Biophysical and structural studies of small molecule, protein, and nucleic-acid interactions with BET bromodomain-containing proteins

Abstract

Bromodomains are ∼110 amino acid structural motifs that recognize specific protein sequences containing acetylated lysines. Many bromodomain modules are found within multidomain proteins that can both interact with chromatin and recruit enzymatic activity. The presence of multiple domains within a single protein leads to context-dependent and selective functions via multivalent protein−protein and protein−nucleic-acid interactions along with enzymatic function. The bromodomain and extra-terminal (BET) domain family has four proteins: BRD2, BRD3, BRD4, and the testis-specific BRDT. Each protein minimally consists of two tandem N-terminal bromodomains (BD1 and BD2) and an extra-terminal domain. The BET proteins have been shown to bind multiple acetylated lysines on histone tails and acetylated regions of various transcription factors. Given their significant role in disease, including cancer, cardiovascular, neurological, and inflammatory diseases, BET bromodomains are a well-studied drug target class. First-generation inhibitors retain high affinities for both the BD1 and BD2 domains of BET proteins. Due to their pan-inhibition of BET bromodomains, it is difficult to segregate the roles of individual BET proteins from each other. Recently, domain-selective inhibitors have been found to have different phenotypic responses and reduced toxicity as compared to the pan-BET inhibitors. Thus, isoform and domain-specific inhibitors will help to dissect the individual roles of each BET bromodomain, and thus new biophysical tools are required to discover them. As BET proteins regulate different cellular processes, it is essential to unravel their chromatin-binding determinants for designing better therapeutic modalities. Recently, epigenetic reader domains have also been found to harbor double-stranded (ds)DNA-binding activity, which is as functionally critical as histone association. Thus, using a suite of biophysical and structural studies, we have explored the dsDNA recognition of the N-terminal bromodomain of the Bromodomain and Extra-Terminal (BET) protein, BRD4. Through these studies, we have inferred that the disordered region connecting the BDs might be playing a significant role in mediating high-affinity interactions with chromatin’s repeatingunit, the nucleosome. These studies also highlight the importance of exploring the multi-domain interactions beyond just the bromodomains in regulating the structure and function of BET proteins. A particular example is the investigation of phosphorylation of disordered regions in BRD4 to regulate the protein’s chromatin recognition and gene-specific function. In this dissertation, I have described various biophysical and structural studies to elucidate the protein, small molecule, and nucleic-acid interactions with multi-domain BET proteins. Chapter 1 discusses various biophysical and structural techniques previously used for studying multi-domain BET proteins and highlights an area for further research in the field. Chapter 2 describes the application of protein-observed fluorine (PrOF) NMR to the tandem bromodomains of BRD4 and BRDT to quantify the selectivity of their interactions with acetylated histones as well as small molecules. This is the first report from the Pomerantz lab on PrOF NMR applied to multi-domain proteins as prior studies have focused on individual bromodomains only. We further determined the selectivity profile of a new class of ligands,1,4-acylthiazepanes, and found them to have ~3−10-fold selectivity for the C-terminal bromodomain of both BRD4 and BRDT. An extension of the previous chapter, chapter 3 focuses on the development of biophysical methods, including AlphaScreen competitive-inhibition assay and structural biology efforts on unraveling the BD2-selectivity of 1,4-acylthiazepanes. It also contains a section on a collaborative screening effort of a second library of 3D fragments to discover newer domain-selective scaffolds. Chapter 4 explores the dsDNA recognition of the N-terminal bromodomain of the Bromodomain and Extra-Terminal (BET) protein, BRD4. Using NMR-based assays, gel-shift assays, and competitive-inhibition assays, we established the binding surface of dsDNA and found it to be largely overlapping with the acetylated-histone (KAc) binding site. Rather than engaging in electrostatic contacts, we found dsDNA to interact competitively within the KAc-binding pocket. These interactions are distinct from the highly homologous BET bromodomain, BRDT. Together, these studies help establish a binding model for dsDNA interactions with BRD4 bromodomains and elucidate the chromatin recognition mechanisms of the BRD4 protein for regulating gene expression.Chapter 5 builds upon chapter 4 and the application of cryo-electron microscopy (cryo-EM) to study the interactions between the tandem domains of BRD4 and BRDT with unmodified nucleosomes. It also describes my efforts toward establishing a structural and quantitative picture of the “phospho-switch mechanism” to regulate BRD4’s native structure and function. Lastly, the appendix chapter explores a genetic code expansion strategy and a cysteine bio-conjugation approach to include alternative 19F NMR probes for protein-observed 19F NMR. These probes bearing a trifluoromethyl (-CF3) group will be useful for studying larger multi-domain bromodomain-containing proteins.