Posttranslational modifications of histones regulate chromatin structure and gene expression. Histone demethylases, members of a newly emerging transcription-factor family, remove methyl groups from the lysine residues of the histone tails and thereby regulate the transcriptional activity of target genes. JmjC-domain-containing proteins have been predicted to be demethylases. For example, the JmjC-containing protein JMJD2A has been characterized as a H3-K9me3- and H3-K36me3-specific demethylase. Here, structures of the catalytic-core domain of JMJD2A with and without alpha-ketoglutarate in the presence of Fe2+ have been determined by X-ray crystallography. The structure of the core domain, consisting of the JmjN domain, the JmjC domain, the C-terminal domain, and a zinc-finger motif, revealed the unique elements that form a potential substrate binding pocket. Sited-directed mutagenesis in conjunction with demethylase activity assays allowed us to propose a molecular model for substrate selection by the JMJD2 histone demethylase family.

The Jumonji C domain is a catalytic motif that mediates histone lysine demethylation. The Jumonji C-containing oxygenase JMJD2A specifically demethylates tri-and dimethylated lysine-9 and lysine-36 of histone 3 (H3K9/36me3/2). Here we present structures of the JMJD2A catalytic core complexed with methylated H3K36 peptide substrates in the presence of Fe(II) and N-oxalylglycine. We found that the interaction between JMJD2A and peptides largely involves the main chains of the enzyme and the peptide. The peptide-binding specificity is primarily determined by the primary structure of the peptide, which explains the specificity of JMJD2A for methylated H3K9 and H3K36 instead of other methylated residues such as H3K27. The specificity for a particular methyl group, however, is affected by multiple factors, such as space and the electrostatic environment in the catalytic center of the enzyme. These results provide insights into the mechanisms and specificity of histone demethylation.demethylase ͉ oxygenase ͉ JmjC ͉ epigenetic ͉ chromatin

JMJD6 is a Jumonji C domain-containing hydroxylase. JMJD6 binds α-ketoglutarate and iron and has been characterized as either a histone arginine demethylase or U2AF65 lysyl hydroxylase. Here, we describe the structures of JMJD6 with and without α-ketoglutarate, which revealed a novel substrate binding groove and two positively charged surfaces. The structures also contain a stack of aromatic residues located near the active center. The side chain of one residue within this stack assumed different conformations in the two structures. Interestingly, JMJD6 bound efficiently to single-stranded RNA, but not to single-stranded DNA, doublestranded RNA, or double-stranded DNA. These structural features and truncation analysis of JMJD6 suggest that JMJD6 may bind and modify single-stand RNA rather than the previously reported peptide substrates.RNA binding proteins | RNA modification | RNA splicing J MJD6 was first characterized as a receptor for phosphatidylserine (PSR), which facilitates the phagocytosis of dead and dying cells by macrophages and fibroblasts (1). Targeted deletion of gene encoding PSR in mice and morpholino knock-downs of PSR in zebrafish resulted in embryonic lethality, with severe defects in hematopoiesis and aberrant development of eye, brain, and heart (2-5). In contrast, knock-down of PSR expression in Caenorhabditis elegans produced only a mild phenotype (5). Somewhat surprisingly, sequence analysis suggested that JMJD6 contains a Jumonji C (JMJC) domain, which places it within a highly conserved, cupin fold-containing enzyme family (6-8). Further analysis demonstrated that the protein is localized specifically in the nucleus (7-9). Despite the significant effects of JMJD6 deficiency, knockout mice engulfed apoptotic cells normally (9). Based on these studies and additional sequence analysis, the protein was recategorized as an α-ketoglutarateand Fe 2þ -dependent hydroxylase and was named JMJD6 (10).Recent studies demonstrated that most JMJC domain-containing proteins function as histone demethylases by specifically acting on lysine residues in histone tails (11)(12)(13)(14). For example, the specific interactions between enzymes from the JMJD2 subfamily and methylated peptides have been structurally characterized (15-18). Interestingly, JMJD6 was reported to demethylate arginine residues in histone tails (10). Several laboratories including ours, however, have been unable to reproduce these results. In other studies, JMJD6 was identified as a lysine hydroxylase that specifically recognizes the protein tail of U2AF65, a mediator of RNA splicing (19).To resolve the disparate results and further elucidate the structure and functions of JMJD6, we determined X-ray crystallographic structures of the protein with and without α-ketoglutarate. To obtain these structures, JMJD6 was cocrystallized with a Fab fragment derived from a JMJD6-specific hamster monoclonal antibody. Intriguingly, the structure of JMJD6 is dramatically different from known structures of other JMJC domain superfamily proteins includ...

