Article

pubs.acs.org/biochemistry

Structural Basis for the BRCA1 BRCT Interaction with the Proteins
ATRIP and BAAT1
Xuying Liu and John A. A. Ladias*
Molecular Medicine Laboratory and Macromolecular Crystallography Unit, Department of Medicine, Harvard Medical School,
Boston Massachusetts 02215, United States

ABSTRACT: The breast and ovarian cancer susceptibility protein 1 (BRCA1) plays a
central role in DNA damage response (DDR). Two tandem BRCA1 C-terminal
(BRCT) domains interact with several proteins that function in DDR and contain the
generally accepted motif pS-X-X-F (pS denoting phosphoserine and X any amino acid),
including the ATR-interacting protein (ATRIP) and the BRCA1-associated protein
required for ATM activation-1 (BAAT1). The crystal structures of the BRCA1 BRCTs
bound to the phosphopeptides ATRIP (235−PEACpSPQFG−243) and BAAT1 (266−
VARpSPVFSS−274) were determined at 1.75 Å and 2.2 Å resolution, respectively. The
pSer and Phe(+3) anchor the phosphopeptides into the BRCT binding groove, with
adjacent peptide residues contributing to the interaction. In the BRCA1−ATRIP
structure, Gln(+2) is accommodated through a conformational change of the BRCA1
E1698 side chain. Importantly, isothermal titration calorimetry experiments showed that
the size and charge of the side chains at peptide positions +1 and +2 contribute
significantly to the BRCA1 BRCT−peptide binding affinity. In particular, the Asp(+1) and Glu(+2) in the human CDC27
peptide 816−HAAEpSDEF−823 abrogate the interaction with the BRCA1 BRCTs due in large part to electrostatic repulsion
between Glu(+2) and E1698, indicating a preference of these domains for specific side chains at positions +1 and +2. These
results emphasize the need for a systematic assessment of the contribution of the peptide residues surrounding pSer and Phe(+3)
to the binding affinity and specificity of the BRCA1 BRCTs in order to elucidate the molecular mechanisms underlying the
hierarchy of target selection by these versatile domains during DDR and tumorigenesis.

T he breast and ovarian cancer susceptibility gene 1 (BRCA1)

encodes a protein that plays a central role in genomic
stability maintenance through its function in a multifaceted
leading to catalytic activation of the kinase, which subsequently
phosphorylates a plethora of proteins participating in DDR.
ATM is recruited at the DSB sites by the Mre11−Rad50−Nbs1
signaling network that involves cell cycle checkpoint control and (MRN) complex, where it phosphorylates histone H2AX at
DNA double-strand break (DSB) repair pathways, collectively serine 139, which is referred to as γH2AX. The γH2AX is
referred to as the DNA damage response (DDR).1−7 Germline recognized by the BRCT domains of the mediator of DNA
mutations in the BRCA1 gene account for the majority of familial damage checkpoint 1 (MDC1), which facilitates recruitment of
breast and ovarian cancers.1−4 The 1,863-residue BRCA1 protein ATM and ATR to propagate γH2AX foci formation. ATR
forms a stable heterodimer with its constitutive interacting responds to DNA replication stress and is recruited to sites of
partner BARD1 that has an essential function in the maintenance stalled DNA replication forks via its obligate partner ATR-
of genomic integrity. The N-terminal region of BRCA1 contains interacting protein (ATRIP), which binds to replication protein
a RING finger that heterodimerizes with the N-terminal BARD1 A (RPA)-coated single-stranded DNA (ssDNA). In response to
RING finger, forming an E3 ubiquitin ligase.1−4 The C-terminal DNA damage, BRCA1 is phosphorylated by ATM, ATR, and
region of BRCA1 contains two tandem BRCT (BRCA1 C- CHK2 and organizes multiple protein complexes that activate
terminal) domains that interact in a phosphorylation-dependent cell cycle checkpoints, DSB repair by homologous recombina-
manner with a number of proteins involved in cell cycle tion (HR),3−5 and repair of DNA interstrand cross-links
checkpoint control and DNA repair.8−13 Mutagenesis or deletion independently of HR.22 Specifically, the BRCA1 BRCT domains
of the BRCT domains predisposes to cancer and suppresses the interact with Abraxas/CCDC98, BACH1/BRIP1, and CtIP,
ability of BRCA1 to inhibit cancer cell growth, indicating that nucleating the assembly of three distinct complexes with specific
these domains are essential for the tumor suppressor activity of functions, designated A, B, and C, respectively.3,4,11 The
BRCA1.1−4,11−16 BRCA1−A complex contains the proteins Abraxas, BRCC36,
DSBs induced by ionizing radiation (IR), radiomimetic BRCC45/BRE, NBA1/MERIT40, and RAP80 and is involved in
chemicals, and endogenous oxygen radicals activate the DDR G2−M checkpoint control. The BRCA1−B complex is made up
signaling pathway.6,7 The ataxia telangiectasia mutated (ATM)
and ataxia telangiectasia and Rad3 related (ATR) protein kinases Received: June 6, 2013
are among the first responders to DNA damage that initiate the Revised: September 21, 2013
DDR.17−21 IR induces autophosphorylation of ATM at Ser1981

