@article{enriquez_krajewski_strahl_rothbart_dowen_rose_2021, title={Binding specificity and function of the SWI/SNF subunit SMARCA4 bromodomain interaction with acetylated histone}, volume={297}, ISSN={["1083-351X"]}, url={https://doi.org/10.1016/j.jbc.2021.101145}, DOI={10.1016/j.jbc.2021.101145}, abstractNote={Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD–H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small-molecule BD inhibitors to treat cancers and other diseases. Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD–H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small-molecule BD inhibitors to treat cancers and other diseases. Bromodomains (BD) are highly conserved epigenetic reader modules that recognize acetyl-lysine (Kac) on histones and other proteins (1Filippakopoulos P. Picaud S. Mangos M. Keates T. Lambert J.P. Barsyte-Lovejoy D. Felletar I. Volkmer R. Muller S. Pawson T. Gingras A.C. Arrowsmith C.H. Knapp S. Histone recognition and large-scale structural analysis of the human bromodomain family.Cell. 2012; 149: 214-231Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar, 2Flynn E.M. Huang O.W. Poy F. Oppikofer M. Bellon S.F. Tang Y. Cochran A.G. A subset of human bromodomains recognizes butyryllysine and crotonyllysine histone peptide modifications.Structure. 2015; 23: 1801-1814Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). (Note: for clarity, histone residues will be referred to in one-letter code, and BD residues will be referred to in three-letter code.) In the nearly 30 years since BDs were first identified, the chromatin field has accumulated a wealth of biophysical, structural, and biochemical data on BDs and other epigenetic readers (3Haynes S.R. Dollard C. Winston F. Beck S. Trowsdale J. Dawid I.B. The bromodomain: A conserved sequence found in human, Drosophila and yeast proteins.Nucleic Acids Res. 1992; 20: 2603Crossref PubMed Scopus (308) Google Scholar, 4Tamkun J.W. Deuring R. Scott M.P. Kissinger M. Pattatucci A.M. Kaufman T.C. Kennison J.A. Brahma: A regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SWI2.Cell. 1992; 68: 561-572Abstract Full Text PDF PubMed Scopus (751) Google Scholar). Structures from eight human BD families have been solved (1Filippakopoulos P. Picaud S. Mangos M. Keates T. Lambert J.P. Barsyte-Lovejoy D. Felletar I. Volkmer R. Muller S. Pawson T. Gingras A.C. Arrowsmith C.H. Knapp S. Histone recognition and large-scale structural analysis of the human bromodomain family.Cell. 2012; 149: 214-231Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar), and small molecules are now available for BD inhibition (5Clegg M.A. Tomkinson N.C.O. Prinjha R.K. Humphreys P.G. Advancements in the development of non-BET bromodomain chemical probes.ChemMedChem. 2019; 14: 362-385Crossref PubMed Scopus (17) Google Scholar). Most BDs and their binding partners have been well characterized in vitro and in various cell lines. The precise functional role and mechanistic underpinnings of BD–histone target specificity at the organismal level, however, remain largely unknown. To date, only a handful of studies have examined BDs in vivo and have done so only in the context of chemical-probe inhibition (6Faivre E.J. McDaniel K.F. Albert D.H. Mantena S.R. Plotnik J.P. Wilcox D. Zhang L. Bui M.H. Sheppard G.S. Wang L. Sehgal V. Lin X. Huang X. Lu X. Uziel T. et al.Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer.Nature. 2020; 578: 306-310Crossref PubMed Scopus (110) Google Scholar, 7Filippakopoulos P. Qi J. Picaud S. Shen Y. Smith W.B. Fedorov O. Morse E.M. Keates T. Hickman T.T. Felletar I. Philpott M. Munro S. McKeown M.R. Wang Y. Christie A.L. et al.Selective inhibition of BET bromodomains.Nature. 