Segmental Isotope Labelling of an Individual Bromodomain of a Tandem Domain BRD4 Using Sortase A

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.溴结构域与额外末端结构域（BET）蛋白家族是表观遗传标记的主要识别蛋白之一，也是肿瘤学及其他疾病领域的重要靶标类别。BET蛋白家族的重要性可通过针对该类靶点的抑制剂爆发式涌入临床试验的现象得以体现。BET家族的重要成员之一为含溴结构域蛋白4（BRD4）。BRD4的三级结构由两个溴结构域组成，二者通过一段约180个氨基酸的柔性域间连接区相连，这使得针对BRD4的结构与生物物理研究变得复杂。深入理解这两个溴结构域之间的相互作用，是开展BRD4合理药物设计的关键，但目前尚无多结构域BRD4的三维结构报道，且串联结构域的核磁共振（NMR）研究受限于蛋白的分子尺寸而难以开展。本研究报道了一种基于分选酶A（Sortase A）介导的BRD4各溴结构域分段标记方法，该策略可实现配体-溴结构域相互作用的原子级分辨率核磁共振研究。在本次标记策略中，我们对BRD4的C端溴结构域进行了U-[2H,15N]-全同位素标记，并在异亮氨酸（δ1位）、缬氨酸和亮氨酸上选择性引入13CH3甲基基团，而N端溴结构域则保持未标记状态。该标记方案可显著简化核磁共振谱图，使得在配体存在的条件下，能够开展高分辨率的相互作用、结构及动力学研究。