The tumour necrosis factor (TNF) ligand TALL-1 and its cognate receptors, BCMA, TACI and BAFF-R, were recently identified as members of the TNF superfamily, which are essential factors contributing to B-cell maturation. The functional, soluble fragment of TALL-1 (sTALL-1) forms a virus-like assembly for its proper function. Here we determine the crystal structures of sTALL-1 complexed with the extracellular domains of BCMA and BAFF-R at 2.6 and 2.5 A, respectively. The single cysteine-rich domain of BCMA and BAFF-R both have saddle-like architectures, which sit on the horseback-like surface formed by four coil regions on each individual sTALL-1 monomer. Three novel structural modules, D2, X2 and N, were revealed from the current structures. Sequence alignments, structural modelling and mutagenesis revealed that one disulphide bridge in BAFF-R is critical for determining the binding specificity of the extracellular domain eBAFF-R to TALL-1 instead of APRIL, a closely related ligand of TALL-1, which was confirmed by binding experiments in vitro.

Glycan-binding proteins are important for a wide variety of basic research and clinical applications, but proteins with high affnity and selectivity for carbohydrates are diffcult to obtain. Here we describe a facile and cost-effective strategy to generate monoclonal lamprey antibodies, called lambodies, that target glycan determinants. We screened a library of yeast surface-displayed (YSD) lamprey variable lymphocyte receptors (VLR) for clones that can selectively bind various biomedically important glycotopes. These glycoconjugates included tumor-associated carbohydrate antigens (Tn and TFα), Lewis antigens (LeA and LeX), N-glycolylneuraminic acid, targets of broadly neutralizing HIV antibodies (poly-Man9 and the HIV gp120), and the glycoproteins asialo-ovine submaxillary mucin (aOSM) and asialo-human glycophorin A (aGPA). We isolated clones that bind each of these targets in a glycan-dependent manner and with very strong binding constants, for example, 6.2 nM for Man9 and 44.7 nM for gp120, determined by surface plasmon resonance (SPR). One particular lambody, VLRB.aGPA.23, was shown by glycan array analysis to be selective for the blood group H type 3 trisaccharide (BG-H3, Fucα1-2Galβ1-3GalNAcα), aGPA, and TFα (Galβ1-3GalNAcα), with affnity constants of 0.2, 1, and 8 nM, respectively. In human tissue microarrays this lambody selectively detected cancer-associated carbohydrate antigens in 14 different types of cancers. It stained 27% of non-small cell lung cancer (NSCLC) samples in a pattern that correlated with poor patient survival. Lambodies with exquisite affnity and selectivity for glycans may find myriad uses in glycobiology and biomedical research.

Background: Variable lymphocyte receptors (VLRs) bind tumor-associated carbohydrates, such as Thomsen-Friedenreich antigen (TF␣), with high selectivity. Results:The crystal structure of a VLR-TF␣ complex coupled with thermodynamic analysis revealed the basis for selectivity. Conclusion: VLRs utilize leucine-rich repeats to recognize glycans with affinity comparable to that of lectins and antibodies. Significance: The VLR-TF␣ structure provides a template for engineering VLRs to bind biomedically relevant glycans.

The freak oscillation in one-dimensional dusty plasma is studied numerically by particle-in-cell method. Using a perturbation method, the basic set of fluid equations is reduced to a nonlinear Schrödinger equation (NLSE). The rational solution of the NLSE is presented, which is proposed as an effective tool for studying the rogue waves in dusty plasma. Additionally, the application scope of the analytical solution of the rogue wave described by the NLSE is given.

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.