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. Study holds ProTherm entries: 25716, 25717, 25718, 25719 Extra Details: BRAC1: Breast Cancer gene 1 protein; conserved C-terminal domain; 6xHis-tag Chemical unfolding; Thermodynamic stability; ITC binding experiments
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:56 p.m.
|Number of data points||4|
|Proteins||BRCA1-associated RING domain protein 1 ; BRCA1-associated RING domain protein 1|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O ionic:NaCl: 300 mM, pH:9.0, buffers:Borate: 50 mM, temp:10.0 C ; Experimental Assay: dG_H2O ionic:NaCl: 300 mM, temp:20.0 C, pH:9.0, buffers:Borate: 50 mM ; Experimental Assay: dG_H2O pH:7.5, temp:20.0 C, ionic:NaCl,Calcium chloride: 300 mM,1 mM, buffers:Tris-HCl: 50 mM|
|Libraries||Mutations for sequence GIDPFTRDGPLVLIGSGLSSEQQKMLSELAVILKAKKYTEFDSTVTHVVVPGDAVQSTLKCMLGILNGCWILKFEWVKACLRRKVCEQEEKYEIPEGPRRSRLNREQLLPKLFDGCYFYLWGTFKHHPKDNLIKLVTAGGGQILSRKPKPDSDVTQTINTVAYHARPDSDQRFCTQYIIYEDLCNYHPERVRQGKVWKAPSSWFIDCVMSFELLPLDS|