2010; 468: 1067-1073Crossref PubMed Scopus (2528) Google Scholar, 8Matzuk M.M. McKeown M.R. Filippakopoulos P. Li Q. Ma L. Agno J.E. Lemieux M.E. Picaud S. Yu R.N. Qi J. Knapp S. 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Despite the advent of genome editing, no research group has—to our knowledge — disrupted BD–histone interactions in a complex multicellular organism to investigate the contributions of BD binding on cell differentiation and development. Nor have researchers begun to mine the vast structural and sequence data already available to elucidate potential global patterns of specificity and plasticity common among BD subfamilies targeting the same marks. This paper addresses these gaps in studies of chromatin regulation by epigenetic readers. SMARCA4/BRG1 is an essential catalytic core subunit of the roughly two-megadalton Switch/Sucrose Nonfermenting (SWI/SNF) multiprotein complex, which uses the energy of ATP hydrolysis to remodel chromatin by perturbing interactions between histone core particles and DNA (14Kwon C.S. Wagner D. Unwinding chromatin for development and growth: A few genes at a time.Trends Genet. 2007; 23: 403-412Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 15Sudarsanam P. Winston F. The SWI/SNF family nucleosome-remodeling complexes and transcriptional control.Trends Genet. 2000; 16: 345-351Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). SMARCA4 can remodel nucleosomal substrates by itself in vitro (16Phelan M.L. Schnitzler G.R. Kingston R.E. Octamer transfer and creation of stably remodeled nucleosomes by human SWI-SNF and its isolated ATPases.Mol. Cell. Biol. 2000; 20: 6380-6389Crossref PubMed Scopus (91) Google Scholar, 17Phelan M.L. Sif S. Narlikar G.J. Kingston R.E. Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits.Mol. Cell. 1999; 3: 247-253Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar) and is functionally and structurally conserved among eukaryotes (14Kwon C.S. Wagner D. Unwinding chromatin for development and growth: A few genes at a time.Trends Genet. 2007; 23: 403-412Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 18Treand C. du Chene I. Bres V. Kiernan R. Benarous R. Benkirane M. Emiliani S. Requirement for SWI/SNF chromatin-remodeling complex in tat-mediated activation of the HIV-1 promoter.EMBO J. 2006; 25: 1690-1699Crossref PubMed Scopus (133) Google Scholar). This remodeling enzyme possesses intrinsic ATPase and helicase activity and has a C-terminal BD motif capable of recognizing lysine 14 acetylation on histone H3 (H3K14ac) (19Morrison E.A. Sanchez J.C. Ronan J.L. Farrell D.P. Varzavand K. Johnson J.K. Gu B.X. Crabtree G.R. Musselman C.A. DNA binding drives the association of BRG1/hBRM bromodomains with nucleosomes.Nat. Commun. 2017; 8: 16080Crossref PubMed Scopus (34) Google Scholar) (Fig. 1A). Malfunction or loss of the SMARCA4 subunit has been implicated in numerous cancers, aberrant patterns of cell differentiation, inflammatory responses, and metabolic dysfunction (20Khavari P.A. Peterson C.L. Tamkun J.W. Mendel D.B. Crabtree G.R. BRG1 contains a conserved domain of the SWI2/SNF2 family necessary for normal mitotic growth and transcription.Nature. 1993; 366: 170-174Crossref PubMed Scopus (520) Google Scholar, 21Medina P.P. Romero O.A. Kohno T. Montuenga L.M. Pio R. Yokota J. Sanchez-Cespedes M. Frequent BRG1/SMARCA4-inactivating mutations in human lung cancer cell lines.Hum. Mutat. 