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Article

of BACH1 and TopBP1 and controls the DNA damage-induced EXPERIMENTAL PROCEDURES
G2 accumulation checkpoint. The BRCA1−C complex is
Protein Purification and Crystallization. The human
composed of CtIP and the MRN complex and is formed in a
BRCA1 BRCT protein (residues 1646−1859) was expressed in
cell cycle-dependent manner during S and G2 phases of the cell
Escherichia coli BL21(DE3) cells as a glutathione S-transferase
cycle. BRCA1−C is involved in DNA end resection to generate
(GST) fusion and was purified as described previously.30 Briefly,
ssDNA needed for HR-mediated DNA repair. In addition to
bacterial cells carrying the BRCA1(1646−1859)−pGEX-KT
these well-studied complexes, the BRCA1 BRCTs also interact
plasmid were grown at 37 °C, induced by the addition of 0.3 mM
with ATRIP23 and the BRCA1-associated protein required for
IPTG when Abs600 reached 0.6−0.8, and incubated for 15 h at 22
ATM activation 1 (BAAT1), 24 also known as BRAT1.
°C. Cells were harvested by centrifugation and resuspended in 50
Phosphorylation of Ser239 in human ATRIP is recognized
mM Tris-Cl (pH 7.5), 0.5 M NaCl, 5 mM DTT, 0.2% Triton X-
specifically by the BRCA1 BRCT domains, whereas a S239A
100, 0.5 mg/mL lysozyme, and protease inhibitor cocktail tablets
substitution abrogates the BRCA1 binding to ATRIP and leads
to a G2−M checkpoint defect, indicating that this interaction is (Roche). The cells were lysed by three freeze−thaw cycles, and
essential for the ATR function in checkpoint control.23 BAAT1 soluble proteins were collected by centrifugation at 11000g at 4
localizes to DSBs, associates with ATM, and is required for ATM °C. The supernatant was applied to a glutathione−Sepharose
autophosphorylation at Ser1981, suggesting that a coordinated column (GE Healthcare), and the BRCA1(1646−1859) moiety
assembly of BRCA1, BAAT1, and ATM is critical for DSB- was released with thrombin digestion and further purified by size
induced ATM activation.24 Importantly, mutations in the exclusion chromatography on Superdex 75 (GE Healthcare).
BAAT1/BRAT1 gene have been recently associated with the The purified protein was concentrated to 20 mg/mL by
lethal neonatal rigidity and multifocal seizure syndrome ultrafiltration (Millipore).
(RMFSL), a lethal neurologic disorder characterized by episodic Crystallization of the BRCT−Phosphopeptide Com-
jerking in utero, lack of psychomotor development, axial and limb plexes. The BRCA1 BRCT protein (20 mg/mL) was incubated
rigidity, and multifocal seizures, suggesting a possible role of the with either ATRIP (PEACpSPQFG) or BAAT1
BAAT1-associated DNA damage repair in neural develop- (VARpSPVFSS) peptides at a 1:1.2 molar ratio for 1 h on ice,
ment.25,26 To date, the BAAT1 region that mediates the and the resulting complexes were crystallized by the hanging-
interaction with the BRCA1 BRCTs has not been reported. drop vapor diffusion method at 20 °C. Crystals of the BRCT−
A number of crystal and solution structures of the BRCA1 ATRIP complex were obtained in 0.2 M ammonium acetate, 0.1
BRCT domains bound to phosphopeptides from BACH1,27−29 M HEPES (pH 7.5), and 17−19% (w/v) PEG 3350. The crystals
CtIP,30 ACC1,31 and high-affinity peptides32,33 showed that were cryoprotected by the addition of 25% (v/v) glycerol prior to
these domains recognize the consensus sequence pS-X-X-F (pS flash-cooling in liquid nitrogen. The BRCT−BAAT1 complex
denoting phosphoserine and X any residue) in a two-pronged was crystallized in 1 M lithium chloride, 0.1 M sodium acetate
mode, with pSer and Phe(+3) inserting into two pockets, P1 and (pH 4.8), and 30% (w/v) PEG 6000, and the crystals were frozen
P2, respectively, and providing the main interactions with in liquid nitrogen.
BRCA1 residues. In addition, these structures revealed how Data Collection and Processing. X-ray diffraction data sets
certain cancer-associated mutations affect the ability of the of the BRCT−ATRIP crystals were collected at the NE-CAT 24-
BRCTs to interact with target proteins, leading to major defects ID-E beamline at the Advanced Photon Source (Argonne
in the DDR pathway. For example, the tumor-associated National Laboratory, Argonne, IL). Data were indexed, scaled,
mutations M1775R and M1775K abrogate the interaction of and integrated using HKL2000.36 The crystals belong to space
the BRCA1 BRCT repeats with their targets, and the structural group P2 with unit cell dimensions a = 65.666 Å, b = 50.309 Å, c =
analysis of these domains have provided the molecular 73.977 Å, α = γ = 90°, and β = 116.03°. Diffraction data sets of the
explanation for how this is achieved at the atomic BRCT−BAAT1 crystals were collected at the X25 beamline at
level.27,32,34,35 However, the structural mechanisms underlying the National Synchrotron Light Source (Brookhaven National
the hierarchy of target selection by the BRCA1 BRCTs, which is Laboratory, Upton, NY). Data were indexed, scaled, and
dictated by the sequence variation around the anchoring residues integrated using HKL2000. These crystals belong to space
pSer and Phe(+3), are not well characterized. group P3221 with unit cell dimensions a = b = 65.22 Å, c = 91.356
Here, we present the molecular basis for the BRCA1 BRCT Å, α = β = 90°, and γ = 120°.
interaction with the proteins ATRIP and BAAT1. Isothermal Structure Determination and Refinement. The BRCT−
titration calorimetry (ITC) experiments showed that these ATRIP and BRCT−BAAT1 structures were determined by
domains interact with the phosphopeptides PEACpSPQFG and molecular replacement using PHASER37 and the BRCA1
VARpSPVFSS, corresponding to residues 235−243 and 266− BRCT−CtIP structure [Protein Data Bank (PDB) entry 1Y98]
274 of human ATRIP and BAAT1, respectively. The crystal with the CtIP peptide removed, as the search model. The
structures of the BRCA1 BRCT repeats bound to the ATRIP and BRCT−ATRIP structure was refined by a combination of
BAAT1 phosphopeptides reveal how these domains accom- restrained refinement, three groups of TLS refinement, and
modate the side chains of Gln(+2) and Val(+2), respectively, and noncrystallographic symmetry in REFMAC5,38 as implemented
how residues surrounding the pSer and Phe(+3) contribute to in the CCP4 suite.39 Manual model correction and ligand
the interaction. Unexpectedly, ITC experiments showed that the building in COOT40 yielded a complete model with the
BRCA1 BRCTs do not interact with a peptide carrying Asp(+1) exception of residues 1816−1818 whose density is missing.
and Glu(+2), indicating a preference of these domains for The crystallized BRCT protein includes the vector-derived
specific side chains at positions +1 and +2. These results point to residues GS at its N terminus, which are disordered. For the
the need for a redefinition of the BRCA1 BRCT recognition BRCT−BAAT1 structure, refinement was done with rigid body
motif pS-X-X-F through a precise determination of the refinement using REFMAC, followed by restrained refinement
contribution of the residues surrounding pSer and Phe(+3) to with REFMAC and manual model building using COOT. Water
the binding affinity and specificity. molecules were built manually. The data collection and structure
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Biochemistry Article

