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Short Article Structure Short Article Structural Insights into Acetylated-Histone H4 Recognition by the Bromodomain-PHD Finger Module of Human Transcriptional Coactivator CBP Alexander N. Plotnikov,1 Shuai Yang,1 Thomas Jiachi Zhou,1 Elena Rusinova,1 Antonio Frasca,1 and Ming-Ming Zhou1,* 1Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.10.021 SUMMARY Acetylation at site-specific lysine residues in nucleosomal his- tones represents distinct biological functions to direct ordered Bromodomain functions as the acetyl-lysine bind- gene transcription. For instance, single acetylation of histone ing domains to regulate gene transcription in chro- H3 at Lys14 (H3K14ac) or Lys18 (H3K18ac) marks for chromatin matin. Bromodomains are rapidly emerging as new remodeling, whereas diacetylation of histone H4 at Lys5 and Lys8 epigenetic drug targets for human diseases. How- (H4K5ac/K8ac) or Lys12 and Lys16 (H4K12/K16ac) signals an ever, owing to their transient nature and modest active state of gene transcription. In contrast to histone methyl- affinity, histone-binding selectivity of bromodomains lysine binding protein modules such as chromodomains and PHD fingers (Patel and Wang, 2013; Yap and Zhou, 2010), BrD/ has remained mostly elusive. Here, we report high- acetyl-lysine interactions are typically transient and of modest resolution crystal structures of the bromodomain- tens-to-hundreds micromolar affinity (Filippakopoulos et al., PHD tandem module of human transcriptional 2012). As such, histone binding selectivity of human BrDs has coactivator CBP bound to lysine-acetylated histone remained mostly elusive. The human BrD family consists of 61 H4 peptides. The structures reveal that the PHD members residing in 46 proteins of different chromatin- and finger serves a structural role in the tandem module transcription-associated functions (Figure 1A). Of these are two and that the bromodomain prefers lysine-acetylated distinct subgroups representing the histone acetyltransferase motifs comprising a hydrophobic or aromatic residue transcriptional coactivators, including CBP/p300 and PCAF, at À2 and a lysine or arginine at À3orÀ4 position and the BET family proteins that are characteristic of two tandem from the acetylated lysine. Our study further provides BrDs such as BRD4. The latter have been extensively character- structural insights into distinct modes of singly and ized recently (Filippakopoulos et al., 2012; Morinie` re et al., 2009). However, despite their equally important functions for lysine diacetylated histone H4 recognition by the bromodo- acetylation in gene activation, the histone binding selectivity of mains of CBP and BRD4 that function differently as a the former subgroup of BrDs is not well understood. transcriptional coactivator and chromatin organizer, Notably, it has been previously reported by Ragvin and respectively, explaining their distinct roles in control coworkers that the BrD-PHD finger tandem module of human co- of gene expression in chromatin. activator p300 binds to nucleosomes with a high degree of lysine acetylation and that this binding requires the presence of both the bromodomain and the PHD finger (Ragvin et al., 2004). Further- INTRODUCTION more, our recent nuclear magnetic resonance (NMR) binding analysis revealed that the BrD of CBP, which shares 98% The bromodomain (BrD) recognition of acetylated lysine resi- sequence identity to that of p300, exhibits a preference for bind- dues in histones is a fundamental molecular mechanism in ing to Lys20-acetylated histone H4 (H4K20ac; GGARKRHR-Kac- control of gene transcription in chromatin (Dhalluin et al., 1999; VLRDNIQ, residues 13–27) over other lysine-acetylated histone Sanchez and Zhou, 2009). BrD and acetyl-lysine binding serves peptides, of which the molecular underpinning is not well under- to modulate chromatin structure opening, facilitate the recruit- stood (Zeng et al., 2008b). Determination of histone binding spec- ment of transcription factors to target gene promoter and ificity of BrDs would require detailed knowledge of the molecular enhancer sites, and promote the assembly and activation of basis of BrD recognition of functionally relevant acetylated the paused RNA polymerase II machinery complex for histone peptides. In this study, we report two crystal structures productive gene transcription (Chiang, 2009). Owing to their of the BrD-PHD finger tandem module of CBP in complex with key functions in gene activation, the BrDs of transcription-asso- lysine-acetylated histone H4 peptides. ciated proteins such as BET (bromodomain and extra-terminal domain) proteins (Dawson et al., 2011; Delmore et al., 2011; Fil- RESULTS AND DISCUSSION ippakopoulos et al., 2010; Nicodeme et al., 2010; Zhang et al., 2012; Zuber et al., 2011) and transcription coactivators CBP Structures of the CBP BrD-PHD Module/Acetylated H4 (Borah et al., 2011) and PCAF (Zeng et al., 2005) have been re- Peptide Complexes ported as new