2008; 29: 617-622Crossref PubMed Scopus (191) Google Scholar, 22Ramos P. Karnezis A.N. Craig D.W. Sekulic A. Russell M.L. Hendricks W.P. Corneveaux J.J. Barrett M.T. Shumansky K. Yang Y. Shah S.P. Prentice L.M. Marra M.A. Kiefer J. Zismann V.L. et al.Small cell carcinoma of the ovary, hypercalcemic type, displays frequent inactivating germline and somatic mutations in SMARCA4.Nat. Genet. 2014; 46: 427-429Crossref PubMed Scopus (201) Google Scholar, 23Tian W. Xu H. Fang F. Chen Q. Xu Y. Shen A. Brahma-related gene 1 bridges epigenetic regulation of proinflammatory cytokine production to steatohepatitis in mice.Hepatology. 2013; 58: 576-588Crossref PubMed Scopus (77) Google Scholar, 24Wong A.K. Shanahan F. Chen Y. Lian L. Ha P. Hendricks K. Ghaffari S. Iliev D. Penn B. Woodland A.M. Smith R. Salada G. Carillo A. Laity K. Gupte J. et al.BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines.Cancer Res. 2000; 60: 6171-6177PubMed Google Scholar). How SMARCA4 targets, is recruited to, and interacts with chromatin substrates to induce ATP-dependent nucleosome remodeling has been a long-standing question. Recent structures of the SMARCA4 catalytic-core domain in complex with a nucleosome core particle, as well as a nucleosome-bound SWI/SNF complex comprising SMARCA4 and nine auxiliary subunits, provided insights into the mechanism of SWI/SNF remodeling (25He S. Wu Z. Tian Y. Yu Z. Yu J. Wang X. Li J. Liu B. Xu Y. Structure of nucleosome-bound human BAF complex.Science. 2020; 367: 875-881Crossref PubMed Scopus (69) Google Scholar, 26Liu X. Li M. Xia X. Li X. Chen Z. Mechanism of chromatin remodelling revealed by the Snf2-nucleosome structure.Nature. 2017; 544: 440-445Crossref PubMed Scopus (125) Google Scholar). But the structural basis underlying SMARCA4 recognition of its histone tail target has remained elusive despite the existence of SMARCA4 structures in the apo state (1Filippakopoulos P. Picaud S. Mangos M. Keates T. Lambert J.P. Barsyte-Lovejoy D. Felletar I. Volkmer R. Muller S. Pawson T. Gingras A.C. Arrowsmith C.H. Knapp S. Histone recognition and large-scale structural analysis of the human bromodomain family.Cell. 2012; 149: 214-231Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar, 27Shen W. Xu C. Huang W. Zhang J. Carlson J.E. Tu X. Wu J. Shi Y. Solution structure of human Brg1 bromodomain and its specific binding to acetylated histone tails.Biochemistry. 2007; 46: 2100-2110Crossref PubMed Scopus (86) Google Scholar, 28Singh M. Popowicz G.M. Krajewski M. Holak T.A. Structural ramification for acetyl-lysine recognition by the bromodomain of human BRG1 protein, a central ATPase of the SWI/SNF remodeling complex.Chembiochem. 2007; 8: 1308-1316Crossref PubMed Scopus (43) Google Scholar) and in complex with a chemical probe (PFI-3) selective for four family VIII BD (29Fedorov O. Castex J. Tallant C. Owen D.R. Martin S. Aldeghi M. Monteiro O. Filippakopoulos P. Picaud S. Trzupek J.D. Gerstenberger B.S. Bountra C. Willmann D. Wells C. Philpott M. et al.Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance.Sci. Adv. 2015; 1e1500723Crossref PubMed Scopus (74) Google Scholar). More recently, a structure of the Saccharomyces cerevisiae Sth1 (ScSth1) BD in complex with H3K14ac (30Chen G. Li W. Yan F. Wang D. Chen Y. The structural basis for specific recognition of H3K14 acetylation by Sth1 in the RSC chromatin remodeling complex.Structure. 2020; 28: 111-118.e113Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) revealed the histone binding mode for the catalytic core of the yeast RSC (Remodel the Structure of Chromatin) complex—a paralog that is at least ten times more abundant than SWI/SNF and differs in both component organization and physiological function (31Chen G. Wang D. Wu B. Yan F. Xue H. Wang Q. Quan S. Chen Y. Taf14 recognizes a common motif in transcriptional machineries and facilitates their clustering by phase separation.Nat. Commun. 