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ABSTRACT: The crystal structure of a conserved hypothetical protein, GK0453, from Geobacillus kaustophilus has been determined to 2.2 Å resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 75.69, c = 64.18 Å. The structure was determined by the molecular-replacement method and was refined to a final R factor of 22.6% (Rfree = 26.3%). Based on structural homology, the GK0453 protein possesses two independent binding sites and hence it may simultaneously interact with two proteins or with a protein and a nucleic acid.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2013; 69(Pt 4):342-5. · 0.51 Impact Factor
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Tamaki Ito,
Takashi Umehara,
Kazuki Sasaki,
Yoshihiro Nakamura,
Norikazu Nishino,
Takaho Terada,
Mikako Shirouzu, Balasundaram Padmanabhan,
Shigeyuki Yokoyama,
Akihiro Ito,
Minoru Yoshida
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ABSTRACT: Histone acetylation constitutes an epigenetic mark for transcriptional regulation. Here we developed a fluorescent probe to visualize acetylation of histone H4 Lys12 (H4K12) in living cells using fluorescence resonance energy transfer (FRET) and the binding of the BRD2 bromodomain to acetylated H4K12. Using this probe designated as Histac-K12, we demonstrated that histone H4K12 acetylation is retained in mitosis and that some histone deacetylase (HDAC) inhibitors continue to inhibit cellular HDAC activity even after their removal from the culture. In addition, a small molecule that interferes with ability of the bromodomain to bind to acetylated H4K12 could be assessed using Histac-K12 in cells. Thus, Histac-K12 will serve as a powerful tool not only to understand the dynamics of H4K12-specific acetylation but also to characterize small molecules that modulate the acetylation or interaction status of histones.
Chemistry & biology 04/2011; 18(4):495-507. · 6.52 Impact Factor
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ABSTRACT: The BET family proteins recognize acetylated chromatin through their two bromodomains, acting as transcriptional activators or tethering viral genomes to the mitotic chromosomes of their host. The structural mechanism for how the N-terminal bromodomain of human BRD2 (BRD2-BD1) deciphers the mono-acetylated status of histone H4 tail was recently reported. Here we show the crystal structure of the second bromodomain of BRD2 (BRD2-BD2) in complex with the di-acetylated histone H4 tail (H4K5ac/K12ac). To our surprise, a single K5ac/K12ac peptide interacts with two BRD2-BD2 molecules simultaneously: the K5ac residue binds to one BRD2-BD2 molecule while the K12ac residue binds to another. These results provide a structural basis for the recognition of two different patterns of the histone acetylation status by a single bromodomain.
FEBS letters 09/2010; 584(18):3901-8. · 3.54 Impact Factor
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ABSTRACT: Recognition of acetylated chromatin by the bromodomains and extra-terminal domain (BET) family proteins is a hallmark for transcriptional activation and anchoring viral genomes to mitotic chromosomes of the host. One of the BET family proteins BRD2 interacts with acetylated chromatin during mitosis and leads to transcriptional activation in culture cells. Here, we report the crystal structures of the N-terminal bromodomain of human BRD2 (BRD2-BD1; residues 74-194) in complex with each of three different Lys-12-acetylated H4 peptides. The BRD2-BD1 recognizes the H4 tail acetylated at Lys-12 (H4K12ac), whereas the side chain of hypoacetylated Lys-8 of H4 binds at the cavity of the dimer interface of BRD2-BD1. From binding studies, we identified the BRD2-BD1 residues that are responsible for recognition of the Lys-12-acetylated H4 tail. In addition, mutation to Lys-8 in the Lys-12-acetylated H4 tail decreased the binding to BRD2-BD1, implicating the critical role of Lys-8 in the Lys-12-acetylated H4 tail for the recognition by BRD2-BD1. Our findings provide a structural basis for deciphering the histone code by the BET bromodomain through the binding with a long segment of the histone H4 tail, which presumably prevents erasure of the histone code during the cell cycle.