determination statistics for each complex are shown in Table 1. located on a flexible loop was modeled as Ala because of poor
In the BRCT−ATRIP structure, F1798, L1800, and T1802 are electron density. The A1817 and Y1845 also fall into the
disallowed region of the Ramachandran plot, although the
Table 1. Data Collection and Refinement Statistics modeled conformation of Y1845 is supported by clear electron
density.
ATRIP BAAT1
Isothermal Titration Calorimetry. Binding constants for
resolution (Å)a 50−1.75 (1.81−1.75) 50−2.20 (2.28−2.20) the BRCA1 BRCTs interaction with various phosphopeptides
observed reflections 158123 108750 were measured using a VP-ITC microcalorimeter (MicroCal).
unique reflections 43821 11813 Briefly, each synthetic phosphopeptide was titrated against the
completeness (%) 99.5 (100.0) 99.5 (99.0) purified BRCA1 BRCT protein in PBS (137 mM NaCl, 2.7 mM
Rsym (%)b 5.9 (17.8) 7.3 (53.3) KCl, 10 mM Na2HPO4, and 2 mM KH2PO4 at pH 7.4) at 20 °C.
overall ⟨I/σ(I)⟩ 18.2 (7.5) 31.2 (3.3) Each protein sample was dialyzed against PBS overnight at 4 °C,
Rcryst (%)c 15.9 19.7 followed by further dialysis against fresh PBS for 2 h. Dilution
Rfree (%)d 20.5 26.0 curves of the background heat were obtained by titrating the
bond lengthe (Å) 0.020 0.014 peptides into PBS under similar conditions and were subtracted
bond anglee (deg) 1.980 1.705 from the protein−peptide curves to determine the corrected heat
average B value (Å2) 22.37 39.32 for each reaction. Each experiment was repeated at least twice.
Ramachandran plot Protein and peptide concentration was determined by
(%)
quantitative amino acid analysis on an ABI 420A derivatizer/
preferred 95.9 95.7
analyzer and an ABI 130A separation system (Applied
allowed 3.3 3.3
disallowed 0.7 0.9
Biosystems), where extinction coefficients were determined
based on amino acid analysis. The integrated heat data were fit
a
Values in parentheses are for the highest resolution shell. bRsym = Σ|(I with a one-site binding model using the program ORIGIN 7.0
− ⟨I⟩)|/Σ(I), where I is the observed integrated intensity, ⟨I⟩ is the
(OriginLab).

■
average integrated intensity obtained from multiple measurements,
and the summation is over all observed reflections. cRcryst = Σ||Fo| − k|
Fc||/Σ|Fo|, where Fo and Fc are the observed and calculated structure RESULTS AND DISCUSSION
factors, respectively. dRfree is calculated as Rcryst using 5% of the BRCA1 BRCT Domains Interact with the ATRIP−
reflections chosen randomly and omitted from the refinement pSer239 and BAAT1−pSer269 Phosphopeptides. To
calculations. eBond lengths and angles are rms deviations from ideal study the in vitro physical interactions of the BRCA1 BRCTs
values. with ATRIP and BAAT1, we performed ITC experiments. The
ATRIP phosphopeptide PEACpSPQFG corresponding to
located on a flexible loop and are in the disallowed region of the residues 235−243 of human ATRIP (NCBI entry
Ramachandran plot. In the BRCT−BAAT1 structure, E1817 NP_569055) and carrying the phospho-Ser239 interacted with

Figure 1. Interaction of the BRCA1 BRCT domains with ATRIP and BAAT1 phosphopeptides. (A to D) Representative ITC results obtained for the
BRCA1 BRCT interaction with the phosphopeptides ATRIP, BAAT(pS156), BAAT(pS269), and BAAT(pS604), respectively. For the isotherms in
panels A−D, the BRCA1 BRCT concentration was 56.7 μM, 56.7 μM, 22.7 μM, and 33.3 μM, whereas the peptide concentration was 1.006 mM, 0.375
mM, 0.36 mM, and 0.683 mM, respectively. (E) Thermodynamic parameters of the BRCA1 BRCT interaction with the shown peptides. ND: not
determined.

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Figure 2. Structure of the BRCA1 BRCT−ATRIP complex. (A) Ribbon representation of the BRCA1 BRCTs (wheat) bound to the ATRIP peptide
(green stick model). The α helices and β strands of the BRCT1 and BRCT2 repeats are labeled (those of BRCT2 are labeled with primes). The pSer and
Phe(+3) are denoted. (B) A weighted 2Fo − Fc electron density map enveloping the peptide and two conserved water molecules (W1 and W2)
calculated at 1.75 Å resolution and contoured at 1.2σ. The figure was created using PyMOL (www.pymol.org). (C) Two-dimensional representation of
the interactions between BRCA1 (orange) and ATRIP (purple) residues. Water molecules (W) are shown as cyan spheres, hydrogen bonds as dashed
lines, and hydrophobic interactions as arcs with radial spokes. The figure was created using LIGPLOT+.50

the BRCA1 BRCT domains with a Kd of 28.2 μM (Figures 1A BRCT interaction with the ATRIP phosphopeptide, we
and E), in agreement with a previous report.23 The human determined the crystal structure of this complex at 1.75 Å
BAAT1 protein sequence (NCBI entry NP_689956) contains resolution. As described previously, the BRCA1(1646−1859)
three regions around serine residues 156, 269, and 604 that have region forms two tandem domains, BRCT1 and BRCT2, each
the motif S-X-X-F, which is recognized by the BRCA1 BRCT consisting of four parallel β strands (β1 to β4) flanked by two α
domains when serine is phosphorylated.2,5,14,35 To identify the helices (α1 and α3) on one side and a single α helix (α2) on the
BAAT1 region that interacts with the BRCA1 BRCTs, we other (Figure 2A). A head-to-tail packing of BRCT1 and BRCT2
performed ITC experiments with the phosphopeptides BAAT- buries a large hydrophobic interface and creates a surface groove
(pS156), BAAT(pS269), and BAAT(pS604) having the with two pockets, P1 and P2. The ATRIP peptide inserts into this
sequences LQGDpSSLFVA, VARpSPVFSS, and groove with phospho-Ser239 and Phe242 (+3) entering the P1
LSVDpSEGFPR, corresponding to human BAAT1 residues and P2 pockets, respectively, and forming the main interactions
152−161, 266−274, and 600−609, respectively. The peptide with the BRCT1 and BRCT2 modules (Figure 2B and C). The
BAAT(pS156) did not interact strongly with the BRCA1 BRCT present structure is consistent with previous biochemical and
protein, and the Kd value could not be determined accurately functional studies on the importance of phospho-Ser239 for the
(Figure 1B and E). By contrast, BAAT(pS269) interacted with BRCA1−ATRIP association.23 The O1P atom of pSer forms a
the BRCA1 BRCTs with a Kd of 3.3 μM (Figures 1C and E). hydrogen bond with the Nζ of K1702, O2P hydrogen bonds with
BAAT(pS604) also failed to interact strongly with these domains the amide nitrogen of G1656, and O3P forms hydrogen bonds
(Figure 1D and E). Although the role of phospho-Ser269 in the directly with the Oγ of S1655 and through a water-mediated
BRCA1−BAAT1 interaction in vivo awaits future studies, based interaction with the amide nitrogen of K1702 (Figure 2C). The
on the ITC results we consider that the BAAT1 region phenyl ring of Phe(+3) is inserted into the hydrophobic pocket
encompassing phospho-Ser269 is the major interaction site of P2 formed by R1699, L1701, F1704, N1774, M1775, R1835, and
the BRCA1 BRCTs. The inability of these domains to interact L1839. The amide nitrogen of Phe(+3) hydrogen bonds with the
with BAAT(pS156) and BAAT(pS604) is rather unexpected carbonyl oxygen of R1699. The carbonyl oxygen of Phe(+3)
because these peptides contain the pS-X-X-F recognition motif. hydrogen bonds with the Nε and Nη2 of R1699, and through a
Taken together, these results provide evidence that certain amino water-mediated interaction with the amide nitrogen of R1699
acids surrounding the anchoring residues pSer and Phe(+3) and the Oε2 of E1698. The carbonyl oxygen of Pro(+1)
reduce the binding affinity for the BRCA1 BRCT domains. participates in a water-mediated hydrogen bond network with
Structural Determinants of the BRCA1 BRCTs Binding the O3P of pSer and the amide nitrogens of L1701 and K1702
to ATRIP. To elucidate the structural basis for the BRCA1 (Figure 2C). The Oε1 atom of Gln(+2) forms a hydrogen bond
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Figure 3. BAAT1 recognition by the BRCA1 BRCT domains. (A and B) Ribbon representation of the BRCA1 BRCTs (brown) bound to the BAAT1
peptide (blue stick model) with a weighted 2Fo − Fc electron density map enveloping the peptide and two water molecules (W1 and W2) calculated at
2.2 Å resolution and contoured at 1.2σ. In B, the model is rotated by 180° along the vertical axis. (C) Two-dimensional representation of the interactions
between BRCA1 (orange) and BAAT1 (purple) residues. The hydrogen bond between the pSer O1P and the Nζ of K1702 is not shown. The figure was
created using LIGPLOT+.