2020; 11: 4206Crossref PubMed Scopus (4) Google Scholar, 32Ye Y. Wu H. Chen K. Clapier C.R. Verma N. Zhang W. Deng H. Cairns B.R. Gao N. Chen Z. Structure of the RSC complex bound to the nucleosome.Science. 2019; 366: 838-843Crossref PubMed Scopus (46) Google Scholar). Unlike SMARCA4, which exclusively targets mono- and di-acetylated H3 tails, Sth1 binds more promiscuously to H3K14ac and other mono-acetylated lysine posttranslational modifications (PTMs) on histones H3 and H4, including H4K20ac, H3K18ac, and H3K27ac (30Chen G. Li W. Yan F. Wang D. Chen Y. The structural basis for specific recognition of H3K14 acetylation by Sth1 in the RSC chromatin remodeling complex.Structure. 2020; 28: 111-118.e113Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Thus, a more complete understanding of the specific contributions of the SMARCA4 BD to SWI/SNF biology requires structural and, more importantly, in vivo validation in a complex multicellular organism, which is sorely lacking in the field. Here, we report findings from structural, physicochemical, and in vivo genetic investigations of the SMARCA4 epigenetic-reader domain. We use the reference nematode Caenorhabditis elegans and its SMARCA4 ortholog (SWSN-4 or CeSMARCA4)—which is conserved among eukaryotes and constitutes the only SWI/SNF ATPase in the worm—as a model. Using peptide microarrays, we show that the CeSMARCA4 BD is, like its human ortholog, highly selective for H3K14ac. We analyzed the basis for H3K14ac-binding selectivity by solving a 1.29 Å resolution structure of the CeSMARCA4 BD–H3K14ac complex and comparing it to other BDs that bind the mark. Based on the premise that binding specificity is evolutionarily conserved within each BD ortholog, we identify residues distant from the H3 binding site that contribute to selective H3K14ac recognition, which could be exploited to create highly specific, next-generation BD chemical probes. To examine the functional significance of the CeSMARCA4 BD–H3K14ac interaction in vivo, we engineered specific BD mutations into the C. elegans swsn-4 gene using CRISPR-Cas9 genome editing (33Enriquez P. CRISPR-mediated epigenome editing.Yale J. Biol. Med. 2016; 89: 471-486PubMed Google Scholar). While BD mutations that abolish acetyl-lysine binding in vitro only modestly impact C. elegans viability, we found that a combination of SMARCA4 BD binding mutants with genetic inactivation of the pbrm-1 gene, which encodes an accessory SWI/SNF subunit, resulted in enhanced embryonic lethality and fertility defects. These data suggest that the SMARCA4 BD plays a significant and redundant role with other members of the SWI/SNF complex in vivo. Collectively, our findings underscore a pressing need for in vivo validation of studies employing BD inhibitors and in vitro-derived data to interpret the functional roles of epigenetic readers in chromatin regulation and signaling. To assess whether SMARCA4 BD–H3K14 binding is conserved between mammals and C. elegans, we screened a recombinantly expressed and purified Glutathione S–Transferase (GST)–bromodomain fusion protein against a microarrayed library of 300+ biotinylated histone peptides (34Rothbart S.B. Krajewski K. Strahl B.D. Fuchs S.M. Peptide microarrays to interrogate the “histone code”.Methods Enzymol. 