Thermodynamic analysis of acetylation-dependent Pb1 bromodomain–histone H3 interactions

Abstract

An acetyl-histone peptide library was used to determine the thermodynamic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains interact with histones by binding acetylated lysines. The bromodomain used in this study, BrD3, is derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin remodeling complex. Steady-state fluorescence anisotropy was used to examine the variations in specificity and affinity that drive molecular recognition. Temperature and salt concentration dependence studies demonstrate that the hydrophobic effect is the primary driving force, consistent with lysine acetylation being required for binding. An electrostatic effect was observed in only two complexes where the acetyl-lysine was adjacent to an arginine. The large change in heat capacity determined for the specific complex suggests that the dehydrated BrD3-histone interface forms a tightly bound, high-affinity complex with the target site. These explorations into the thermodynamic driving forces that confer acetylation site-dependent BrD3-histone interactions improve our understanding of how individual bromodomains work in isolation. Furthermore, this work will permit the development of hypotheses regarding how the native Pb1, and the broader class of bromodomain proteins, directs multisubunit chromatin remodeling complexes to specific acetyl-nucleosome sites in vivo. KeywordsAcetyl-lysine; Histone acetylation; Polybromo; Histone code; Protein-protein interactions; Thermodynamics Histone acetylation is a key regulator of transcription; however, it is still a matter of debate as to whether the regulatory features are influenced by modification at specific lysine sites or it is the cumulative electrostatic consequences of random lysine acetylation. Mounting evidence indicates that large multiprotein chromatin remodeling complexes regulate transcription by localizing to certain chromatin sites through the interaction of bromodomain (BrD) 1 proteins with acetylated nucleosomes [1][2][3][4][5][6][7]. The BrD is a protein motif, approximately 100 amino acids in length, primarily associated with targeting subunits of chromatin remodeling complexes [8]. The biological role of the BrD centers on its ability to discriminate the acetylation state of lysine residues in histone proteins. In fact, there is growing evidence that the position of the acetyl-lysine is not random, but instead, the BrD requires acetylation at specific sites for highaffinity binding [9]. For example, fluorescence resonance energy transfer assays were used to show that the bromodomain protein Brd2 requires the interaction between its bromodomain and an acetyl-histone to amplify transcription in vivo [10]. To activate transcription after SAGA (Spt-Ada-Gcn5 acetyl-transferase) complex directed acetylation at specific nucleosomal locations, BrD proteins of the Swi2/Snf2 complex are required to displace acetyl-histones [11]. Likewise, the yeast RSC (remodels the structure of chromatin) complex requires multiple BrD...

Structural insights into selective histone H3 recognition by the human Polybromo bromodomain 2

“…This is a weak interaction but falls in the range of binding affinity previously reported for other BRD/lysine-acetylated peptide interactions such as human BRG1/H3K14ac ( K d of 1 mM) [17] and PCAF BRD/H3K14ac ( K d of 128 μM) [18]. It is noted that several recent papers reported single-digit micromolar affinities for BRD-acetylated histone peptide binding obtained using steady-state fluorescence anisotropy measurements [19, 20]. However, in our hands, we have never been able to obtain an affinity this high, including using a fluorescence polarization assay with a FITC-tagged H3K14ac peptide.…”

Binding specificity and function of the SWI/SNF subunit SMARCA4 bromodomain interaction with acetylated histone H3K14

“…The lower affinity of the Hs SMARCA4 BC may result from the different measurement techniques. In addition, different length H3 peptides are utilized in these studies ranging from H3 8–18 K14ac to H3 1–25 K14ac ( 30 , 59 ). Similar discrepancies with NMR measurements have been reported elsewhere, for example the K d for polybromo BRD2 was reported as ∼500 μM by NMR ( 60 ), and single-digit μM values by fluorescence anisotropy ( 59 , 61 ).…”

“…In addition, different length H3 peptides are utilized in these studies ranging from H3 8–18 K14ac to H3 1–25 K14ac ( 30 , 59 ). Similar discrepancies with NMR measurements have been reported elsewhere, for example the K d for polybromo BRD2 was reported as ∼500 μM by NMR ( 60 ), and single-digit μM values by fluorescence anisotropy ( 59 , 61 ). A future study is required that systematically compares binding affinity of BDs binding H3K14ac.…”

Histones: At the Crossroads of Peptide and Protein Chemistry

“… 50 Moreover, thermodynamic analyses performed on the binding of a typical BD to H3K9ac confirm that such interactions are primarily driven by the hydrophobic effect. 51 …”