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Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family

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Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family Resource Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family Panagis Filippakopoulos,1,* Sarah Picaud,1 Maria Mangos,3 Tracy Keates,1 Jean-Philippe Lambert,5 Dalia Barsyte-Lovejoy,3 Ildiko Felletar,1 Rudolf Volkmer,6 Susanne Mu¨ller,1 Tony Pawson,5,7 Anne-Claude Gingras,5,7 Cheryl H. Arrowsmith,3,4 and Stefan Knapp1,2,8,* 1Nuffield Department of Clinical Medicine, Structural Genomics Consortium 2Department of Oncology University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7LD, UK 3Structural Genomics Consortium 4Ontario Cancer Institute, Campbell Family Cancer Research Institute and Department of Medical Biophysics University of Toronto, Toronto, Ontario M5G 1L7, Canada 5Centre for Systems Biology, Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada 6Institut fu¨ r Medizinische Immunologie, Charite´ -Universita¨ tsmedizin Berlin, Hessische Str. 3-4, 10115 Berlin, Germany 7Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada 8Department of Biochemistry and Molecular Biology, George Washington University, School of Medicine and Health Sciences, 2300 Eye Street, NW, Suite 530, Washington, DC, 20037, USA *Correspondence: panagis.fi[email protected] (P.F.), [email protected] (S.K.) DOI 10.1016/j.cell.2012.02.013 SUMMARY effect on the physiochemical properties of modified lysine resi- dues neutralizing the positive charge of the ε-amino group Bromodomains (BRDs) are protein interaction (Kouzarides, 2000). Lysine acetylation is abundant in large modules that specifically recognize ε-N-lysine acety- macromolecular complexes that function in chromatin remodel- lation motifs, a key event in the reading process of ing, DNA damage, and cell-cycle control (Choudhary et al., 2009) epigenetic marks. The 61 BRDs in the human and particularly in histones. Cellular acetylation levels are genome cluster into eight families based on struc- stringently controlled by two enzyme families: the histone acetyl- ture/sequence similarity. Here, we present 29 high- transferases (HATs) and histone deacetylases (HDACs) (Shahba- zian and Grunstein, 2007). Histone acetylation has been associ- resolution crystal structures, covering all BRD fami- ated with transcriptional activation, but specific marks have also lies. Comprehensive crossfamily structural analysis been linked to DNA repair (Kouzarides, 2007). identifies conserved and family-specific structural Bromodomains (BRDs) are protein interaction modules that features that are necessary for specific acetylation- exclusively recognize acetylation motifs. BRDs are evolutionarily dependent substrate recognition. Screening of more conserved and present in diverse nuclear proteins comprising than 30 representative BRDs against systematic HATs (GCN5, PCAF), ATP-dependent chromatin-remodeling histone-peptide arrays identifies new BRD sub- complexes (BAZ1B), helicases (SMARCA), methyltransferases strates and reveals a strong influence of flanking (MLL, ASH1L), transcriptional coactivators (TRIM/TIF1, TAFs) posttranslational modifications, such as acetylation transcriptional mediators (TAF1), nuclear-scaffolding proteins and phosphorylation, suggesting that BRDs recog- (PB1), and the BET family (Muller et al., 2011)(Figure 1A and nize combinations of marks rather than singly acety- Table 1). Despite large sequence variations, all BRD modules share a conserved fold that comprises a left-handed bundle of lated sequences. We further uncovered a structural four a helices (a , a , a , a ), linked by loop regions of variable mechanism for the simultaneous binding and recog- Z A B C length (ZA and BC loops), which line the Kac binding site and nition of diverse diacetyl-containing peptides by determine binding specificity. Cocrystal structures with peptides BRD4. These data provide a foundation for struc- have demonstrated that Kac is recognized by a central deep ture-based drug design of specific inhibitors for this hydrophobic cavity, where it is anchored by a hydrogen bond emerging target family. to an asparagine residue present in most BRDs (Owen et al., 2000). INTRODUCTION Dysfunction of BRD proteins has been linked to development of several diseases. For instance, recurrent t(15;19) chromo- ε-N-acetylation of lysine residues (Kac) is one of the most somal translocations that result in a fusion protein that frequently occurring posttranslational modifications (PTMs) in comprises both BRD4 or BRD3 and the NUT (nuclear protein in proteins (Choudhary et al., 2009). Acetylation has a profound testis) lead to an aggressive form of human squamous 214 Cell 149, 214–231, March 30, 2012 ª2012 Elsevier Inc. carcinoma (French, 2010a; French