Kinetic analysis of interaction of BRCA1 tandem breast cancer C-terminal domains with phosphorylated peptides reveals two binding conformations

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

Research output: Contribution to journalArticle

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Abstract

Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants (Kd) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the Kd values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.

Original languageEnglish (US)
Pages (from-to)9866-9879
Number of pages14
JournalBiochemistry
Volume47
Issue number37
DOIs
StatePublished - Sep 16 2008

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Conformations
Phosphopeptides
Surface plasmon resonance
Breast Neoplasms
Peptides
Kinetics
BRCA1 Protein
Ubiquitin-Protein Ligases
Phosphoproteins
X ray crystallography
Fluorescence spectroscopy
Calorimetry
Titration
Nuclear magnetic resonance spectroscopy
Surface Plasmon Resonance
Proteins
Anisotropy
Repair
Fluorescence
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

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, Zeljko; Owen, Whyte G.; Caride, Ariel J.; Wasielewski, Emeric; Mer, Georges.

In: Biochemistry, Vol. 47, No. 37, 16.09.2008, p. 9866-9879.

Research output: Contribution to journalArticle

Nominé, Yves ; Botuyan, Maria Victoria ; Bajzer, Zeljko ; Owen, Whyte G. ; Caride, Ariel J. ; Wasielewski, Emeric ; Mer, Georges. / Kinetic analysis of interaction of BRCA1 tandem breast cancer C-terminal domains with phosphorylated peptides reveals two binding conformations. In: Biochemistry. 2008 ; Vol. 47, No. 37. pp. 9866-9879.
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