The EMBO Journal Vol.17 No.21 pp.6404–6411, 1998 Structure of an XRCC1 BRCT domain: a new protein–protein interaction module Xiaodong Zhang1, Solange More´ ra1,2, and cell cycle control (Koonin et al., 1996; Bork et al., Paul A.Bates3, Philip C.Whitehead4, 1997; Callebaut and Mornon, 1997). It was first identified Arnold I.Coffer4, Karl Hainbucher1,5, in the C-terminal region of the breast cancer suppressor Rachel A.Nash6, Michael J.E.Sternberg3, protein BRCA1 and was thus named BRCT domain Tomas Lindahl6 and Paul S.Freemont1,7 (Koonin et al., 1996). Two mammalian proteins which contain BRCT domains and have functions in DNA repair, 1Molecular Structure and Function, 3Biomolecular Modelling, and DNA ligase III and XRCC1 protein, bind each other 4Protein Isolation and Cloning Laboratories, Imperial Cancer Research strongly (Ljungquist et al., 1994; Caldecott et al., 1995) 6 Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX and ICRF Clare to form a heterodimer through specific interactions Hall Laboratories, South Mimms, Herts EN6 3LD, UK between their C-terminal BRCT domains (Nash et al., 2Present address: Laboratoire d’Enzymologie et de Biochimie 1997). Thus, the C-terminal stretch of 96 amino acids of Structurales, UPR9063 CNRS-Universite´ Paris-Sud, Baˆt. 34, 1 Avenue de la Terrasse, 91198-Gif-sur-Yvette, France XRCC1 is necessary and sufficient to bind DNA ligase 5Present address: Institut fu¨r Physikalische Chemie, Universita¨t Graz, III efficiently. A second, more N-terminal BRCT domain Heinrichstraße 28, 8010 Graz, Austria in XRCC1 interacts with poly(ADP-ribose) polymerase 7Corresponding author (Masson et al., 1998). Similar results have been obtained e-mail: [email protected] for the C-terminal BRCT region of DNA ligase IV, which X.Zhang and S.More´ra contributed equally to this work forms a heterodimer with the XRCC4 protein and is involved in DNA double-strand break repair (Critchlow The BRCT domain (BRCA1 C-terminus), first identi- et al., 1997). These data identify BRCT domains as fied in the breast cancer suppressor protein BRCA1, is protein–protein interaction entities, which can either bind an evolutionarily conserved protein–protein interaction different BRCT domains specifically or interact with other region of ~95 amino acids found in a large number of unknown protein folds. proteins involved in DNA repair, recombination and XRCC1 (633 amino acids) has no known enzymatic cell cycle control. Here we describe the first three- activity (Thompson et al., 1990) but apparently functions dimensional structure and fold of a BRCT domain as a scaffolding protein in the mammalian base excision- determined by X-ray crystallography at 3.2 Å reso- repair pathway. Thus, XRCC1 promotes the efficiency of lution. The structure has been obtained from the the repair process and serves to bring together DNA C-terminal region of the human DNA repair protein polymerase β and DNA ligase III, since these two enzymes XRCC1, and comprises a four-stranded parallel β-sheet do not interact directly (Kubota et al., 1996; Cappelli surrounded by three α-helices, which form an auto- et al., 1997). As observed for several proteins involved nomously folded domain. The compact XRCC1 struc- in the correction of abasic sites in DNA, deletion of the ture explains the observed sequence homology between XRCC1 gene in mice results in an embryonic lethal different BRCT motifs and provides a framework for phenotype (Tebbs et al., 1996). This is consistent with an modelling other BRCT domains. Furthermore, the essential role in removal of endogenous DNA damage. established structure of an XRCC1 BRCT homodimer However, two Chinese hamster ovary cell lines with suggests potential protein–protein interaction sites for mutations in XRCC1 have been isolated; these cells show the complementary BRCT domain in DNA ligase III, reduced ability to join single-strand breaks in DNA, with since these two domains form a stable heterodimeric concomitant cellular hypersensitivity to ionizing radiation complex. Based on the XRCC1 BRCT structure, we and alkylating agents (Thompson et al., 1990; Cappelli have constructed a model for the C-terminal BRCT et al., 1997; Shen et al., 1998). domain of BRCA1, which frequently is mutated in The BRCT domain was first identified by a database familial breast and ovarian cancer. The model allows search, comparing regions of the BRCA1 protein with insights into the effects of such mutations on the fold other available protein sequences (Koonin et al., 1996). of the BRCT domain. The BRCA1 gene encodes a 220 kDa nuclear phospho- Keywords: BRCA1/BRCT/protein–protein interaction/ protein in which structural changes confer susceptibility X-ray crystallography/XRCC1 to familial breast and ovarian cancer (Miki et al., 1994). Thus, inherited mutations associated with loss of activity of BRCA1 