Balasundaram Padmanabhan

Different Electrostatic Potentials Define ETGE and DLG Motifs as Hinge and Latch in Oxidative Stress Response

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.

Structural Basis for Acetylated Histone H4 Recognition by the Human BRD2 Bromodomain.

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.

Crystal Structure of the Human BRD2 Bromodomain INSIGHTS INTO DIMERIZATION AND RECOGNITION OF ACETYLATED HISTONE H4

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.

Real-Time Imaging of Histone H4K12–Specific Acetylation Determines the Modes of Action of Histone Deacetylase and Bromodomain Inhibitors

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.

Crystal structures of fission yeast histone chaperone Asf1 complexed with the Hip1 B-domain or the Cac2 C terminus.

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.

Solution structure of the extraterminal domain of the bromodomain‐containing protein BRD4

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 Å 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 α‐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.

Structural implications for K5/K12-di-acetylated histone H4 recognition by the second bromodomain of BRD2.

MINT‐7989882, MINT‐7989824, MINT‐7989846, MINT‐7989865: H4 (uniprotkb:P62805) binds (MI:0407) to BRD2 (uniprotkb:P25440) by surface plasmon resonance (MI:0107) MINT‐7989539: H4 (uniprotkb:P62805) and BRD2 (uniprotkb:P25440) bind (MI:0407) by X‐ray crystallography (MI:0114)

Structural analysis of the complex of Keap1 with a prothymosin alpha peptide.

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.

Bromodomain and extra-terminal (BET) family proteins: New therapeutic targets in major diseases

The bromodomains and extra-terminal domain (BET) family proteins recognize acetylated chromatin through their bromodomains (BDs) and help in regulating gene expression. BDs are chromatin ‘readers’: by interacting with acetylated lysines on the histone tails, they recruit chromatin-regulating proteins on the promoter region to regulate gene expression and repression. Extensive efforts have been employed by scientific communities worldwide to identify and develop potential inhibitors of BET family BDs to regulate protein expression by inhibiting acetylated histone (H3/H4) interactions. Several small molecule inhibitors have been reported, which not only have high affinity but also have high specificity to BET BDs. These developments make BET family proteins an important therapeutic targets for major diseases such as cancer, neurological disorders, obesity and inflammation. Here, we review and discuss the structural biology of BET family BDs and their applications in major diseases.

The Keap1–Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases

The overproduction of reactive oxygen species (ROS) generates oxidative stress in cells. Oxidative stress results in various pathophysiological conditions, especially cancers and neurodegenerative diseases (NDD). The Keap1–Nrf2 [Kelch-like ECH-associated protein 1–nuclear factor (erythroid-derived 2)-like 2] regulatory pathway plays a central role in protecting cells against oxidative and xenobiotic stresses. The Nrf2 transcription factor activates the transcription of several cytoprotective genes that have been implicated in protection from cancer and NDD. The Keap1–Nrf2 system acts as a double-edged sword: Nrf2 activity protects cells and makes the cell resistant to oxidative and electrophilic stresses, whereas elevated Nrf2 activity helps in cancer cell survival and proliferation. Several groups in the recent past, from both academics and industry, have reported the potential role of Nrf2-mediated transcription to protect from cancer and NDD, resulting from mechanisms involving xenobiotic and oxidative stress. It suggests that the Keap1–Nrf2 system is a potential therapeutic target to combat cancer and NDD by designing and developing modulators (inhibitors/activators) for Nrf2 activation. Herein, we review and discuss the recent advancement in the regulation of the Keap1–Nrf2 system, its role under physiological and pathophysiological conditions including cancer and NDD, and modulators design strategies for Nrf2 activation.