Journal of Biological Chemistry 03/2010; 285(10):7610-8. · 4.77 Impact Factor
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ABSTRACT: In bacteria and plants, dihydrodipicolinate synthase (DHDPS) plays a key role in the (S)-lysine biosynthesis pathway. DHDPS catalyzes the first step of the condensation of (S)-aspartate-beta-semialdehyde and pyruvate to form an unstable compound, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. The activity of DHDPS is allosterically regulated by (S)-lysine, a feedback inhibitor. The crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) was solved by the molecular-replacement method and was refined to 2.2 A resolution. The structure revealed that MjDHDPS forms a functional homotetramer, as also observed in Escherichia coli DHDPS, Thermotoga maritima DHDPS and Bacillus anthracis DHDPS. The binding-site region of MjDHDPS is essentially similar to those found in other known DHDPS structures.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2009; 65(Pt 12):1222-6. · 0.51 Impact Factor
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ABSTRACT: The X-ray crystal structure of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj-GAPDH) was determined to 1.81 A resolution. The crystal belonged to space group C222(1), with unit-cell parameters a = 83.4, b = 152.0, c = 118.6 A. The structure was solved by molecular replacement and was refined to a final R factor of 17.1% (R(free) = 19.8%). The final structure included the cofactor NADP(+) at the nucleotide-binding site and featured unoccupied inorganic and substrate phosphate-binding sites. A comparison with GAPDH structures from mesophilic sources suggested that Mj-GAPDH is stabilized by extensive electrostatic interactions between the C-terminal alpha-helices and various distal loop regions, which are likely to contribute to thermal stability. The key phosphate-binding residues in the active site of Mj-GAPDH are conserved in other archaeal GAPDH proteins. These residues undergo a conformational shift in response to occupancy of the inorganic phosphate site.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2009; 65(Pt 12):1227-33. · 0.51 Impact Factor
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ABSTRACT: The pyridoxal 5'-phosphate-dependent enzyme 4-amino-4-deoxychorismate lyase converts 4-amino-4-deoxychorismate to p-aminobenzoate and pyruvate in one of the crucial steps in the folate-biosynthesis pathway. The primary structure of the hypothetical protein TTHA0621 from Thermus thermophilus HB8 suggests that TTHA0621 is a putative 4-amino-4-deoxychorismate lyase. Here, the crystal structure of TTHA0621 is reported at 1.93 A resolution. The asymmetric unit contained four NCS molecules related by 222 noncrystallographic symmetry, in which the formation of intact dimers may be functionally important. The cofactor pyridoxal 5'-phosphate (PLP) binds to the protein in the large cleft formed by the N-terminal and C-terminal domains of TTHA0621. The high structural similarity and the conservation of the functional residues in the catalytic region compared with 4-amino-4-deoxychorismate lyase (PabC; EC 4.1.3.38) from Escherichia coli suggest that the TTHA0621 protein may also possess 4-amino-4-deoxychorismate lyase activity.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2009; 65(Pt 12):1234-9. · 0.51 Impact Factor
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ABSTRACT: BRD4, which is a member of the BET (bromodomains and extraterminal) protein family, interacts preferentially with acetylated chromatin and possesses multiple cellular functions in meiosis, embryonic development, the cell cycle, and transcription. BRD4 and its family members contain two bromodomains known to bind acetylated lysine, and a conserved ET domain whose function is unclear. Here we show the solution structure of the ET domain of mouse BRD4, which provides the first three-dimensional structure of an ET domain in the BET family. We determined the NMR structure of BRD4-ET with a root-mean-square deviation of 0.41 A for the backbone atoms in the structured region of residues 608-676 on the basis of 1793 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure of the BRD4-ET domain comprises three alpha-helices and a characteristic loop region of an irregular but well-defined structure. A DALI search revealed no close structural homologs in the current Protein Data Bank. The BRD4-ET structure has an acidic patch that forms a continuous ridge with a hydrophobic cleft, which may interact with other proteins and/or DNA.