with the Oε1 of E1698 through a water-mediated interaction. BRCT domains and the backbones of five residues of the two
Additional van der Waals contacts between ATRIP residues peptides (positions 0 to +4) are superimposed well with a root-
Pro(+1), Gln(+2), Phe(+3), and Gly(+4) contribute to the mean-square (rms) deviation of 0.097 Å for all Cα atoms, whereas
interaction, whereas Cys(−1), Ala(−2), Glu(−3), and Pro(−4) the N- and C-terminal regions of the peptides are not
do not seem to interact with BRCA1 residues (Figure 2C). superimposable. As described above, there are several similarities
Structural Basis for BAAT1 Recognition by the BRCA1 in the mode of interaction of the common residues pSer,
BRCTs. To determine the molecular mechanisms underlying the Pro(+1), and Phe(+3) with the BRCT domains but also notable
BRCA1 BRCT interaction with the BAAT(pS269) phosphopep- differences in the mode of interaction of residues at position +2.
tide (henceforth referred to as BAAT1 since it is the main Previous crystal structures of these domains bound to
interaction site of BRCA1), we solved the crystal structure of this phosphopeptides with a threonine27,28,31,33 or a valine30,32
complex at 2.2 Å resolution. As expected, the phospho-Ser269 residue at position +2 revealed how the side chains of these
and Phe272 (+3) form the main interactions with the BRCT residues participated in the interaction. In the BRCT−ATRIP
domains (Figure 3A and B). The O1P atom of pSer forms a complex, the longer side chain of Gln(+2) is accommodated
hydrogen bond with the Nζ of K1702, the O2P of pSer hydrogen through a ∼100° rotation of the E1698 side chain, as compared
bonds with the amide nitrogen of G1656, and the O3P forms with the BRCT−BAAT1 structure (Figure 4A). The conforma-
hydrogen bonds with the Oγ of S1655 and the amide nitrogen of tional change of E1698 may account, at least in part, for the lower
K1702 through a water-mediated interaction (Figure 3A−C). affinity of the BRCA1 BRCTs for the ATRIP peptide.
The phenyl ring of Phe(+3) is inserted into the P2 pocket with Previous structural studies of the BRCT domains of BRCA1,
the carbonyl oxygen of Phe(+3) hydrogen bonding with the Nη1 MDC1,41 and microcephalin (MCPH1)42,43 bound to their
of R1699 and through a water-mediated interaction with the cognate phosphopeptides revealed two crystallographic water
amide nitrogen of R1699. The amide nitrogen of Phe(+3) molecules (corresponding to W1 and W2) at the interfaces of
hydrogen bonds with the carbonyl oxygen of R1699. To a lesser these complexes. Superposition of the BRCA1 BRCTs bound to
degree, the BAAT1 residues Pro(+1), Val(+2), and Ser(+4) also the phosphopeptides ATRIP, BAAT1, BACH1, and CtIP with
contribute to the interaction through van der Waals contacts and the MDC1 BRCT−γH2AX and MCPH1 BRCT−γH2AX
water-mediated hydrogen bonds, whereas Arg(−1), Ala(−2), complexes shows that these water molecules are conserved and
Val(−3), and Ser(+5) do not participate in interactions with form a hydrogen bonding network between BRCT and peptide
BRCA1 residues (Figure 3C). residues (Figure 4B). In the BRCA1 structures, W1 participates
Structural Comparison of the BRCT−ATRIP and BRCT− in hydrogen bonding with the O3P of pSer, the amide nitrogen of
BAAT1 Complexes. Superposition of the BRCA1 BRCTs K1702, and W2, which hydrogen bonds with the carbonyl oxygen
bound to ATRIP and BAAT1 phosphopeptides shows that the of Pro(+1) and the amide nitrogen of L1701 (Figures 2C and
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the binding affinity and derived the generally accepted

