title: Thermodynamic study of the BRCT domain of BARD1 and its interaction with the -pSER-X-X-Phe- motif-containing BRIP1 peptide.
abstract: The BRCA1-associated RING domain protein 1 (BARD1) is the heterodimeric partner of BRCA1. The BRCA1/BARD1 complex demonstrates ubiquitin ligase activity and has been implicated in genomic stability and tumor suppression. Both proteins possess a structurally conserved C-terminal domain (BRCT). While BRCA1-BRCT has been shown to mediate BRCA1 interactions with phosphoproteins such as BRIP1 by recognizing the pSer-X-X-Phe motif, attempts to demonstrate analogous interactions of its dimeric counterpart BARD1-BRCT, have so far been unsuccessful. In this study, chemical-denaturation experiments of BARD1-BRCT domain suggest that its low thermodynamic stability (DeltaG=2.5 kcal/mol) at room temperature, may affect some of its biochemical properties, such as its interaction with phosphopeptides. The stability of BARD1-BRCT domain at 10 degrees C, increases to 7.5 kcal/mol and isothermal titration calorimetry (ITC) experiments at this lower temperature showed binding to the BRIP1 phosphopeptide via an enthalpy-driven interaction, which appears to be specific to the pSer-X-X-Phe peptide-binding motif. Substitution of either pSer at position 0 with Ser (non-phosphorylated peptide) or Phe with Val at position +3, leads to no-binding ITC results. While these findings are indicative that BRIP1 is a potential BARD1 binding partner, it becomes evident that in vitro binding assays involving the entire BARD1 protein and in vivo experiments are also needed to establish its binding partners and its potential role in tumor suppression pathways.
authors.affiliation: Biomolecular Physics Laboratory, IRRP, National Centre for Scientific Research "Demokritos", 153 10 Aghia Paraskevi, Greece.
authors.forename: Angelos
authors.initials: A
authors.lastname: Thanassoulas
authors.forename: Michail
authors.initials: M
authors.lastname: Nomikos
authors.forename: Maria
authors.initials: M
authors.lastname: Theodoridou
authors.forename: Drakoulis
authors.initials: D
authors.lastname: Yannoukakos
authors.forename: Dimitris
authors.initials: D
authors.lastname: Mastellos
authors.forename: George
authors.initials: G
authors.lastname: Nounesis
mesh_terms: D020816:Amino Acid Motifs; D002942:Circular Dichroism; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D025521:Tumor Suppressor Proteins; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases