Molecular basis of chromatin remodelling by DDM1 involved in plant DNA methylation.

Eukaryotic gene regulation occurs at the chromatin level, which requires changing the chromatin structure by a group of ATP-dependent DNA translocases-namely, the chromatin remodellers1. In plants, chromatin remodellers function in various biological processes and possess both conserved and plant-specific components2-5. DECREASE IN DNA METHYLATION 1 (DDM1) is a plant chromatin remodeller that plays a key role in the maintenance DNA methylation6-11. Here we determined the structures of Arabidopsis DDM1 in complex with nucleosome in ADP-BeFx-bound, ADP-bound and nucleotide-free conformations. We show that DDM1 specifically recognizes the H4 tail and nucleosomal DNA. The conformational differences between ADP-BeFx-bound, ADP-bound and nucleotide-free DDM1 suggest a chromatin remodelling cycle coupled to ATP binding, hydrolysis and ADP release. This, in turn, triggers conformational changes in the DDM1-bound nucleosomal DNA, which alters the nucleosome structure and promotes DNA sliding. Together, our data reveal the molecular basis of chromatin remodelling by DDM1.

Molecular basis of the plant ROS1-mediated active DNA demethylation.

Active DNA demethylation plays a crucial role in eukaryotic gene imprinting and antagonizing DNA methylation. The plant-specific REPRESSOR OF SILENCING 1/DEMETER (ROS1/DME) family of enzymes directly excise 5-methyl-cytosine (5mC), representing an efficient DNA demethylation pathway distinct from that of animals. Here, we report the cryo-electron microscopy structures of an Arabidopsis ROS1 catalytic fragment in complex with substrate DNA, mismatch DNA and reaction intermediate, respectively. The substrate 5mC is flipped-out from the DNA duplex and subsequently recognized by the ROS1 base-binding pocket through hydrophobic and hydrogen-bonding interactions towards the 5-methyl group and Watson-Crick edge respectively, while the different protonation states of the bases determine the substrate preference for 5mC over T:G mismatch. Together with the structure of the reaction intermediate complex, our structural and biochemical studies revealed the molecular basis for substrate specificity, as well as the reaction mechanism underlying 5mC demethylation by the ROS1/DME family of plant-specific DNA demethylases.

The H3K9me2-binding protein AGDP3 limits DNA methylation and transcriptional gene silencing in Arabidopsis.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

A novel protein complex that regulates active DNA demethylation in Arabidopsis.

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base-excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at 1 000s of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl-DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo. Loss-of-function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.

The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor.

In plants, flowering time is controlled by environmental signals such as day-length and temperature, which regulate the floral pathway integrators, including FLOWERING LOCUS T (FT), by genetic and epigenetic mechanisms. Here, we identify an H3K27me3 demethylase, JUMONJI 13 (JMJ13), which regulates flowering time in Arabidopsis. Structural characterization of the JMJ13 catalytic domain in complex with its substrate peptide reveals that H3K27me3 is specifically recognized through hydrogen bonding and hydrophobic interactions. Under short-day conditions, the jmj13 mutant flowers early and has increased FT expression at high temperatures, but not at low temperatures. In contrast, jmj13 flowers early in long-day conditions regardless of temperature. Long-day condition and higher temperature induce the expression of JMJ13 and increase accumulation of JMJ13. Together, our data suggest that the H3K27me3 demethylase JMJ13 acts as a temperature- and photoperiod-dependent flowering repressor.

Arabidopsis AGDP1 links H3K9me2 to DNA methylation in heterochromatin.

Heterochromatin is a tightly packed form of chromatin that is associated with DNA methylation and histone 3 lysine 9 methylation (H3K9me). Here, we identify an H3K9me2-binding protein, Agenet domain (AGD)-containing p1 (AGDP1), in Arabidopsis thaliana. Here we find that AGDP1 can specifically recognize the H3K9me2 mark by its three pairs of tandem AGDs. We determine the crystal structure of the Agenet domain 1 and 2 cassette (AGD12) of Raphanus sativus AGDP1 in complex with an H3K9me2 peptide. In the complex, the histone peptide adopts a unique helical conformation. AGD12 specifically recognizes the H3K4me0 and H3K9me2 marks by hydrogen bonding and hydrophobic interactions. In addition, we find that AGDP1 is required for transcriptional silencing, non-CG DNA methylation, and H3K9 dimethylation at some loci. ChIP-seq data show that AGDP1 preferentially occupies long transposons and is associated with heterochromatin marks. Our findings suggest that, as a heterochromatin-binding protein, AGDP1 links H3K9me2 to DNA methylation in heterochromatin regions.

Structure of the Arabidopsis JMJ14-H3K4me3 Complex Provides Insight into the Substrate Specificity of KDM5 Subfamily Histone Demethylases.

In chromatin, histone methylation affects the epigenetic regulation of multiple processes in animals and plants and is modulated by the activities of histone methyltransferases and histone demethylases. The jumonji domain-containing histone demethylases have diverse functions and can be classified into several subfamilies. In humans, the jumonji domain-containing Lysine (K)-Specific Demethylase 5/Jumonji and ARID Domain Protein (KDM5/JARID) subfamily demethylases are specific for histone 3 lysine 4 trimethylation (H3K4me3) and are important drug targets for cancer treatment. In Arabidopsis thaliana, the KDM5/JARID subfamily H3K4me3 demethylase JUMONJI14 (JMJ14) plays important roles in flowering, gene silencing, and DNA methylation. Here, we report the crystal structures of the JMJ14 catalytic domain in both substrate-free and bound forms. The structures reveal that the jumonji and C5HC2 domains contribute to the specific recognition of the H3R2 and H3Q5 to facilitate H3K4me3 substrate specificity. The critical acidic residues are conserved in plants and animals with the corresponding mutations impairing the enzyme activity of both JMJ14 and human KDM5B, indicating a common substrate recognition mechanism for KDM5 subfamily demethylases shared by plants and animals and further informing efforts to design targeted inhibitors of human KDM5.

Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin.

Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acid-adenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATP-dependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3.

Structural Basis for the Unique Multivalent Readout of Unmodified H3 Tail by Arabidopsis ORC1b BAH-PHD Cassette.

DNA replication initiation relies on the formation of the origin recognition complex (ORC). The plant ORC subunit 1 (ORC1) protein possesses a conserved N-terminal BAH domain with an embedded plant-specific PHD finger, whose function may be potentially regulated by an epigenetic mechanism. Here, we report structural and biochemical studies on the Arabidopsis thaliana ORC1b BAH-PHD cassette which specifically recognizes the unmodified H3 tail. The crystal structure of ORC1b BAH-PHD cassette in complex with an H3(1-15) peptide reveals a strict requirement for the unmodified state of R2, T3, and K4 on the H3 tail and a novel multivalent BAH and PHD readout mode for H3 peptide recognition. Such recognition may contribute to epigenetic regulation of the initiation of DNA replication.

Mechanism of DNA methylation-directed histone methylation by KRYPTONITE.

In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide, and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the ε-ammonium of K9 positioned adjacent to bound SAH. These structural insights, complemented by functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation.

Molecular mechanism of action of plant DRM de novo DNA methyltransferases.

DNA methylation is a conserved epigenetic gene-regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homodimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the small interfering RNA (siRNA) effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for RNA polymerase V-mediated noncoding RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts.

SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation.

RNA-directed DNA methylation in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA. The SU(VAR)3-9 homologues SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown. Here we show that genome-wide Pol V association with chromatin redundantly requires SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases, a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM)-binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins. SUVH2 and SUVH9 both contain SRA (SET- and RING-ASSOCIATED) domains capable of binding methylated DNA, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH9 [corrected] with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results indicate that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.

Structural basis for recognition of H3T3ph and Smac/DIABLO N-terminal peptides by human Survivin.

Survivin is an inhibitor of apoptosis family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone H3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes and identify intermolecular hydrogen-bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.

Expression, purification, crystallization, and preliminary X-ray diffraction analysis of the human TLE1 Q domain.

Human transducin-like enhancer of split 1 (TLE1) plays crucial roles in a number of developmental processes and is involved in pathogenesis of malignancy tumors. The N-terminal glutamine-rich domain (Q domain) of TLE1 mediates its tetramerization and interactions with different DNA-binding transcription factors to regulate Notch and Wnt signaling pathways. To better understand the molecular mechanism of TLE1's functions in these pathways, we cloned, purified, and crystallized the TLE1 Q domain (TLE1-Q). The crystals belong to space group C222(1), with the complete diffraction data of the native and Se-Met TLE1-Q collected to 3.5 and 4.1 Å resolutions, respectively. The phasing-solving and model building are in progress.