recognition motif pS-X-X-F.2,5,14,35 However, since all of the
BRCA1 BRCT interaction partners contain these anchoring
residues, the affinity and specificity of these interactions should
be determined by amino acids surrounding these two residues, as
was observed previously.47 We therefore hypothesized that the
residues at positions +1 and +2 might be responsible for the low
affinity of the BRCA1 BRCTs for the BAAT1(pS156) and
BAAT1(pS604) phosphopeptides. To explore the role of these
residues in the BRCA1 BRCT−peptide interaction, we
performed ITC experiments with variant BAAT1(pS156) and
BAAT1(pS604) phosphopeptides that have Pro(+1) and
Val(+2), residues that are present in the high-affinity peptides
BAAT1(pS269) and CtIP.30 As expected, replacement of
Ser(+1) with Pro(+1) in the peptide BAAT1(pS156)S/P and
of Leu(+2) with Val(+2) in BAAT1(pS156)L/V improved the
binding of these peptides to BRCA1 BRCTs (Figure 5A and B),
whereas substitution of both residues in the peptide BAAT1-
(pS156)SL/PV resulted in stronger binding with a Kd of 30 μM
(Figure 5C). Modeling of the peptide BAAT1(pS156) on the
BRCA1 BRCT structure shows that the side chain of Ser(+1)
likely abolishes the van der Waals contacts that are observed
between N1774 and the Cβ and Cγ atoms of Pro(+1) (Figures 2C
and 5D). In addition, Pro(+1) likely contributes to the binding
affinity by affecting the conformation of the peptide. Although
Leu(+2) seems to be accommodated in the binding pocket at a
distance of ∼2.6 Å from E1698 (Figure 5D), it is likely that the
isobutyl group of Leu(+2) imposes a larger steric strain
compared to the shorter isopropyl group of Val(+2).
In a similar series of experiments, substitution of Glu(+1) with
Figure 4. (A) Conformational change of the E1698 side chain in the Pro(+1) in the peptide BAAT1(pS604)E/P improved the
BRCA1−ATRIP complex. Superposition of the crystal structures of the binding (Figure 5E). Likewise, replacement of Gly(+2) with
BRCA1 BRCTs (wheat) bound to ATRIP peptide (green) and the
BRCTs (brown) bound to BAAT1 (blue) complexes. A dashed arc Val(+2) in the peptide BAAT1(pS604)G/V also increased the
indicates the rotation of the E1698 side chain. (B) Stereoview of binding affinity (Figure 5F), whereas the substitution of both
superposition of the BRCA1 BRCT complexes with ATRIP, BAAT1, residues in BAAT1(pS604)EG/PV resulted in strong binding
BACH1 (PDB entry 1T15), CtIP (PDB entry 1Y98), the MDC1 with an estimated Kd of 4.9 μM (Figure 5G). Modeling of the
BRCTs bound to γH2AX (PDB entry 2AZM), and the MCPH1 BRCTs peptide BAAT1(pS604) on the BRCA1 BRCT structure shows a
complexed with γH2AX (PDB entry 3U3Z). The structurally conserved steric clash between Glu(+1) and N1774 (Figure 5H), whereas
water molecules W1 and W2 that mediate the BRCT−peptide the lack of a side chain in Gly(+2) abolishes the van der Waals
interaction are shown. contacts between the Cγ2 of Val(+2) and E1698 (Figures 3C and
5H), providing a possible structural explanation for the lower
3C). In the MDC1 complex, W1 hydrogen bonds with the O1P of affinity of BAAT1(pS604) as compared to that of the mutated
pSer, W2, and the amide nitrogen of K1936, whereas W2 forms peptides. Notably, although the peptides BAAT(pS156)SL/PV
hydrogen bonds with the carbonyl oxygen of Gln(+1) and the and BAAT(pS604)EG/PV share an identical core sequence
amide nitrogen of V1935.41 In the MCPH1−γH2AX complex, (DpSPVF), their affinities for the BRCA1 BRCTs differ by ∼6-
W1 hydrogen bonds with the O3P of pSer, W2, and the amide fold (Figure 5C and G), indicating that residues outside the
nitrogen of N696, whereas W2 forms hydrogen bonds with the common sequence contribute significantly to the binding affinity.
carbonyl oxygen of Gln(+1) and the amide nitrogen of L695.42 Taken together, these results demonstrate that the failure of the
Since these water molecules hydrogen bond with an OP atom of BRCA1 BRCTs to interact strongly with the BAAT1(pS156)
pSer (an obligatory feature of the phosphopeptides recognized and BAAT1(pS604) phosphopeptides is due mainly to the side
by the BRCA1, MDC1, and MCPH1 BRCT domains) and with chains at positions +1 and +2, with residues outside the motif pS-
main chain atoms of the BRCTs and peptide ligands, their X-X-F also playing a role in the affinity of the interaction.
arrangement is independent of the BRCT and peptide To further explore the contribution of the size and charge of
sequences. In fact, these water molecules are also observed in the side chains at +1 and +2 in the binding affinity, we performed
the crystal structures of the BRCT domains of TopBP1,44 ITC experiments with the phosphopeptide CDC27(pS820)
Rtt107,45 and BARD146 (data not shown), and therefore appear having the sequence HAAEpSDEF that corresponds to residues
to constitute an integral component of the BRCT recognition 816−823 of human CDC27 (cell division cycle 27), a
interface. component of the anaphase-promoting complex (GenBank
BRCA1 BRCT Recognition Motif. The plethora of BRCA1 entry AAA60471). Strikingly, the BRCA1 BRCTs did not
BRCT binding partners3−5 poses a challenge for the elucidation interact with CDC27(pS820) despite the presence of the pS-X-
of the molecular mechanisms governing the hierarchy of target X-F motif (Figure 6A). However, replacement of Asp(+1) with
selection by these domains. Initial studies recognized the critical Pro(+1) in the peptide CDC27(pS820)D/P resulted in binding
contribution of the two anchoring residues pSer and Phe(+3) in with an apparent Kd of 50.5 μM (Figure 6B), whereas
F dx.doi.org/10.1021/bi400714v | Biochemistry XXXX, XXX, XXX−XXX
Biochemistry Article