2012; 512: 107-135Crossref PubMed Scopus (53) Google Scholar). The library comprises peptides from all core histones, as well as the major histone variants, in single and combinatorial modification states. Each peptide contains a terminal biotin tag for immobilization on streptavidin-coated glass slides. The CeSMARCA4 GST–BD fusion bound specifically to H3K14ac-containing peptides. Co-occurrence of H3K9ac or H3K18ac weakened the interaction, and CeSMARCA4 did not bind to unmodified histone H3 N-terminal tails (Fig. 1, B–D). Overall, the CeSMARCA4 BD is highly selective for mono-acetylated H3K14 tails, suggesting functional and structural conservation between the human and worm proteins. We also tested the human BAZ2B BD, a family V BD, and found that, like the CeSMARCA4 (family VIII) BD, it is highly selective for H3K14ac-modified peptides; however, BAZ2B also recognizes poly-acetylated tails, binding to di- (H3K9acK14ac) and tri-acetylated (H3K9acK14acK18ac) peptides (Fig. 1D) (35Ferguson F.M. Dias D.M. Rodrigues J.P. Wienk H. Boelens R. Bonvin A.M. Abell C. Ciulli A. Binding hotspots of BAZ2B bromodomain: Histone interaction revealed by solution NMR driven docking.Biochemistry. 2014; 53: 6706-6716Crossref PubMed Scopus (15) Google Scholar, 36Philpott M. Yang J. Tumber T. Fedorov O. Uttarkar S. Filippakopoulos P. Picaud S. Keates T. Felletar I. Ciulli A. Knapp S. Heightman T.D. Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery.Mol. Biosyst. 2011; 7: 2899-2908Crossref PubMed Scopus (109) Google Scholar, 37Tallant C. Valentini E. Fedorov O. Overvoorde L. Ferguson F.M. Filippakopoulos P. Svergun D.I. Knapp S. Ciulli A. Molecular basis of histone tail recognition by human TIP5 PHD finger and bromodomain of the chromatin remodeling complex NoRC.Structure. 2015; 23: 80-92Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We next sought to quantitate the CeSMARCA4 GST–BD binding affinity for H31–20K14ac and H37–20K14ac histone peptides (Fig. 2A) in solution via isothermal titration calorimetry (ITC). We calculated single-digit and low double-digit μM dissociation constants—KD = 9.3 ± 0.2 and 11.6 ± 0.1 μM, respectively—for these modified histone peptides (Fig. 2B). To further assess whether GST interferes with binding affinity of the complex, we cleaved off the GST tag and retested binding for H37–20K14ac via ITC, which yielded KD = 23.4 ± 0.8 μM and confirmed that GST does not interfere with BD binding. Our results contrast sharply with previous studies of the binding affinities between the Homo sapiens SMARCA4/2 (HsSMARCA4/2) ortholog and titrated H3K14ac-modified histone peptides via NMR perturbation experiments, which reported KDs of approximately 1.2 mM for H39–18K14ac (27Shen W. Xu C. Huang W. Zhang J. Carlson J.E. Tu X. Wu J. Shi Y. Solution structure of human Brg1 bromodomain and its specific binding to acetylated histone tails.Biochemistry. 2007; 46: 2100-2110Crossref PubMed Scopus (86) Google Scholar), 500 μM for H33–17K9acK14ac (28Singh M. Popowicz G.M. Krajewski M. Holak T.A. Structural ramification for acetyl-lysine recognition by the bromodomain of human BRG1 protein, a central ATPase of the SWI/SNF remodeling complex.Chembiochem. 2007; 8: 1308-1316Crossref PubMed Scopus (43) Google Scholar), and 900 μM for H39–19K14ac (19Morrison E.A. Sanchez J.C. Ronan J.L. Farrell D.P. Varzavand K. Johnson J.K. Gu B.X. Crabtree G.R. Musselman C.A. DNA binding drives the association of BRG1/hBRM bromodomains with nucleosomes.Nat. Commun. 