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Segmental Isotope Labelling of an Individual Bromodomain of a Tandem Domain BRD4 Using Sortase A
Felix P Williams1, Alexander G Milbradt1, Kevin J Embrey1
1Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Alderley Park, Macclesfield SK10 4TF, United Kingdom.
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.
Insights
Researchers developed a novel method for labeling individual bromodomains of bromodomain and extra-terminal (BET) protein 4 (BRD4). This technique enables detailed NMR studies of ligand-BRD4 interactions, advancing drug design for cancer and other diseases.
Area of Science:
- Epigenetics and Molecular Biology
- Structural Biology
- Drug Discovery
Background:
- Bromodomain and extra-terminal (BET) proteins are key epigenetic readers and crucial oncology targets.
- Bromodomain-containing protein 4 (BRD4) is a significant BET family member, but its multi-domain structure complicates structural and biophysical studies.
- Existing methods for studying BRD4's tandem bromodomains using NMR are limited by protein size.
Purpose of the Study:
- To develop a method for segmental labeling of BRD4's individual bromodomains.
- To enable high-resolution NMR studies of ligand-BRD4 interactions.
- To facilitate rational drug design targeting BRD4.
Main Methods:
- Utilized Sortase A-mediated segmental labeling for rapid labeling of BRD4 bromodomains.
- Employed U-[2H,15N]-isotope labeling on the C-terminal bromodomain with selective 13CH3 methyl group introduction on specific amino acids (Ile, Val, Leu).
- Kept the N-terminal bromodomain unlabelled to simplify NMR spectra.
Main Results:
- Achieved simplified NMR spectra through a specific isotopic labeling strategy.
- Demonstrated the potential for high-resolution studies of BRD4 interactions.
- Enabled detailed investigation of ligand binding and its effects on BRD4 structure and dynamics.
Conclusions:
- The developed labeling strategy is a powerful tool for studying BRD4.
- This method overcomes limitations of traditional NMR studies for large, multi-domain proteins.
- Facilitates atomic-level understanding of ligand-BRD4 interactions, crucial for therapeutic development.