Structural modeling and biochemical studies reveal insights into the molecular basis of the recognition of beta-2-microglobulin by antibody BBM.1.

Human beta-2-microglobulin (beta2m) is the light chain of human leucocyte antigen-I (HLA-I). It can disassociate from HLA-I and accumulate to cause serious dialysis-related amyloidosis (DRA) in long-term hemodialysis patients. Monoclonal antibody (mAb) BBM.1 can recognize both free-form and HLA-I associated beta2m. It can be used for specific elimination of beta2m from serum and can induce apoptosis of several types of tumor cells, and thus has great therapeutic potential. In this study, we constructed structural models of the BBM.1 Fv (fragment of the variable domain) and the BBM.1 Fv-beta2m complex, followed by biochemical evaluation. Analysis of the optimal complex model reveals that the previously identified immunodominant residues Glu(44) and Arg(45) of beta2m have direct interactions with BBM.1, while Asp(38) exerts its function mainly via stabilization of Arg(45). In addition, Arg(81) of beta2m is a newly identified immunodominant residue to have direct interaction with BBM.1. Further modeling study shows no steric conflict between the antibody and the HLA-I heavy chain. These results provide insights into the molecular basis of the recognition of beta2m by BBM.1 and explain why BBM.1 can bind both free-form and HLA-1 associated beta2m. This information could be exploited in the engineering and improvement of BBM.1 and the development of other beta2m-targeting mAbs for therapeutic purposes.

Structure of the Fab fragment of therapeutic antibody Ofatumumab provides insights into the recognition mechanism with CD20.

CD20 is an important drug target for B-cell depletion therapy against certain B-cell lymphomas and autoimmune diseases. The success of anti-CD20 antibody drugs such as Rituximab, Ibritumomab, and Tositumomab has promoted the development of new generation of anti-CD20 antibodies for therapeutic applications. Ofatumumab is a fully human anti-CD20 antibody that is currently in phase III clinical trial for several types of malignancies and autoimmune diseases and is one of the most promising anti-CD20 drugs. Here we report the crystal structure of the Fab fragment of Ofatumumab at 2.2A resolution. The antigen combining site is composed of a large, deep pocket formed by six CDR loops. The pocket has a hydrophobic periphery and a positively charged bottom. Structure analysis and comparison with other antibodies suggest that the hydrophobic periphery might interact with the epitope on CD20 that is enriched with hydrophobic residues and very close to cell membrane, and the positively charged bottom might interact with Glu(150) of CD20 which is the only negatively charged residue within the epitope. These results provide some insights into the recognition of Ofatumumab with CD20 and explain how the antibody can recognize an epitope so close to the cell membrane.

Emodin targets the beta-hydroxyacyl-acyl carrier protein dehydratase from Helicobacter pylori: enzymatic inhibition assay with crystal structural and thermodynamic characterization.

BACKGROUND: The natural product Emodin demonstrates a wide range of pharmacological properties including anticancer, anti-inflammatory, antiproliferation, vasorelaxant and anti-H. pylori activities. Although its H. pylori inhibition was discovered, no acting target information against Emodin has been revealed to date. RESULTS: Here we reported that Emodin functioned as a competitive inhibitor against the recombinant beta-hydroxyacyl-ACP dehydratase from Helicobacter pylori (HpFabZ), and strongly inhibited the growth of H. pylori strains SS1 and ATCC 43504. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) based assays have suggested the kinetic and thermodynamic features of Emodin/HpFabZ interaction. Additionally, to inspect the binding characters of Emodin against HpFabZ at atomic level, the crystal structure of HpFabZ-Emodin complex was also examined. The results showed that Emodin inhibition against HpFabZ could be implemented either through its occupying the entrance of the tunnel or embedding into the tunnel to prevent the substrate from accessing the active site. CONCLUSION: Our work is expected to provide useful information for illumination of Emodin inhibition mechanism against HpFabZ, while Emodin itself could be used as a potential lead compound for further anti-bacterial drug discovery.

Molecular basis of recognition of human osteopontin by 23C3, a potential therapeutic antibody for treatment of rheumatoid arthritis.

