Kinetic analysis of interaction of BRCA1 tandem breast cancer c-terminal domains with phosphorylated peptides reveals two binding conformations.
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 ( K d) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the K d 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.
Nominé
Yves
Y
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, Minnesota 55905, USA.
Botuyan
Maria Victoria
MV
Bajzer
Zeljko
Z
Owen
Whyte G
WG
Caride
Ariel J
AJ
Wasielewski
Emeric
E
Mer
Georges
G
D019313:BRCA1 Protein / Q000737:chemistry* / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D051856:Fanconi Anemia Complementation Group Proteins / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D013696:Temperature; D013816:Thermodynamics
C102108:BACH1 protein, human; D019313:BRCA1 Protein; D050976:Basic-Leucine Zipper Transcription Factors; D051856:Fanconi Anemia Complementation Group Proteins; D010455:Peptides