et al., 2001). Deregulation of BRDs. BRD-containing proteins are large multidomain proteins transcription as a consequence of altered protein acetylation associated with chromatin remodeling, transcriptional control, patterns is a hallmark of cancer, a mechanism that is currently methyl or acetyltransferase activity, or helicases (Figure 1A and targeted by HDAC inhibitors (Lane and Chabner, 2009). It is likely Table 1). The domain organization in BRD-containing proteins that selective inhibitors capable of targeting BRDs will find broad is evolutionarily highly conserved, and the BRD motif is often application in medicine and basic research as exemplified by the flanked by other epigenetic reader domains. Most frequently recent development of highly specific and potent acetyl-lysine observed combinations include the presence of plant homeodo- competitive BET BRD inhibitors (Chung et al., 2011; Dawson mains (PHDs) N terminal to the BRD, multiple BRDs, as well as et al., 2011; Delmore et al., 2011; Filippakopoulos et al., 2010; various other domains that generally mediate protein interac- Mertz et al., 2011; Nicodeme et al., 2010). tions such as bromo-adjacent homology (BAH) domains (Good- To date, only a small number of lysine acetylation marks have win and Nicolas, 2001). been identified to specifically interact with individual BRDs, and Phylogenetic analysis of the BRD family outside the two central the often weak affinities reported for BRD interactions with their core helices was complicated by the low-sequence homology potential target sites have been determined by a variety of and nonconserved insertions in BRD loop regions. We therefore different techniques making data comparison difficult (Muller used three-dimensional structure-based alignments including et al., 2011). Reported affinities range from nano- to millimolar available NMR models together with secondary structure predic- dissociation constant (KD) values raising the issue of which tion (Jones, 1999) and manual curation of the aligned sequences affinity window is relevant for specific BRD-peptide interactions. to establish an alignment of all human BRDs (Figure S1). The For instance, the BRDs of BRD2 have been shown to bind derived phylogram clustered into eight major BRD families desig- histone 4 acetylated at lysine 12 (H4K12ac)(Kanno et al., 2004), nated by Roman numerals (I–VIII) (Figure 1B). Key references for with KD values that range from 360 mM for the diacetylated all BRD-containing proteins are included in Table 1. peptide H4K5acK12ac (Umehara et al., 2010) to 2.9 mM for the Multiple protein interaction modules can be tightly linked to monoacetylated H4K12ac peptide (Huang et al., 2007). Closely form a single stable interaction domain, or they can be con- spaced multiple Kac sites have also been shown to significantly nected by flexible linker sequences allowing conformational increase affinity of the histone H4 N terminus for BRDT by simul- adaptation to diverse sequences motifs. An example of a tightly taneous binding to the same BRD (Morinie` re et al., 2009). Thus, linked dual-domain reader is the PHD-BRD of TRIM24, in which the field would greatly benefit from a more systematic analysis both domains interact through a large interface that orients both of BRD structure and peptide binding properties in order to peptide binding cavities to the same side of the protein (Tsai better understand acetylation-mediated signaling as interpreted et al., 2010). In contrast, the two BRDs in TAF1 are free to orient through BRDs. independently as shown by the different domain orientations Here, we present a comprehensive structural characterization in the dual-domain structure determined here and a previously of the human BRD family together with identified Kac-specific published model (Jacobson et al., 2000)(Figure 1C). The fre- interaction sites of these essential protein recognition modules quent combination of multiple interaction modules in the same with their target sites in histones. Using available sequence data- protein suggests that the epigenetic reading process involves bases, we identified 61 BRDs in the human proteome that are concomitant recognition of several PTMs. present in 46 diverse proteins. High-throughput cloning led to the establishment of 171 expression systems that yielded func- Structural Analysis tional recombinant proteins. Using these reagents, we crystal- In order to establish a platform of recombinant BRDs for func- lized and determined the structures of 29 BRDs, including 25 tional and structural studies, we subcloned all human BRDs structures that had not been previously published. We per- into bacterial expression systems in frame with a cleavable formed a SPOT blot analysis that covered all possible Kac sites N- (or C-) terminal
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