Figure 5. (A to C) Representative ITC results obtained for the BRCA1 BRCT interaction with the phosphopeptides BAAT(pS156)S/P,
BAAT(pS156)L/V, and BAAT(pS156)SL/PV, respectively. The sequences of the peptides with phosphoserine (red) and the substituted residues
(white letters on blue background) are shown at the bottom. For the isotherms in panels A−C, the BRCA1 BRCT concentration was 44.0 μM, 56.7 μM,
and 22.7 μM, whereas the peptide concentration was 0.728 mM, 0.753 mM, and 0.676 mM, respectively. (D) Surface model of the BRCA1 BRCTs
bound to the BAAT(pS156) peptide. (E to G) ITC results obtained for the BRCA1 BRCT interaction with the phosphopeptides BAAT(pS604)E/P,
BAAT(pS604)G/V, and BAAT(pS604)EG/PV, respectively. The sequences of the peptides are shown at the bottom. For the isotherms in panels E−G,
the BRCA1 BRCT concentration was 33.3 μM, 33.3 μM, and 18.9 μM, whereas the peptide concentration was 0.694 mM, 0.74 mM, and 0.413 mM,
respectively. (H) Surface model of the BRCA1 BRCTs bound to the BAAT(pS604) peptide.

substitution of Glu(+2) with Thr(+2) in CDC27(pS820)E/T pockets, respectively. It will therefore be important to determine
promoted strong binding with a Kd of 0.73 μM (Figure 6C). The in future studies whether the observed biphasic interaction
∼69-fold higher affinity of CDC27(pS820)E/T compared to profiles could be accounted for by models that involve two
CDC27(pS820)D/P indicates that Glu(+2) has a more binding sites and determine the thermodynamic and structural
detrimental effect on the binding affinity than Asp(+1). mechanisms underlying the observed exothermic-to-endother-
Moreover, simultaneous substitutions of both acidic residues in mic transitions. Taken together, these results indicate that
the peptide CDC27(pS820)DE/PT restored very strong binding Glu(+2) is not favored by the BRCA1 BRCTs, whereas the
to the BRCTs with a Kd of 0.14 μM (Figure 6D). The extremely concomitant presence of Asp(+1) and Glu(+2) adversely affects
tight binding of the BRCA1 BRCTs to CDC27(pS820)DE/PT the affinity of these domains for the peptide.
compared to that of other peptides harboring an internal pSPTF To obtain structural insight into the BRCA1 BRCT selection
motif is likely due to recognition of the peptide C terminus, as has against Glu(+2), we compared the BRCA1−ATRIP,
been shown previously.33,43 Notably, the BRCA1 BRCT MDC1−γH2AX,41 and MCPH1−Cdc2743 structures. The
interaction with CDC27(pS820)D/P and CDC27(pS820)E/T R1932 in MDC1 generates a basic region that favors acidic
produced biphasic thermograms composed of an initial side chains at position +2 (Figure 6E). The corresponding area of
exothermic component followed by an endothermic one (Figure MCPH1 (occupied by L692) also generates a positive electro-
6B and C), whereas the CDC27(pS820)DE/PT generated an static potential that accommodates the Glu(+2) of Cdc27
exothermic thermogram (Figure 6D). Although the obtained (Figure 6F). By contrast, BRCA1 has E1698 at the equivalent
binding profiles were not studied further in the present work, position, which creates an electrostatic repulsion with acidic side
these and similar peptides may be useful tools for the chains at position +2 (Figure 6G). Steric hindrance with E1698
investigation of the driving forces, potential conformational may also contribute to lowering the affinity, as described for the
changes during the BRCT−phosphopeptide interaction, and the side chain of Gln(+2). It is likely that accommodation of acidic
order of the pSer and Phe(+3) insertion into the BRCT P1 and P2 and perhaps other unfavorable side chains at positions +1 and +2
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Biochemistry Article

Figure 6. (A to D) ITC results obtained for the BRCA1 BRCT interaction with the phosphopeptides CDC27(pS820), CDC27(pS820)D/P,
CDC27(pS820)E/T, and CDC27(pS820)DE/PT, respectively. For the isotherms in panels A−D, the BRCA1 BRCT concentration was 56.7 μM, 56.7
μM, 56.7 μM, and 29.5 μM, whereas the peptide concentration was 1.13 mM, 1.5 mM, 1.39 mM, and 0.369 mM, respectively. (E to G) Surface
electrostatic potential of the MDC1, MCPH1, and BRCA1 BRCTs complexed with γH2AX, Cdc27, and ATRIP peptides (white stick models),
respectively. The MDC1 R1932, MCPH1 L692, and BRCA1 E1698 residues that participate in the selection of Glu(+2) are labeled and encircled.
Electrostatic potentials were calculated with PyMol and are colored red (acidic, −1 kBT), white (neutral, 0 kBT), and blue (basic, 1 kBT).

depends on the number and strength of interactions between interaction with the BRCA1 BRCTs. On the basis of these
residues flanking the pS-X-X-F motif and BRCT residues. findings, we suggest that certain residues at these positions may
Nevertheless, it appears that the presence of Asp(+1)/Glu(+2) impose severe electrostatic and/or steric constraints on the
in CDC27 is sufficient to exclude the BRCA1 protein from the
interaction with these domains. It is therefore imperative that the
CDC27-mediated signaling pathways. Clearly, further studies are
warranted to delineate the structural determinants underlying roles of the residues at positions +1 and +2 of the recognition
the complex recognition of phosphopeptides by these domains. motif pS-X-X-F, as well as those flanking this sequence, be

