Kinetic Analysis of Interaction of BRCA1 Tandem Breast Cancer C-Terminal Domains with Phosphorylated Peptides Reveals Two Binding Conformations

Nominé, Yves; Botuyan, Maria Victoria; Bajzer, Željko; Owen, Whyte G.; Caride, Ariel J.; Wasielewski, Emeric; Mer, Georges

doi:10.1021/bi702247d

Cited by 15 publications

(9 citation statements)

References 58 publications

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“…The similar thermodynamic profile in all four tetrapeptides ( 1 , 7 , 18 and 19 , Figure 3 ) suggests that the binding is anchored by pS and F contacts, consistent with reported studies of the longer peptides. 17, 24 …”

Section: Resultsmentioning

confidence: 99%

Structure–Activity Relationship Studies To Probe the Phosphoprotein Binding Site on the Carboxy Terminal Domains of the Breast Cancer Susceptibility Gene 1

et al. 2011

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The carboxy terminal BRCT domains of the breast cancer susceptibility gene 1 (BRCA1) bind to a plethora of phosphorylated proteins through a pSXXF consensus recognition motif. BRCT-protein binding regulates key cellular functions such as lipogenesis, cell-cycle checkpoint control and DNA damage response. Identification of the minimal binding sequence and defining the key interactions responsible for biological activity are critical steps in the peptidomimetic design process. Here, we report a systematic structure activity relationship study that maps the BRCT(BRCA1)-pSXXF binding interface. The study has led to the identification of peptides with nanomolar binding affinities comparable to the previously reported 13-mer peptides. The results also provide a clear description of the pSXXF-BRCT interface, which is essential for developing small molecule inhibitors via the peptidomimetic approach.

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Section: Resultsmentioning

confidence: 99%

Structure–Activity Relationship Studies To Probe the Phosphoprotein Binding Site on the Carboxy Terminal Domains of the Breast Cancer Susceptibility Gene 1

et al. 2011

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“…The ϩ3/ϩ4 positions of this target ( 1159 pSNLQWPS) are not conserved with the BACH1 target sequence, and it may be that recognition of this peptide involves a further rearrangement of the TopBP1 BRCT7/8 specificity pocket. Although TopBP1 is more flexible than the other tandem BRCT proteins, a certain degree of flexibility in packing of the tandem repeats of BRCA1 likely also exists in solution, as suggested by both NMR (61) and thermodynamic stability studies (62,63). A BRCT interface rotation is also observed in the Nbs1 BRCT domains, although it appears to be initiated by a mechanism dependent on the neighboring FHA domain.…”

Section: Discussionmentioning

confidence: 96%

Molecular Basis of BACH1/FANCJ Recognition by TopBP1 in DNA Replication Checkpoint Control

Leung

Gong

Chen

et al. 2011

Journal of Biological Chemistry

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The diverse roles of TopBP1 in DNA replication and checkpoint signaling are associated with the scaffolding ability of TopBP1 to initiate various protein-protein interactions. The recognition of the BACH1/FANCJ helicase by TopBP1 is critical for the activation of the DNA replication checkpoint at stalled replication forks and is facilitated by the C-terminal tandem BRCT7/8 domains of TopBP1 and a phosphorylated Thr 1133 binding motif in BACH1. Here we provide the structural basis for this interaction through analysis of the x-ray crystal structures of TopBP1 BRCT7/8 both free and in complex with a BACH1 phospho-peptide. In contrast to canonical BRCT-phospho-peptide recognition, TopBP1 BRCT7/8 undergoes a dramatic conformational change upon BACH1 binding such that the two BRCT repeats pivot about the central BRCT-BRCT interface to provide an extensive and deep peptide-binding cleft. Additionally, we provide the first structural mechanism for Thr(P) recognition among BRCT domains. Together with systematic mutagenesis studies, we highlight the role of key contacts in governing the unique specificity of the TopBP1-BACH1 interaction.DNA damage checkpoints coordinate the cellular events necessary to ensure that DNA is repaired and faithfully replicated before cell cycle progression. A critical checkpoint triggered by DNA damage encountered during DNA replication (1, 2) involves ATR (Ataxia telangiectasia and Rad3 related), a Ser/Thr kinase that phosphorylates an array of proteins including CHK1 to regulate checkpoint control (3). The ability of ATR to function at the replication fork is dependent on the assembly of a growing list of proteins, including replication protein A, the ATR-ATRIP heterodimer, the trimeric Rad9-Hus1-Rad1 (9-1-1) clamp complex, BACH1/FANCJ (BRCA1-associated C-terminal helicase/Fanconi anemia group J protein), and topoisomerase II␤-binding protein 1 (TopBP1) 2 (4 -6). In particular, the emergence of TopBP1 as a key regulator in the DNA replication checkpoint pathway is underscored by its multiple roles contributing to the activation of ATR.TopBP1 possesses nine BRCT domains, the most of any BRCT domain-containing protein (7,8). Originally identified with eight BRCT domains using sequence analysis, recent structural studies have confirmed an additional cryptic BRCT domain (BRCT0) at the extreme N terminus of TopBP1 (9, 10). The roles of BRCT domains as phosphorylated proteinbinding modules have been demonstrated in studies of the BRCT domains in BRCA1 (breast cancer-associated protein 1) and other 12). Although the phospho-peptide binding ability of single BRCTs is still poorly defined, the role of tandem BRCT domains in recognizing phospho-peptide motifs is well established. For example, the tandem BRCT domains of BRCA1 form a functional unit to recognize the Ser(P)-Xaa-Xaa-Phe binding motif in a number of proteins involved in the DNA damage response (11-16), and the structural principles governing these interactions have been elucidated through a number of structural studies (17)(18)(19)(20)(21...

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“…These additional contacts may explain the large, negative Δ H values describing the binding of 8.6, which are significantly larger in magnitude (24.6 kcal mol −1 vs 15 to 18 kcal mol −1 ) than our pSer mutant and other pSer peptide binders. 19,45 The unfavorable entropy term describing the binding of peptide 8.6 does not appear to be due to the additional contacts at the N-terminus, since the pSer mutant led to a favorable increase in entropy, even though it presumably maintains many of the same contacts. The additional contacts mediated by the inhibitor peptide are, however, likely responsible for the high degree of specificity of this inhibitor, which is unable to bind to the highly related MDC1 and TopBP1 (BRCT) 2 domains, which both bind related phosphopeptides.…”

Section: Resultsmentioning

confidence: 99%

Peptide Library Approach to Uncover Phosphomimetic Inhibitors of the BRCA1 C-Terminal Domain

White

Sun

Ma³

et al. 2015

ACS Chem. Biol.

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Many intracellular protein–protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein–protein interactions mediated by serine phosphorylation.

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Kinetic Analysis of Interaction of BRCA1 Tandem Breast Cancer C-Terminal Domains with Phosphorylated Peptides Reveals Two Binding Conformations

Cited by 15 publications

References 58 publications

Structure–Activity Relationship Studies To Probe the Phosphoprotein Binding Site on the Carboxy Terminal Domains of the Breast Cancer Susceptibility Gene 1

Structure–Activity Relationship Studies To Probe the Phosphoprotein Binding Site on the Carboxy Terminal Domains of the Breast Cancer Susceptibility Gene 1

Molecular Basis of BACH1/FANCJ Recognition by TopBP1 in DNA Replication Checkpoint Control

Peptide Library Approach to Uncover Phosphomimetic Inhibitors of the BRCA1 C-Terminal Domain

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