2017; 8: 16080Crossref PubMed Scopus (34) Google Scholar) peptides. To understand how SMARCA4 interacts with modified histones, we determined the crystal structure of the C. elegans SMARCA4 BD (residues 1176–1296) in complex with an H37–20K14ac modified peptide at 1.29 Å resolution (Table 1). The CeSMARCA4 BD exhibits the canonical fold of a left-handed bundle of four α-helices (αZ, αA, αB, αC) linked by one long (ZA) and two short (AB and BC) interhelical loops (Fig. 3A). The ZA loop includes two short helices (αZ′ and αA′) and a 310 helical turn preceding αA’. The four amphipathic α-helices are antiparallel and pack tightly against each other to define the hydrophobic cavity for acetyl-lysine recognition (38Owen D.J. Ornaghi P. Yang J.C. Lowe N. Evans P.R. Ballario P. Neuhaus D. Filetici P. Travers A.A. The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p.EMBO J. 2000; 19: 6141-6149Crossref PubMed Scopus (395) Google Scholar). Residues Tyr1196–Ile1204 fold into a β-hairpin structure characteristic of family VIII BD.Table 1Crystallographic statisticsData statistics PDB Code7LHY Wavelength (Å)1.0 Resolution range (Å)48.8–1.29 (1.34–1.29) Space groupP 41 21 2 Unit cell69.06 69.06 55.3290.00 90.00 90.00 Unique reflections34,008 (3336) Multiplicity13.8 (12) Completeness (%)99.6 (100) Mean I/sigma(I)13.9 (2.1) Wilson B-factor (Å2)17.2 R-sym0.075 (0.93)Refinement statistics Resolution (Å)26.97–1.29 (1.33–1.29) R-work0.1792 (0.2679) R-free0.1938 (0.2703) Number nonhydrogen atoms1022Macromolecules868H3 peptide39Water115 Protein residues110 RMS(bonds)0.007 RMS(angles)0.935 Ramachandran favored (%)100 Ramachandran outliers (%)0 Average B-factor (Å2)23.3Macromolecules22.3H3 peptide29.6Solvent31.3 Open table in a new tab The structure of the CeSMARCA4 BD–H37–20K14ac complex reveals the molecular histone–tail interactions of the SWI/SNF enzymatic core. Residues H313–17 of the modified histone peptide could be unequivocally traced in the electron density map (Fig. 3B). The histone peptide lysine acetylamide binds within the central, largely hydrophobic cavity, as observed for other BDs (Fig. 3, A and C), including the recently reported family VIII, yeast RSC ScSth1 BD–H3K14ac complex (30Chen G. Li W. Yan F. Wang D. Chen Y. The structural basis for specific recognition of H3K14 acetylation by Sth1 in the RSC chromatin remodeling complex.Structure. 2020; 28: 111-118.e113Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 38Owen D.J. Ornaghi P. Yang J.C. Lowe N. Evans P.R. Ballario P. Neuhaus D. Filetici P. Travers A.A. The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p.EMBO J. 2000; 19: 6141-6149Crossref PubMed Scopus (395) Google Scholar). The acetyl carbonyl group of H3K14ac forms hydrogen bonds with the conserved Asn1263 residue and a water molecule that bridges to the conserved Tyr1220 residue. These residues stabilize acetyl-lysine binding to BDs (38Owen D.J. Ornaghi P. Yang J.C. Lowe N. Evans P.R. Ballario P. Neuhaus D. Filetici P. Travers A.A. The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p.EMBO J. 2000; 19: 6141-6149Crossref PubMed Scopus (395) Google Scholar). Site-directed mutations of Tyr1220Ala or Asn1263Ala in the CeSMARCA4 BD independently abolish binding to H3 N-terminal tails in vitro, as confirmed by ITC (Fig. 2, A and B). A network of six water molecules, one of which also mediates a hydrogen bond between K14 Nε and the Val1207 backbone carbonyl, are buried within the hydrophobic cleft. Four of the six waters are conserved across BD families. The hydroxyl of Tyr1287 in the ScSth1 structure forms a hydrogen bond with the H3K14ac amide; however, this interaction is absent in the CeSMARCA4 structure. Additional histone peptide residues also interact with CeSMARCA4, either through the ZA- and BC-loop residues, or the αB- and αC-helices flanking the cleft. The H3G13 carbonyl is anchored to the αC-helix by a water-mediated hydrogen bond that bridges the main-chain amides of Glu1268 (Leu1545 in HsSMARCA4/2) and Ile1269 (Fig. 3E). The H3G13 carbonyl interacts with Trp1338 in the ScSth1 structure. H3A15 and H3P16 are hydrophobic (Fig. 3D) and mutating them to hydrophilic residues reduced binding to the ScSth1 BD (30Chen G. Li W. Yan F. Wang D. Chen Y. The structural basis for specific recognition of H3K14 acetylation by Sth1 in the RSC chromatin remodeling complex.Structure. 