Structural characterization of BRCT–tetrapeptide binding interactions
Introduction
BRCT(BRCA1) interacts with several cell cycle related proteins, and plays an essential role in the DNA-damage response and repair pathway [1], [2], [3], [4], [5], [6], [7]. Mutations in BRCT(BRCA1) lead to breast cancer, and also sensitize cells to DNA damaging agents. Therefore inhibitors targeting this domain offer a novel approach to cancer therapy.
Structural and biochemical studies show that BRCT(BRCA1) recognizes and binds a conserved sequence pSXXF in these proteins [8], [9], [10], [11], [12], [13]. We have recently shown that peptides containing the pSXXF sequence as the minimal structural unit required for inhibitor design [14]. The small size (4-amino acids) and the micromolar (μM) binding affinity of these peptides make them an excellent template for a peptidomimetic approach for designing more potent nanomolar (nM) inhibitors. The knowledge of the structural and thermodynamic basis of the binding interactions is essential for such an approach. Towards this goal, we have now characterized the binding of a series of peptides, SRSTpSPTFNK, pSPTF, pSPAF and Flu-βA-pSPTF to BRCT(BRCA1) using NMR spectroscopy and isothermal titration calorimetry.
Our results indicate that the binding of pSXXF tetrapeptides is accompanied by global dynamic changes, and that the tetrapeptide captures all of the interactions observed for the longer peptides. We further show that the fluorescein moiety in Flu-βA-pSPTF binds to the previously identified hydrophobic site adjacent to the pSXXF binding site, and that its higher binding affinity should be due to favored interactions at this site. Most importantly, correlating the NMR and calorimetry data show that the binding affinities of the tetrapeptides are intimately linked to structural and dynamic changes both in BRCT(BRCA1) and the tetrapeptides.
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Materials and methods
Protein purification and NMR sample preparation. The BRCT(BRCA1) construct was expressed, and purified as discussed previously [14]. 15N labeling was achieved by growing the transformed cells in M9 minimal media containing 15NH4Cl (Cambridge Isotope Laboratories) as the sole nitrogen source. The peptides were synthesized using standard Fmoc chemistry and HPLC purified either in house or by the Tufts University Core Facility [14]. The NMR sample consisted of ∼150 μM BRCT in 50 mM KH2PO4, pH 7.0
Results and discussion
Previous structural studies of BRCT(BRCA1)–phosphopeptide complexes reveal a well defined binding pocket for pSer and for Phe(P + 3) residues (Fig. 1), and mutational studies have also shown that these residues are critical for binding, and so pSXXF sequence has been implicated as the recognition motif for BRCT(BRCA1) interaction and function [8], [9], [10], [11], [12], [13]. We have recently shown that the tetrapeptides containing the pSXXF bind only with slightly lower affinities compared to
Acknowledgments
This work was supported in part by the grants NIH R01CA127239 (A.N.), ACB G218000116 (A.N.) and John Sealy Endowment fund (K.R.).
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