epigenetic drug targets for a wide array of human To determine histone binding selectivity of the CBP BrD and how diseases, including cancer and inflammation. it functions together with the adjacent PHD finger, we solved the Structure 22, 353–360, February 4, 2014 ª2014 Elsevier Ltd All rights reserved 353 Structure Structure and H4 Recognition of CBP BrD-PHD Module Figure 1. Crystal Structure of CBP BrD-PHD in Complex with H4K20ac Peptide (A) Domain organization of CBP and other human BrD proteins (left). Phylogenetic tree of human BrD proteins (right). (B) Ribbon depiction of 3D structure of the tandem BrD (green) and PHD finger (orange) of CBP bound to a histone H4K20ac peptide (shown in sticks with carbon atoms in yellow). The linker of the BrD-PHD module is colored in light cyan and regions that lack electron density are indicated by dots. Zinc atoms are shown in magenta spheres. (C) Surface-filled representation of the CBP BrD-PHD/H4K20ac complex structure. (D) Electrostatic surface potential representation of the CBP BrD-PHD/H4K20ac structure. See also Figure S1. crystal structures of the BrD-PHD tandem module of human CBP S1A and S1B available online) or H4K12ac/K16ac (KGGKGLG- in complex with a lysine-acetylated histone H4 peptide (residues Kac-GGA-Kac-RHRKVLRDN) at 1.9 A˚ and 1.83 A˚ resolution, 5–25) containing either H4K20ac (KGGKG-LGKGGAKRHR-Kac- respectively (Table 1). The new structures revealed that the VLRDN where Kac is acetylated lysine; Figures 1B–1D; Figures BrD and the PHD finger in the tandem domain establish direct 354 Structure 22, 353–360, February 4, 2014 ª2014 Elsevier Ltd All rights reserved Structure Structure and H4 Recognition of CBP BrD-PHD Module Table 1. Crystallography Data and Refinement Statistics unreported insights into the molecular functions of CBP in con- trol of gene transcription in chromatin. CBP-BP/H4K20ac CBP-BP/H4K12ac/K16ac PDB code 4N3W 4N4F BrD-PHD Finger Interactions Data collection The PHD finger of CBP has the canonical PHD finger fold that is Space group C121 C121 composed of a small two-stranded b sheet packaged on one Cell dimensions side by a short a helix that is stabilized by two zinc atoms a, b, c (A˚ ) 92.46, 59.31, 92.01, 59.46, 53.88 coordinated by Cys1199, Cys1200, His1291, and Cys1294 and 53.44 Cys1283, Cys1286, Cys1308, and Cys1311, respectively (Fig- a, b, g () 90, 102.96, 90 90, 102.68, 90 ure 2A). The two zinc coordination centers also serve as a stable Resolution (highest 30.00–1.9 30.00–1.83 (1.88–1.83) base for an extended interface established between the PHD resolution shell) (A˚ ) (1.95–1.9) finger and the BrD. This interdomain interface comprises a network of hydrophobic/aromatic and electrostatic interac- Rmerge (%) 5.1 (32.2) 4.9 (48.9) I/sI 35.9 (5.6) 26.7 (2.7) tions involving Asp1149 with Lys1289, Trp1151 with Phe1280, Asp1155 with Lys1203, Trp1158 with Tyr1204, and Tyr1198 with Completeness (%) 98.7 (93.8) 98.4 (96.9) Arg1288 of the BrD and the PHD finger, respectively (Figure 2B). Redundancy 7.4 (7.1) 4.9 (4.4) Notably, while many of these residues involved in interdomain Refinement interactions are conserved in CBP or p300 from different species Resolution (A˚ ) 19.6–1.9 19.69–1.83 from human to mouse, they are not all present in the BrDs from No. of reflections 20,884 23,279 many different types of transcription-associated proteins, partic- Rwork/Rfree (%) 23.2/28.3 20.9/24.2 ularly BET proteins and those that do not contain BrD and PHD No. of atoms 1,709 1,785 finger tandem modules (Figure S2). Domain-domain interactions Rmsd or orientation even in the BrD-PHD finger or PHD finger-BrD tandem modules varies widely, as shown in comparison of such Bond length (A˚ ) 0.016 0.02 tandem module structures from BPTF (Li et al., 2006), TRIM24 Bond angle ( ) 1.8 1.7 (Tsai et al., 2010), TRIM28 (Zeng et al., 2008a), and TRIM33 (Xi et al., 2011)(Figure 2C). This suggests that domain-domain orien- interactions forming a single structural unit. Specifically, the CBP tation in the tandem modules is likely related to specific functions BrD adopts the classical left-handed four-helix bundle structure of the individual domains or of the overall tandem modules, which in complex with the acetylated H4 peptide. Either H4-peptide- have diverse functionality in molecular interactions with histones bound structure of the BrD is nearly identical to that of the protein in control of gene transcription. Indeed, unlike the other tandem in the free state with root-mean-square deviations (rmsd) of modules that interact with histones, the tandem PHD finger-BrD 0.46 A˚ and 0.47 A˚ for all Ca atoms, respectively. The PHD finger of human corepressor TRIM28 (also known as KRAB-associated is packaged against the BrD between helix B and helix C at one protein or KAP1) functions as a unique small ubiquitin-like modi- end of the helical bundle opposite from the acetyl-lysine binding fier E3 ligase for gene transcription silencing (Zeng et al., 2008a).
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