Protein Science 10/2008; 17(12):2174-9. · 2.80 Impact Factor
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ABSTRACT: Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6(4)22, with unit-cell parameters a = b = 148.49, c = 106.96 A, gamma = 120.0 degrees . Assuming the presence of two molecules in the asymmetric unit, the calculated V(M) is 2.84 A(3) Da(-1), which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 A resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2008; 64(Pt 6):498-500. · 0.51 Impact Factor
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ABSTRACT: The cytoplasmic repressor Keap1 regulates the function of transcription factor Nrf2 which plays critical roles in oxidative and xenobiotic stresses. The Neh2 domain of Nrf2 interacts with Keap1 at the bottom region of the Kelch/beta-propeller domain which is formed by double-glycine repeat and C-terminal region domains (Keap1-DC). The structure of Keap1-DC complexed with an Nrf2 peptide containing a conserved DLG motif has been determined at 1.9 A resolution. The Keap1-bound DLG peptide possesses a hairpin conformation, and it binds to the Keap1 protein at the bottom region of the beta-propeller domain. The intermolecular interaction occurs through their complementary electrostatic interactions. Comparison of the present structure with the recently reported Keap1-DC complex structure suggests that the DLG and ETGE motifs of Neh2 in Nrf2 bind to Keap1 in a similar manner but with different binding potencies.
Journal of Synchrotron Radiation 06/2008; 15(Pt 3):273-6. · 2.73 Impact Factor
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ABSTRACT: The assembly of core histones onto eukaryotic DNA is modulated by several histone chaperone complexes, including Asf1, CAF-1, and HIRA. Asf1 is a unique histone chaperone that participates in both the replication-dependent and replication-independent pathways. Here we report the crystal structures of the apo-form of fission yeast Asf1/Cia1 (SpAsf1N; residues 1-161) as well as its complexes with the B-domain of the fission yeast HIRA orthologue Hip1 (Hip1B) and the C-terminal region of the Cac2 subunit of CAF-1 (Cac2C). The mode of the fission yeast Asf1N-Hip1B recognition is similar to that of the human Asf1-HIRA recognition, suggesting that Asf1N recognition of Hip1B/HIRA is conserved from yeast to mammals. Interestingly, Hip1B and Cac2C show remarkably similar interaction modes with Asf1. The binding between Asf1N and Hip1B was almost completely abolished by the D37A and L60A/V62A mutations in Asf1N, indicating the critical role of salt bridge and van der Waals contacts in the complex formation. Consistently, both of the aforementioned Asf1 mutations also drastically reduced the binding to Cac2C. These results provide a structural basis for a mutually exclusive Asf1-binding model of CAF-1 and HIRA/Hip1, in which Asf1 and CAF-1 assemble histones H3/H4 (H3.1/H4 in vertebrates) in a replication-dependent pathway, whereas Asf1 and HIRA/Hip1 assemble histones H3/H4 (H3.3/H4 in vertebrates) in a replication-independent pathway.
Journal of Biological Chemistry 06/2008; 283(20):14022-31. · 4.77 Impact Factor
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ABSTRACT: The Nrf2 transcription factor, which plays important roles in oxidative and xenobiotic stress, is negatively regulated by the cytoplasmic repressor Keap1. The beta-propeller/Kelch domain of Keap1, which is formed by the double-glycine repeat and C-terminal region domains (Keap1-DC), interacts directly with the Neh2 domain of Nrf2. The nuclear oncoprotein prothymosin alpha (ProTalpha) also interacts directly with Keap1 and may play a role in the dissociation of the Keap1-Nrf2 complex. The structure of Keap1-DC complexed with a ProTalpha peptide (amino acids 39-54) has been determined at 1.9 A resolution. The Keap1-bound ProTalpha peptide possesses a hairpin conformation and binds to the Keap1 protein at the bottom region of the beta-propeller domain. Complex formation occurs as a consequence of their complementary electrostatic interactions. A comparison of the present structure with recently reported Keap1-DC complex structures revealed that the DLG and ETGE motifs of the Neh2 domain of Nrf2 and the ProTalpha peptide bind to Keap1 in a similar manner but with different binding potencies.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 05/2008; 64(Pt 4):233-8. · 0.51 Impact Factor
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ABSTRACT: Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress.