Osteopontin plays an important role in the development and perpetuation of rheumatoid arthritis (RA). Antibodies targeting osteopontin have shown promising therapeutic benefits against this disease. We have previously reported a novel anti-RA monoclonal antibody, namely, 23C3, and shown it capable of alleviating the symptoms of RA in a murine collagen-induced arthritis model, restoring the cytokine production profile in joint tissues, and reducing T-cell recall responses to collagen type II. We describe here the crystal structure of 23C3 in complex with its epitope peptide. Analyses of the complex structure reveal the molecular mechanism of osteopontin recognition by 23C3. The peptide folds into two tandem beta-turns, and two key residues of the peptide are identified to be critical for the recognition by 23C3: TrpP43 is deeply embedded into a hydrophobic pocket formed by AlaL34, TyrL36, LeuL46, TyrL49, PheL91, and MetH102 and therefore has extensive hydrophobic interactions with 23C3, while AspP47 has a network of hydrophilic interactions with residues ArgH50, ArgH52, SerH53, and AsnH56 of the antibody. Besides the complementarity-determining region loops, the framework region L2 of 23C3 is also shown to interact with the epitope peptide, which is not common in the antibody-antigen interactions and thus could be exploited in the engineering of 23C3. These results not only provide valuable information for further improvement of 23C3 such as chimerization or humanization for its therapeutic application, but also reveal the features of this specific epitope of osteopontin that may be useful for the development of new antibody drugs against RA.

Crystal structure of HAb18G/CD147: implications for immunoglobulin superfamily homophilic adhesion.

CD147, a member of the immunoglobulin superfamily (IgSF), plays fundamental roles in intercellular interactions in numerous pathological and physiological processes. Importantly, our previous studies have demonstrated that HAb18G/CD147 is a novel hepatocellular carcinoma (HCC)-associated antigen, and HAb18G/CD147 stimulates adjacent fibroblasts and HCC cells to produce elevated levels of several matrix metalloproteinases, facilitating invasion and metastasis of HCC cells. In addition, HAb18G/CD147 has also been shown to be a novel universal cancer biomarker for diagnosis and prognostic assessment of a wide range of cancers. However, the structural basis underlying the multifunctional character of CD147 remains unresolved. We report here the crystal structure of the extracellular portion of HAb18G/CD147 at 2.8A resolution. The structure comprises an N-terminal IgC2 domain and a C-terminal IgI domain, which are connected by a 5-residue flexible linker. This unique C2-I domain organization is distinct from those of other IgSF members. Four homophilic dimers exist in the crystal and adopt C2-C2 and C2-I dimerization rather than V-V dimerization commonly found in other IgSF members. This type of homophilic association thus presents a novel model for homophilic interaction between C2 domains of IgSF members. Moreover, the crystal structure of HAb18G/CD147 provides a good structural explanation for the established multifunction of CD147 mediated by homo/hetero-oligomerizations and should represent a general architecture of other CD147 family members.

Crystal structure of chimeric antibody C2H7 Fab in complex with a CD20 peptide.

Anti-CD20 monoclonal antibodies have been proven to be efficient in the treatment of certain B-cell lymphomas and autoimmune diseases. Intriguingly, these antibodies seem to exert diverse functions with narrow epitope specificity. This study is to investigate the molecular basis of the fine specificity of 2H7 derived antibodies which are of great therapeutic potential. We show that chimeric 2H7 (C2H7) can mediate complement dependent cytotoxicity and antibody-dependent cellular cytotoxicity effects on CD20 positive human Burkitt lymphoma cells and the Fab fragment can well recognize and bind to an epitope peptide of the extracellular loop of CD20. The crystal structure of C2H7 in complex with the CD20 epitope peptide was determined at 2.6A resolution. The bound peptide displays a circular conformation and the binding specificity is mainly contributed by the (170)ANPS(173) motif and the disulfide bond of the peptide which maintains the unique conformation of the peptide. Compared with the complex structure of another anti-CD20 monoclonal antibody Rituximab with the same epitope peptide which was previously determined, the major differences lie in the CDR loop H3 of C2H7 which stretches outward against the interface. Correspondingly, the pocket which accommodates the peptide becomes wider and the peptide moves toward loop H3 and thus is more distant from loops H1 and H2. The hydrogen-bonding interactions are also quite different from those observed in the Rituximab-epitope peptide complex, and both the hydrophilic and hydrophobic interactions are less intense. Our data not only reveal the molecular basis for the fine specificity of C2H7 to CD20, but also provide valuable information for further improvement of antibodies derived from 2H7.