2020; 28: 111-118.e113Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). The H3A15 sidechain packs adjacent to the BC loop, anchored by hydrogen bonds between the peptide main chain with the carbonyl δ1-oxygen of the conserved Asn1263 (Fig. 3C) and the backbone amide of Glu1264 (Leu1573 in HsSMARCA4/2) (Fig. 3E). The H3P16 cyclic sidechain packs closely with the Tyr1262 benzene ring (Phe1571 in HsSMARCA4/2) (Fig. 3D). While KacXXR occurs multiple times within H3 and H4 tails, the KacXPR sequence is unique to H3K14ac (39Sabari B.R. Zhang D. Allis C.D. Zhao Y. Metabolic regulation of gene expression through histone acylations.Nat. Rev. Mol. Cell Biol. 2017; 18: 90-101Crossref PubMed Scopus (364) Google Scholar). H3P16 appears to provide binding specificity for CeSmarca4 BD binding and positions H3R17 around the αB-helix to facilitate BD contacts (Fig. 3, B and E). H3R17 contributes essential interactions for binding to H3K14ac—the H3R17A mutation abolishes binding in ScSth1 (30Chen G. Li W. Yan F. Wang D. Chen Y. The structural basis for specific recognition of H3K14 acetylation by Sth1 in the RSC chromatin remodeling complex.Structure. 2020; 28: 111-118.e113Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) and HsBAZ2B (35Ferguson F.M. Dias D.M. Rodrigues J.P. Wienk H. Boelens R. Bonvin A.M. Abell C. Ciulli A. Binding hotspots of BAZ2B bromodomain: Histone interaction revealed by solution NMR driven docking.Biochemistry. 2014; 53: 6706-6716Crossref PubMed Scopus (15) Google Scholar). The backbone amide nitrogen of H3R17 forms a hydrogen bond with the Tyr1262 backbone carbonyl (Fig. 3E). The H3R17 sidechain is tethered to the BD αB-helix and BC loop via hydrogen bonds between the sidechain nitrogen atoms and the main-chain Gln1260, Thr1261, and Asn1263 carbonyl groups—all conserved in HsSMARCA4/2 (Fig. 3E). Water-mediated hydrogen bonds stabilize the interaction between H3R17 and Tyr1270 on the αC-helix. The guanidinium group of H3R17 adopts a single conformation in the CeSMARCA4 structure, oriented toward Glu1265. The electron density for CeSMARCA4 Glu1265 indicates the sidechain adopts multiple conformations (Fig. 3F). We modeled the population of Glu1265 rotamers with Ringer (40Lang P.T. Ng H.L. Fraser J.S. Corn J.E. Echols N. Sales M. Holton J.M. Alber T. Automated electron-density sampling reveals widespread conformational polymorphism in proteins.Protein Sci. 2010; 19: 1420-1431Crossref PubMed Scopus (107) Google Scholar), which identifi}, number={4}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={Elsevier BV}, author={Enriquez, Paul and Krajewski, Krzysztof and Strahl, Brian D. and Rothbart, Scott B. and Dowen, Robert H. and Rose, Robert B.}, year={2021}, month={Oct} }
 @article{buhrman_enriquez_dillard_baer_truong_grunden_rose_2021, title={Structure, Function, and Thermal Adaptation of the Biotin Carboxylase Domain Dimer from Hydrogenobacter thermophilus 2-Oxoglutarate Carboxylase}, volume={60}, ISSN={["0006-2960"]}, url={https://doi.org/10.1021/acs.biochem.0c00815}, DOI={10.1021/acs.biochem.0c00815}, abstractNote={2-Oxoglutarate carboxylase (OGC), a unique member of the biotin-dependent carboxylase family from the order Aquificales, captures dissolved CO2 via the reductive tricarboxylic acid (rTCA) cycle. Structure and function studies of OGC may facilitate adaptation of the rTCA cycle to increase the level of carbon fixation for biofuel production. Here we compare the biotin carboxylase (BC) domain of Hydrogenobacter thermophilus OGC with the well-studied mesophilic homologues to identify features that may contribute to thermal stability and activity. We report three OGC BC X-ray structures, each bound to bicarbonate, ADP, or ADP-Mg2+, and propose that substrate binding at high temperatures is facilitated by interactions that stabilize the flexible subdomain B in a partially closed conformation. Kinetic measurements with varying ATP and biotin concentrations distinguish two temperature-dependent steps, consistent with biotin's rate-limiting role in organizing the active site. Transition state thermodynamic values derived from the Eyring equation indicate a larger positive ΔH⧧ and a less negative ΔS⧧ compared to those of a previously reported mesophilic homologue. These thermodynamic values are explained by partially rate limiting product release. Phylogenetic analysis of BC domains suggests that OGC diverged prior to Aquificales evolution. The phylogenetic tree identifies mis-annotations of the Aquificales BC sequences, including the Aquifex aeolicus pyruvate carboxylase structure. Notably, our structural data reveal that the OGC BC dimer comprises a "wet" dimerization interface that is dominated by hydrophilic interactions and structural water molecules common to all BC domains and likely facilitates the conformational changes associated with the catalytic cycle. Mutations in the dimerization domain demonstrate that dimerization contributes to thermal stability.}, number={4}, journal={BIOCHEMISTRY}, publisher={American Chemical Society (ACS)}, author={Buhrman, Greg and Enriquez, Paul and Dillard, Lucas and Baer, Hayden and Truong, Vivian and Grunden, Amy M. and Rose, Robert B.}, year={2021}, month={Feb}, pages={324–345} }
 @misc{enriquez_2017, title={GM-food regulations: engage the public}, volume={548}, ISSN={["1476-4687"]}, DOI={10.1038/548031b}, number={7665}, journal={NATURE}, author={Enriquez, Paul}, year={2017}, month={Aug}, pages={31–31} }
 @article{merchante_brumos_yun_hu_spencer_enriquez_binder_heber_stepanova_alonso_2015, title={Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2}, volume={163}, ISSN={["1097-4172"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84948814371&partnerID=MN8TOARS}, DOI={10.1016/j.cell.2015.09.036}, abstractNote={The central role of translation in modulating gene activity has long been recognized, yet the systematic exploration of quantitative changes in translation at a genome-wide scale in response to a specific stimulus has only recently become technically feasible. Using the well-characterized signaling pathway of the phytohormone ethylene and plant-optimized genome-wide ribosome footprinting, we have uncovered a molecular mechanism linking this hormone's perception to the activation of a gene-specific translational control mechanism. Characterization of one of the targets of this translation regulatory machinery, the ethylene signaling component EBF2, indicates that the signaling molecule EIN2 and the nonsense-mediated decay proteins UPFs play a central role in this ethylene-induced translational response. Furthermore, the 3'UTR of EBF2 is sufficient to confer translational regulation and required for the proper activation of ethylene responses. These findings represent a mechanistic paradigm of gene-specific regulation of translation in response to a key growth regulator.}, number={3}, journal={CELL}, author={Merchante, Catharina and Brumos, Javier and Yun, Jeonga and Hu, Qiwen and Spencer, Kristina R. and Enriquez, Paul and Binder, Brad M. and Heber, Steffen and Stepanova, Anna N. and Alonso, Jose M.}, year={2015}, month={Oct}, pages={684–697} }