Molecular and Cellular Biology 12/2007; 27(21):7511-21. · 5.53 Impact Factor
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ABSTRACT: The 26S proteasome-dependent protein degradation is an evolutionarily conserved process. The mammalian oncoprotein gankyrin, which associates with S6 of the proteasome, facilitates the degradation of pRb, and thus possibly acts as a bridging factor between the proteasome and its substrates. However, the mechanism of the proteasome-dependent protein degradation in yeast is poorly understood. Here, we report the tertiary structure of the complex between Nas6 and a C-terminal domain of Rpt3, which are the yeast orthologues of gankyrin and S6, respectively. The concave region of Nas6 bound to the alpha-helical domain of Rpt3. The stable interaction between Nas6 and Rpt3 was mediated by intermolecular interactions composed of complementary charged patches. The recognition of Rpt3 by Nas6 in the crystal suggests that Nas6 is indeed a subunit of the 26S proteasome. These results provide a structural basis for the association between Nas6 and the heterohexameric ATPase ring of the proteasome through Rpt3.
Biochemical and Biophysical Research Communications 09/2007; 359(3):503-9. · 2.48 Impact Factor
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ABSTRACT: BRD2 is a bromodomain-containing BET-family protein that associates with acetylated histones throughout the cell cycle. Although the tertiary structures of the bromodomains involved in histone acetyl transfer are already known, the structures of the BET-type bromodomains, which are required for tight association with acetylated chromatin, are poorly understood. Here, the expression, purification and crystallization of the C-terminal bromodomain of human BRD2 are reported. The protein was crystallized by the sitting-drop vapour-diffusion method in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 71.78, b = 52.60, c = 32.06 A and one molecule per asymmetric unit. The crystal diffracted beyond 1.80 A resolution using synchrotron radiation.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2007; 63(Pt 7):613-5. · 0.51 Impact Factor
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Yoshihiro Nakamura,
Takashi Umehara,
Hiroaki Hamana,
Yoshihide Hayashizaki,
Makoto Inoue,
Takanori Kigawa,
Mikako Shirouzu,
Takaho Terada,
Akiko Tanaka, Balasundaram Padmanabhan,
Shigeyuki Yokoyama
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ABSTRACT: The Wnt/beta-catenin signaling pathway plays important roles in animal development and cancer. Pygopus (Pygo) and Legless (Lgs) are recently discovered core components of the Wnt/beta-catenin transcription machinery complex, and are crucially involved in the regulation of the transcription of the Arm/beta-catenin and T cell factors (TCF). Lgs/Bcl9 functions as an adaptor between Pygo and Arm/beta-catenin. Here, we report the first crystal structure of the plant homeodomain (PHD) finger of Pygopus (Pygo1 PHD), a Pygo family member, which is essential for the association with Lgs/Bcl9. The Pygo1 PHD structure forms a canonical PHD finger motif, stabilized by two Zn ions coordinated in a cross-brace scheme. Surprisingly, the Pygo1 PHD domain forms a dimer in both the crystals and solution. This is the first structural evidence for dimerization among the known PHD domain structures. The dimer formation occurs by the interactions of antiparallel beta-sheets between the symmetry-related beta3 strands of the monomers. The Pygo1 PHD dimer interface mainly comprises hydrophobic residues. Interestingly, some of the interface residues, such as Met372, Thr373, Ala376 and Leu380, are reportedly important for the association with Lgs/Bcl9 and are also critical for transcriptional activation. The M372A and L380D mutants, and several surrounding mutants such as S385A and A386D, showed decreased ability to form dimers and to interact with the homology domain 1 (HD1) of Lgs/Bcl9. These results suggest that the Pygo1 PHD dimerization is functionally important for Lgs/Bcl9 recognition as well as for the regulation of the Wnt/beta-catenin signaling pathway.
Journal of Molecular Biology 07/2007; 370(1):80-92. · 4.00 Impact Factor
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ABSTRACT: The non-ATPase subunit Nas6, which is the human orthologue of gankyrin, was co-expressed with the C-terminal domain of the ATPase subunit Rpt3 of the yeast 26S proteasome in Escherichia coli, purified to near-homogeneity and crystallized using the hanging-drop vapour-diffusion method. The protein crystallized in space group P2(1), with unit-cell parameters a = 60.38, b = 100.22, c = 72.20 A, beta = 94.70 degrees and with three Nas6-Rpt3C molecules per asymmetric unit. The crystal diffracted to beyond 2.2 A resolution using synchrotron radiation.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2007; 63(Pt 3):190-2. · 0.51 Impact Factor
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ABSTRACT: Gankyrin is an oncoprotein commonly overexpressed in most hepatocellular carcinomas. Gankyrin interacts with S6 ATPase of the 19S regulatory particle of the 26S proteasome and enhances the degradation of the tumor suppressors pRb and p53. Here, we report the structure of gankyrin in complex with the C-terminal domain of S6 ATPase. Almost all of the seven ankyrin repeats of gankyrin interact, through its concave region, with the C-terminal domain of S6 ATPase. The intermolecular interactions occur through the complementary charged residues between gankyrin and S6 ATPase. Biochemical studies based on the structure of the complex revealed that gankyrin interacts with pRb in both the presence and absence of S6 ATPase; however, the E182 residue in gankyrin is essential for the pRb interaction. These results provide a structural basis for the involvement of gankyrin in the pRb degradation pathway, through its association with S6 ATPase of the 26S proteasome.
