Structural basis of BACH1 phosphopeptide recognition by BRCA1 tandem BRCT domains

Abstract

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.

Figure 1. Amino Acid Sequence of BACH1 Phosphopeptide

The numbering scheme adopted for BACH1 phosphopeptide (BACH1p) is 1 to 13. The sequence corresponds to residues 985–998 of BACH1 protein. The phosphorylated Ser is indicated by pSer6. Relative positions of other residues from pSer6 are also indicated.

Figure 2. Variation in BRCA1-BRCT Chemical Shifts upon BACH1p Binding

(A) Overlay of ¹⁵N-HSQC spectra of ¹⁵N/[50%²H]-labeled BRCA1-BRCT, free (black) and in a 1:1 complex with nonlabeled BACH1p (red), illustrating the extent of chemical shift perturbations upon peptide binding. (B–E) Overlay of ¹⁵N-HSQC spectra of BRCA1-BRCT, free (black) and bound to nonlabeled BACH1p (red), selectively ¹⁵N-labeled for Leu, Tyr, Val, and Phe, respectively. (F) Overlay of a selected region of the ¹³C-HSQC spectrum of ¹⁵N/¹³C/²H-labeled BRCA1-BRCT, but protonated at the methyl groups of Val, Leu, and Ile (δ1), in the absence (black) and presence (red) of nonlabeled BACH1p.

Figure 3. Summary of NMR Data for BRCA1-BRCT in Complex with BACH1p

(A) Sequence of BRCA1-BRCT (1646–1859) with a summary of secondary structure elements derived from NMR data of the BACH1p-bound protein: interproton NOEs, solvent protection, TALOS, and chemical shift index (CSI) results. NOEs were measured from 100, 200, and 300 ms mixing times 3D ¹⁵N-edited and ¹³C-edited NOESY data recorded on 50%, 60%, and 100% deuterated ¹⁵N- and ¹⁵N/¹³C-labeled BRCA1-BRCT samples, as well as on a ¹⁵N/¹³C-labeled and deuterated (but protonated at Val, Leu, and Ile δ1 methyls) protein. An NOE is indicated by a bar; the thickness is proportional to intensity. A solid circle represents a residue significantly protected from ²H exchange (i.e., ¹H signal is still visible in the HSQC spectrum 4 hr after exchange in D₂O). Solid and empty squares indicate that Phi and Psi angles predicted by TALOS are compatible with those expected for α helices and β strands, respectively. Negative and positive CSI are designated by a square below and above the line, respectively. β strands (arrows) and α helices (coils) deduced from all the NMR data are summarized (NMR). Thin lines connecting the β strands indicate that long-range HN_i-HN_j and Hα_i-HN_j NOEs have been observed. The NMR data are in very good agreement with the secondary structures found in the crystal structure of free BRCA1-BRCT (X-ray). (B) Mapping of interresidue NOEs on the crystal structure of free BRCA1-BRCT (PDB entry 1JNX). Protons involved in NOEs are connected by red lines; 453 medium-range and 238 long-range NOEs are shown. (C) Residues involved in intermolecular NOEs are connected by red double arrows.

Figure 4. Identification of the BACH1p Binding Site on BRCA1-BRCT

(A) Changes in chemical shifts mapped on the crystal structure of free BRCA-BRCT. Shown in red are residues that are affected upon BACH1p binding. (B) Same as (A) after 180° rotation about the vertical axis. (C) Residues involved in intermolecular NOEs with BACH1p mapped on the crystal structure of free BRCA-BRCT. The region involved in phosphoserine binding is circled in yellow, while the hydrophobic pocket is indicated by a red circle. (D) Stereo ribbon representation of BRCA1-BRCT showing the amino acids involved in intermolecular NOEs, as well as Met1775 and Lys1702. Red and yellow circles are defined as in (C).

Figure 5. Evidence of Conformational Equilibrium in Bound BACH1p

Regions from the 2D ¹H-¹H NOESY (A), F1/F2 double-filtered NOESY (B), F2-filtered NOESY (C), and F2 ¹⁵N-edited NOESY (D) spectra of the free BACH1p peptide (A) and BRCA1-BRCT/BACH1p complex (B–D). BACH1p peptide was nonlabeled in (A) to (D). BRCA1-BRCT was ¹⁵N/¹³C/²H-labeled with protonated methyls (Val, Leu, and Ile δ1) in (B) and (C) and ¹⁵N/¹³C/[60%-²H]-labeled in (D). The F2 dimension in (A)–(C) corresponds to the aromatic region of BACH1p Phe9. The F2 dimension in (D) corresponds to the HN of Thr1700 without ¹⁵N decoupling. Experiments were done in D₂O at 30°C and pH 7.5. Thr1700 HN is protected from exchange with D₂O as seen in (D). (A) and (B) show intrapeptide NOEs. (C) displays both intrapeptide and peptide/protein NOEs. (D) contains intraprotein and peptide/protein NOEs. Note two sets of signals for Phe9 Hα, Thr8 Hβ, and Thr8 Hγ indicating two conformations of bound BACH1p in (B)–(D). In one of these conformations, Phe 9 is close in space to Pro7, as indicated by a box in (B) and (C).

Figure 6. Tyr1703 Connnects the Phophopeptide Binding Site to the Inter-BRCT Linker

(A) Superposition of a selected region from the ¹H spectra of ¹⁵N/¹³C/²H-labeled BRCA1-BRCT, free (black) and bound to nonlabeled BACH1p (red), showing the hydroxyl proton of Tyr1703. Note that other ¹H signals in the spectra are ¹⁵N-coupled. Experiments were recorded at 30°C and pH 7.5. (B) Location of Tyr1703 in the 3D structure of BRCA1-BRCT. Tyr1703 bridges the phosphopeptide binding site to the inter-BRCT linker region.

Figure 7. S1655A and K1702A Mutants of BRCA1 Do Not Interact with BACH1 In Vitro and In Vivo

(A) 293T cell lysates were incubated with wild-type or mutant GST-BRCA1-BRCT proteins immobilized on Sepharose beads. Proteins bound to beads were eluted and separated by SDS-PAGE. Western blots were performed with anti-BACH1 antibody (upper panel). Western blots of whole-cell lysates were also included to demonstrate equal amounts of BACH1 in these samples (lower panel). (B) Myc epitope-tagged wild-type and mutant BRCA1 were transiently expressed in 293T cells. Immunoprecipitation and immunoblotting experiments were performed with anti-myc or anti-BACH1 antibodies as indicated.

Figure 8. Phosphoprotein Binding Activity of BRCA1 Is Required for DNA Damage-Induced G2/M Checkpoint Control and BRCA1 Nuclear Foci Formation

Wild-type or mutant BRCA1 was transiently expressed in HCC1937 cells. (A) G2/M checkpoint assays were conducted as described in the Experimental Procedures. The fraction of cells in M phase is expressed as a percentage of that measured in unirradiated control cells. (B) Three hours after γ-irradiation, immunostaining experiments were performed with the indicated antibodies.