result in a greatly increased risk of women Introduction developing breast cancer (Easton, 1997). The detailed molecular functions of BRCA1 are unknown, but recently The BRCT domain is defined by distinct hydrophobic BRCA1 has been implicated in transcriptional regulation clusters of amino acids and is believed to occur as an and DNA repair (reviewed in Bertwistle and Ashworth, autonomous folding unit of ~95 amino acids. This domain 1998). Specifically, the C-terminal region of BRCA1 is found in proteins involved in DNA repair, recombination containing two BRCT domains acts as a transcriptional 6404 © Oxford University Press Crystal structure of an XRCC1 BRCT domain Table I. Crystallographic statistics a Diffraction Resolution (Å) No. of unique Redundancy I/σ Completeness Rsym Mosaicity data sets reflections (%) (outer shell) (°) Sel-Met 30–3.2 7526 4.7 6.2 99.5 7.2 (28.6) 0.2 Sel-Met 1 Pt 30–3.5 5570 10.5 7.9 100 12.7 (30.2) 0.6 Phasing statistics b c Sites Rcullis Phasing powers SIR FOM FOM after DM (centric/acentric) (centric/acentric) (20–3.5 Å)d (20–3.2 Å)e 2 0.66/0.63 1.44/1.65 0.285 0.81 Refinement statistics f Resolution No. of reflections Atoms Rcryst (%) Rfree (%) r.m.s. bond r.m.s.bond 2σ cutoff (work/free) length (Å) angle (°) 15–3.2 Å 6209/712 808 21.7 26.5 0.012 1.684 a Rsym 5 ΣiΣi|Ii – ,I.|/Σ,I., where Ii and ,I. are the observed and average intensity. |F –|F 1F || b PH P H Rcullis 5 , where FPH and FP are the derivative and native protein structure factors. |FPH – FP | F c P Phasing powers 5 〈〉, where FH is the calculated heavy-atom structure factor. FPH –|FP1FH| dSIR FOM: single isomorphous replacement figure of merit. eFOM after DM: figure of merit after density modifications using real space 2-fold NCS averaging and histogram matching. f10% of the data at 3.2 Å were set aside for free R-factor calculation. activation region in reporter assay systems (Chapman C-terminal BRCT domain to predict the structural con- and Verma, 1996). BRCA1 co-purifies with the RNA sequences of cancer-predisposing mutations found within polymerase II holoenzyme complex (Scully et al., 1997a), the domain. and a direct interaction between the RNA helicase A component of the holoenzyme and BRCA1 has been Results and discussion detected (Anderson et al., 1998). Moreover, BRCA1 is phosphorylated in response to DNA damage and associates The structure of the XRCC1 C-terminal BRCT with hRAD51 (Scully et al., 1997b), the human homologue domain of the Escherichia coli RecA protein, which plays a key The tertiary structure of the C-terminal BRCT domain role in homologous recombination and post-replication from human XRCC1 (residues 538–633; 96 residues) was repair. Recent data indicate that BRCA1 co-immuno- solved by X-ray crystallography to 3.2 Å resolution (see precipitates with p53, and can regulate p53-mediated gene Table I and Materials and methods for details), using expression with an absolute requirement for the most phases obtained from single isomorphous replacement, C-terminal BRCT domain, suggesting that BRCA1 func- further improved through density averaging, and refined tions as a coactivator of p53 (Ouchi et al., 1998). A to a free R-factor of 26.5% (Table I). The relatively large unique involvement of the second of two C-terminal unit cell and highly hydrated nature of the crystals obtained BRCT domains in heterodimer formation and tight binding were limiting factors in the resolution of the data. However, of its protein partner has also been observed for DNA ligase the final electron density map improved by 2-fold non- IV (Herrmann et al., 1998). Many different mutations in crystallographic-symmetry averaging and solvent flat- BRCA1 have been described [see the Breast Cancer tening showed clear density for main chain and most side Information Core (BIC) databases on the World Wide chains. This allowed an unambiguous trace of a model Web: http://www.nhgri.nih.gov/Intramural_research/Lab_ containing residues 538–633. The structure forms a com- transfer/Bic], the positions of which have been correlated pact globular α/β domain (~36 Å326 Å323 Å) consisting with a predisposition to breast and/or ovarian cancer of a four-stranded parallel β-sheet (with strand order (Gayther et al., 1995). β2β1β3β4) surrounded by three α-helices (α1-α3; Figure Here we report the first three-dimensional structure and 1A). The overall topology is β1α1β2β3α2β4α3, with two fold of a BRCT domain. This provides a structural basis α-helices (α1 and α3) on one side of the β-sheet and the to explain the observed sequence conservation of the third helix (α2) on the other (Figure 1A). The β-sheet BRCT family as well as giving insights into the function forms the core of the structure, with helix α1 forming of the XRCC1 BRCT domain in terms of specific protein– hydrophobic interactions with residues from β1 and β2.