex formed by the N-terminal segments of BRCA1 (residues 1−103) and BARD1 (residues 26−122). Within this complex, the core structures (that is, the zinc-binding sequences) of the two RING domains are largely homologous to those of other RING proteins. Thus, the RING sequences of BRCA1 form a central  -helix, a short  -sheet of three antiparallel -strands, and two extended loops containing the first and last pairs of metal-binding residues. This resembles the structurally characterized RING domains of the viral IEEHV polypeptide, the Cbl oncoprotein, and the RAG1 recombination activating polypeptide14,
15,
16. The BARD1 sequence folds into a similar structure but lacks the central -helix, as does the RING domain of the promyelocytic leukemia (PML) oncoprotein17. What is striking about the BRCA1−BARD1 complex is the manner in which the two domains interact (Fig. 1b). The core element of each RING motif is flanked by long -helices that pair in an antiparallel fashion. Heterodimerization is achieved by combining the paired helices of BRCA1 and BARD1 into a stable four-helix bundle, an arrangement that places the core elements of the two RING domains in direct apposition to one another (Fig. 1b). Although the flanking helical sequences of the domains are tightly packed within the bundle, there are few direct contacts between the two juxtaposed zinc-binding cores of BRCA1 and BARD1.
Although RING proteins are numerous, only a few have been shown to interact through their respective motifs to form a dimeric complex. One of these, a homodimer of RAG1, has been analyzed structurally15. RAG1 dimerization is also mediated by helical sequences that flank the core elements of the two interacting RING domains. In this case, however, the flanking helices interact in an orthogonal fashion that is quite distinct from the antiparallel alignment of helices within the four-helix bundle of the BRCA1−BARD1 heterodimer. As a result, the zinc binding elements of the two RING domains are placed distal to one another within the RAG homodimeric complex. Thus, there are now at least two modes of RING−RING dimerization (shown in Fig. 2 of Brzovic et al.4). It will be intriguing to see whether these are also employed by other RING protein dimers. Cancer biologists will be especially keen to see the RING heterodimer formed by MDM2, an E3 ligase responsible for ubiquitination of the p53 tumor suppressor, and its paralog MDMX.
The catalytic mechanism by which RING domains promote ubiquitination is not known10. Consequently, the BRCA1−BARD1 structure raises some important issues that may be difficult to approach at this juncture. In particular, why does dimerization enhance the enzymatic activity of BRCA1 and BARD1 so dramatically? Do the closely positioned RING domains collaborate in the ubiquitin transfer reaction? Likewise, does the distal arrangement of zinc-binding cores within the RAG homodimeric complex influence its enzymatic properties (if indeed this complex also functions as an E3 ligase)? Crucial insights into the mechanism of BRCA1-mediated tumor suppression should emerge from further characterization of the BRCA1−BARD1 complex, and especially from the identification of its natural enzymatic substrates.
The C-terminal end
On page 838, Williams et al.5 present the crystal structure of the two C-terminal BRCT domains of BRCA1 (residues 1646−1859). The BRCT motif usually consists of 90−100 amino acids and is found in many eukaryotic proteins, either as an isolated domain or in tandem repeats of two or more BRCT units18,
19. Although most BRCT proteins are involved in the cellular response to genotoxic stress, a common biochemical function for this motif has not been identified. Nevertheless, tumor-associated missense mutations in the BRCT motifs of BRCA1 predispose carriers to familial breast/ovarian cancer, indicating that these sequences are essential for BRCA1-mediated tumor suppression.
Crystal structures of two other BRCT domains have been reported: an isolated motif from a bacterial NAD-dependent DNA ligase20 and the second (C-terminal) of two tandem BRCT motifs at the C-terminus of XRCC1, a human protein required for single-strand DNA break repair21. The central structures of the BRCA1 motifs presented by Williams et al.5 resemble the published BRCT structures; each forms a four stranded parallel -sheet flanked on one side by two -helices (1 and 3) and on the other by a third helix (2). A unique feature of the new structure is that the two tandem motifs of BRCA1 associate with one another in a head-to-tail manner to form a composite domain that is resistant to limited proteolysis (Fig. 1c). Significantly, two of the best-characterized missense mutations of BRCA1 involve residues at the hydrophobic interface between the two BRCT motifs, and the authors show that these tumor-associated mutations reduce the proteolytic stability of the composite BRCT domain.
It is interesting to note that the mode of association between the two BRCT domains of BRCA1 — a head-to-tail interaction between the 2 helix of the first motif and the 1' and 3' helices of the second (Fig. 1c) — is distinct from that proposed for the interchain BRCT dimer formed by XRCC1 and DNA ligase III (head-to-head 1/1' packing)22. This has important implications for other proteins that use the BRCT motif as an interaction surface. Huyten et al.19 recently noted that the internal linker between tandem BRCT motifs would disrupt 1/1' packing and suggested that the interchain and intrachain modes of BRCT dimerization might be different. Williams et al. now provide the first experimental evidence that tandem BRCT motifs can in fact assemble into a stable structural unit in cis, and they show that this is achieved by a novel mode of BRCT dimerization5. Based on primary sequence similarities, they propose that the tandem repeats of other BRCT proteins may also pack together in a head-to-tail 2/1'3' fashion. Given that BRCA1 exists as a heterodimer with BARD1 in vivo, it will be intriguing to see whether the two BRCT motifs of BARD1 associate with one another in an analogous manner, and whether there are intermolecular interactions between the BRCT domains of BRCA1 and BARD1.
In the absence of an apparent enzymatic function, the BRCT motif is often viewed as a protein interaction surface, and a growing number of cellular factors have already been shown to associate with the BRCT sequences of BRCA1 (ref. 2). Clearly, these factors — as well as proteins that bind other regions of the BRCA1−BARD1 heterodimer−may regulate the enzymatic properties of the RING domains, or may even serve as physiological substrates of BRCA1−BARD1-dependent ubiquitination. As a common form of posttranslational modification that is in some ways akin to protein phosphorylation, ubiquitination has been exploited by the cell as a regulatory signal to control many different biological processes10. Given the versatile nature of this signal, the various functions now attributed to BRCA1 may well be mediated by the E3 ligase activity of the BRCA1−BARD1 heterodimer. Needed insights into the mechanism of BRCA1-mediated tumor suppression should emerge from further structural studies of the BRCA1−BARD1 complex, its associated proteins and the catalytic properties of its RING motifs.
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