title: Segmental Isotope Labelling of an Individual Bromodomain of a Tandem Domain BRD4 Using Sortase A.
abstract: Bromodomain and extra-terminal (BET) family of proteins are one of the major readers of epigenetic marks and an important target class in oncology and other disease areas. The importance of the BET family of proteins is manifested by the explosion in the number of inhibitors against these targets that have successfully entered clinical trials. One important BET family member is bromodomain containing protein 4 (BRD4). Structural and biophysical studies of BRD4 are complicated by its tertiary-structure consisting of two bromodomains connected by a flexible inter-domain linker of approximately 180 amino acids. A detailed understanding of the interplay of these bromodomains will be key to rational drug design in BRD4, yet there are no reported three-dimensional structures of the multi-domain BRD4 and NMR studies of the tandem domain are hampered by the size of the protein. Here, we present a method for rapid Sortase A-mediated segmental labelling of the individual bromodomains of BRD4 that provides a powerful strategy that will enable NMR studies of ligand-bromodomain interactions with atomic detail. In our labelling strategy, we have used U-[2H,15N]-isotope labelling on the C-terminal bromodomain with selective introduction of 13CH3 methyl groups on Ile (δ1), Val and Leu, whereas the N-terminal bromodomain remained unlabelled. This labelling scheme resulted in significantly simplified NMR spectra and will allow for high-resolution interaction, structure and dynamics studies in the presence of ligands.
authors.affiliation: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Alderley Park, Macclesfield SK10 4TF, United Kingdom.
authors.forename: Felix P
authors.initials: FP
authors.lastname: Williams
authors.affiliation: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Alderley Park, Macclesfield SK10 4TF, United Kingdom.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Milbradt
authors.affiliation: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Alderley Park, Macclesfield SK10 4TF, United Kingdom.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Embrey
authors.affiliation: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Alderley Park, Macclesfield SK10 4TF, United Kingdom.
authors.forename: Romel
authors.identifier: 0000-0001-5652-4098
authors.initials: R
authors.lastname: Bobby
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases; D001426:Bacterial Proteins; D055592:Biophysical Phenomena; D018797:Cell Cycle Proteins; D003546:Cysteine Endopeptidases; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D000092468:Bromodomain Containing Proteins
chemical_list: D019881:Aminoacyltransferases; D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D003546:Cysteine Endopeptidases; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C120542:sortase A