■
systematically assessed and their contribution to the affinity and
CONCLUDING REMARKS specificity of the interaction accurately determined in order to
The crystal structures of the BRCA1 BRCT domains bound to obtain a comprehensive understanding of the molecular
phosphopeptides ATRIP and BAAT1 elucidated the molecular mechanisms controlling the hierarchy of target selection by the
determinants of their interaction and provided a structural basis BRCA1 BRCT domains during DDR and breast/ovarian
for the BRCA1 function in ATRIP−ATR and BAAT1−ATM oncogenesis. Knowledge of these mechanisms will also facilitate
signaling, respectively. Notably, the BRCT−ATRIP structure the structure-based design of specific inhibitors of BRCA1 BRCT
revealed for the first time the mode of BRCA1 BRCT interaction
with a phosphopeptide having Gln(+2) since to date, only binding to physiologic targets, with potential applications in
structures of these domains bound to phosphopeptides having cancer treatment through modulation of the DNA repair
Thr(+2) or Val(+2) have been reported. Importantly, in the pathways.48,49

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process of mapping the BRCA1 binding site on the BAAT1
protein we observed that certain side chains at positions +1 and ASSOCIATED CONTENT
+2 of the generally accepted BRCA1 BRCT recognition motif
pS-X-X-F had detrimental effects on the affinity of the Accession Codes
interaction. In particular, the combination of Asp(+1) and PDB entry 4IGK (BRCA1−ATRIP complex) and PDB entry
Glu(+2) in the peptide CDC27(pS820) adversely affected its 4IFI (BRCA1−BAAT1 complex).
H dx.doi.org/10.1021/bi400714v | Biochemistry XXXX, XXX, XXX−XXX
Biochemistry

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Article

AUTHOR INFORMATION (12) Li, M. L., and Greenberg, R. A. (2012) Links between genome
integrity and BRCA1 tumor suppression. Trends Biochem. Sci. 37, 418−
Corresponding Author 424.
*Tel: 617-667-0064. E-mail: [email protected]. (13) Silver, D. P., and Livingston, D. M. (2012) Mechanisms of
Funding BRCA1 tumor suppression. Cancer Discovery 2, 679−684.
(14) di Masi, A., Gullotta, F., Cappadonna, V., Leboffe, L., and Ascenzi,
This work was supported by grant DA030209 from the National
P. (2011) Cancer predisposing mutations in BRCT domains. IUBMB
Institutes of Health, and grants W81XWH1010043,
Life 63, 503−512.
W81XWH1010602, W81XWH1110076, and (15) Shakya, R., Reid, L. J., Reczek, C. R., Cole, F., Egli, D., Lin, C. S.,
W81XWH1110180 from the US Department of Defense to deRooij, D. G., Hirsch, S., Ravi, K., Hicks, J. B., Szabolcs, M., Jasin, M.,
J.A.A.L. Baer, R., and Ludwig, T. (2011) BRCA1 tumor suppression depends on
Notes BRCT phosphoprotein binding, but not its E3 ligase activity. Science
The authors declare no competing financial interest. 334, 525−528.

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(16) Drost, R., Bouwman, P., Rottenberg, S., Boon, U., Schut, E.,
Klarenbeek, S., Klijn, C., van der Heijden, I., van der Gulden, H.,
ACKNOWLEDGMENTS Wientjens, E., Pieterse, M., Catteau, A., Green, P., Solomon, E., Morris, J.
We thank the staff at the APS NE-CAT and NSLS X25 beamlines R., and Jonkers, J. (2011) BRCA1 RING function is essential for tumor
for assistance during data collection, and Dr. Olivier Kocher at suppression but dispensable for therapy resistance. Cancer Cell 20, 797−
Harvard Medical School for providing access to the micro- 809.
calorimeter facility. (17) Derheimer, F. A., and Kastan, M. B. (2010) Multiple roles of ATM

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in monitoring and maintaining DNA integrity. FEBS Lett. 584, 3675−
3681.
ABBREVIATIONS (18) Ditch, S., and Paull, T. T. (2012) The ATM protein kinase and
ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia cellular redox signaling: beyond the DNA damage response. Trends
and Rad3 related; ATRIP, ATR-interacting protein; BAAT1, Biochem. Sci. 37, 15−22.
BRCA1-associated protein required for ATM activation-1; (19) McKinnon, P. J. (2012) ATM and the molecular pathogenesis of
BACH1, BRCA1-associated C-terminal helicase; BRCA1, breast ataxia telangiectasia. Annu. Rev. Pathol. 7, 303−321.
(20) Flynn, R. L., and Zou, L. (2011) ATR: a master conductor of
and ovarian cancer susceptibility gene 1; BRCT, BRCA1 C-
cellular responses to DNA replication stress. Trends Biochem. Sci. 36,
terminal; CDC27, cell division cycle 27; CtIP, CtBP-interacting 133−140.
protein; DDR, DNA damage response; DSB, DNA double- (21) Nam, E. A., and Cortez, D. (2011) ATR signalling: more than
strand break; GST, glutathione S-transferase; ITC, isothermal meeting at the fork. Biochem. J. 436, 527−536.
titration calorimetry; MCPH1, microcephalin; MDC1, mediator (22) Bunting, S. F., Callén, E., Kozak, M. L., Kim, J. M., Wong, N.,
of DNA damage checkpoint 1; MRN, Mre11−Rad50−Nbs1; López-Contreras, A. J., Ludwig, T., Baer, R., Faryabi, R. B., Malhowski,
PDB, Protein Data Bank; RPA, replication protein A; ssDNA, A., Chen, H. T., Fernandez-Capetillo, O., D’Andrea, A., and
single-stranded DNA Nussenzweig, A. (2012) BRCA1 functions independently of homolo-

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gous recombination in DNA interstrand crosslink repair. Mol. Cell 46,
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