Structure 03/2007; 15(2):179-89. · 6.35 Impact Factor
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Yoshihiro Nakamura,
Takashi Umehara,
Kazumi Nakano,
Moon Kyoo Jang,
Mikako Shirouzu,
Satoshi Morita,
Hiroko Uda-Tochio,
Hiroaki Hamana,
Takaho Terada,
Naruhiko Adachi,
Takehisa Matsumoto,
Akiko Tanaka,
Masami Horikoshi,
Keiko Ozato, Balasundaram Padmanabhan,
Shigeyuki Yokoyama
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ABSTRACT: The BET (bromodomains and extra terminal domain) family proteins recognize acetylated chromatin through their bromodomain and act as transcriptional activators. One of the BET proteins, BRD2, associates with the transcription factor E2F, the mediator components CDK8 and TRAP220, and RNA polymerase II, as well as with acetylated chromatin during mitosis. BRD2 contains two bromodomains (BD1 and BD2), which are considered to be responsible for binding to acetylated chromatin. The BRD2 protein specifically recognizes the histone H4 tail acetylated at Lys12. Here, we report the crystal structure of the N-terminal bromodomain (BD1, residues 74-194) of human BRD2. Strikingly, the BRD2 BD1 protein forms an intact dimer in the crystal. This is the first observation of a homodimer among the known bromodomain structures, through the buried hydrophobic core region at the interface. Biochemical studies also demonstrated BRD2 BD1 dimer formation in solution. The two acetyllysine-binding pockets and a negatively charged secondary binding pocket, produced at the dimer interface in BRD2 BD1, may be the unique features that allow BRD2 BD1 to selectively bind to the acetylated H4 tail.
Journal of Biological Chemistry 03/2007; 282(6):4193-201. · 4.77 Impact Factor
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Yoshihiro Nakamura,
Takashi Umehara,
Kazumi Nakano,
Moon Kyoo Jang,
Mikako Shirouzu,
Satoshi Morita,
Hiroko Uda-Tochio,
Hiroaki Hamana,
Takaho Terada,
Naruhiko Adachi,
Takehisa Matsumoto,
Akiko Tanaka,
Masami Horikoshi,
Keiko Ozato, Balasundaram Padmanabhan,
Shigeyuki Yokoyama
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ABSTRACT: The BET (bromodomains and extra terminal domain) family proteins recognize acetylated chromatin through their bromodomain and act as transcriptional activators.
One of the BET proteins, BRD2, associates with the transcription factor E2F, the mediator components CDK8 and TRAP220, and
RNA polymerase II, as well as with acetylated chromatin during mitosis. BRD2 contains two bromodomains (BD1 and BD2), which
are considered to be responsible for binding to acetylated chromatin. The BRD2 protein specifically recognizes the histone
H4 tail acetylated at Lys12. Here, we report the crystal structure of the N-terminal bromodomain (BD1, residues 74-194) of human BRD2. Strikingly, the
BRD2 BD1 protein forms an intact dimer in the crystal. This is the first observation of a homodimer among the known bromodomain
structures, through the buried hydrophobic core region at the interface. Biochemical studies also demonstrated BRD2 BD1 dimer
formation in solution. The two acetyllysine-binding pockets and a negatively charged secondary binding pocket, produced at
the dimer interface in BRD2 BD1, may be the unique features that allow BRD2 BD1 to selectively bind to the acetylated H4 tail.
Journal of Biological Chemistry 02/2007; 282(6):4193-4201. · 4.77 Impact Factor
