Histone lysine methyltransferases and demethylases in Plasmodium falciparum

Dynamic histone lysine methylation, regulated by methyltransferases and demethylases, plays fundamental roles in chromatin structure and gene expression in a wide range of eukaryotic organisms. A large number of SET-domain-containing proteins make up the histone lysine methyltransferase (HKMT) family, which catalyses the methylation of different lysine residues with relatively high substrate specificities. Another large family of Jumonji C (JmjC)-domain-containing histone lysine demethylases (JHDMs) reverses histone lysine methylation with both lysine site and methyl-state specificities. Through bioinformatic analysis, at least nine SET-domain-containing genes were found in the malaria parasite Plasmodium falciparum and its sibling species. Phylogenetic analysis separated these putative HKMTs into five subfamilies with different putative substrate specificities. Consistent with the phylogenetic subdivision, methyl marks were found on K4, K9 and K36 of histone H3 and K20 of histone H4 by site-specific methyl-lysine antibodies. In addition, most SET-domain genes and histone methyl-lysine marks displayed dynamic changes during the parasite asexual erythrocytic cycle, suggesting that they constitute an important epigenetic mechanism of gene regulation in malaria parasites. Furthermore, the malaria parasite and other apicomplexan genomes also encode JmjC-domain-containing proteins that may serve as histone lysine demethylases. Whereas prokaryotic expression of putative active domains of four P. falciparum SET proteins did not yield detectable HKMT activity towards recombinant P. falciparum histones, two protein domains expressed in vitro in a eukaryotic system showed HKMT activities towards H3 and H4, respectively. With the discovery of these Plasmodium SET- and JmjC-domain genes in the malaria parasite genomes, future efforts will be directed towards elucidation of their substrate specificities and functions in various cellular processes of the parasites.     

Structural basis for the product specificity of histone lysine methyltransferases

DIM-5 is a SUV39-type histone H3 Lys9 methyltransferase that is essential for DNA methylation in N. crassa. We report the structure of a ternary complex including DIM-5, S-adenosyl-L-homocysteine, and a substrate H3 peptide. The histone tail inserts as a parallel strand between two DIM-5 strands, completing a hybrid sheet. Three post-SET cysteines coordinate a zinc atom together with Cys242 from the SET signature motif (NHXCXPN) near the active site. Consequently, a narrow channel is formed to accommodate the target Lys9 side chain. The sulfur atom of S-adenosyl-L-homocysteine, where the transferable methyl group is to be attached in S-adenosyl-L-methionine, lies at the opposite end of the channel, approximately 4 A away from the target Lys9 nitrogen. Structural comparison of the active sites of DIM-5, an H3 Lys9 trimethyltransferase, and SET7/9, an H3 Lys4 monomethyltransferase, allowed us to design substitutions in both enzymes that profoundly alter their product specificities without affecting their catalytic activities.     

Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development

Extensive and dynamic chromatin remodeling occurs after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate early embryonic development. Histone H3 lysine 4 methylation (H3K4me) is an important epigenetic mechanism that associates with gene-specific activation and functions in development. However, dynamic regulation of H3K4me during early embryonic development remains unclear. Herein, the authors examined the dynamic changes of H3K4me and its key regulators (Ash1l, Ash2l, Kmt2a, Kmt2b, Kmt2c, Setd1a, Setd7, Kdm1a, Kdm1b, Kdm5a, Kdm5b, Kdm5c, and Kdm5d) in mouse oocytes and preimplantation embryos. An increase in levels of H3K4me2 and me3 was observed at the one- to two-cell stages (P?P?P?相似文献   

Molecular basis for substrate discrimination by glycine transporters

Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors, and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts on the N-methyl-d-aspartate family of receptors. There are two Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2, which control extracellular glycine concentrations and these transporters show differences in substrate selectivity and blocker sensitivity. A bacterial Na(+)-dependent leucine transporter (LeuT(Aa)) has recently been crystallized and its structure determined. When the amino acid residues within the leucine binding site of LeuT(Aa) are aligned with residues of the two glycine transporters there are a number of identical residues and also some key differences. In this report, we demonstrate that the LeuT(Aa) structure represents a good working model of the Na(+)/Cl(-)-dependent neurotransmitters and that differences in substrate selectivity can be attributed to a single difference of a glycine residue in transmembrane domain 6 of GLYT1 for a serine residue at the corresponding position of GLYT2.     

Molecular basis for substrate recognition and septum cleavage by AtlA,the major N-acetylglucosaminidase of Enterococcus faecalis

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved β-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA–substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.     

Structural basis for substrate recognition and dissociation by human transportin 1

Transportin 1 (Trn1) is a transport receptor that transports substrates from the cytoplasm to the nucleus through nuclear pore complexes by recognizing nuclear localization signals (NLSs). Here we describe four crystal structures of human Trn1 in a substrate-free form as well as in the complex with three NLSs (hnRNP D, JKTBP, and TAP, respectively). Our data have revealed that (1) Trn1 has two sites for binding NLSs, one with high affinity (site A) and one with low affinity (site B), and NLS interaction at site B controls overall binding affinity for Trn1; (2) Trn1 recognizes the NLSs at site A followed by conformational change at site B to interact with the NLSs; and (3) a long flexible loop, characteristic of Trn1, interacts with site B, thereby displacing transport substrate in the nucleus. These studies provide deep understanding of substrate recognition and dissociation by Trn1 in import pathways.     

Structural basis and sequence rules for substrate recognition by Tankyrase explain the basis for cherubism disease

The poly(ADP-ribose)polymerases Tankyrase 1/2 (TNKS/TNKS2) catalyze the covalent linkage of ADP-ribose polymer chains onto target proteins, regulating their ubiquitylation, stability, and function. Dysregulation of substrate recognition by Tankyrases underlies the human disease cherubism. Tankyrases recruit specific motifs (often called RxxPDG "hexapeptides") in their substrates via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), each predicted to bind substrate. Here we report crystal structures of a representative ARC of TNKS2 bound to targeting peptides from six substrates. Using a solution-based peptide library screen, we derive a rule-based consensus for Tankyrase substrates common to four functionally conserved ARCs. This 8-residue consensus allows us to rationalize all known Tankyrase substrates and explains the basis for cherubism-causing mutations in the Tankyrase substrate 3BP2. Structural and sequence information allows us to also predict and validate other Tankyrase targets, including Disc1, Striatin, Fat4, RAD54, BCR, and MERIT40.     

Enabling lead discovery for histone lysine demethylases by high-throughput RapidFire mass spectrometry

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.     

Structural basis for substrate recognition and hydrolysis by mouse carnosinase CN2

L-carnosine is a bioactive dipeptide (beta-alanyl-L-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of L-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn(2+), and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn(2+) at a resolution of 1.7 A and that with Mn(2+) at 2.3 A CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn(2+) and Zn(2+) complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.     

Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases

2,3-Butanediol dehydrogenase (BDH) catalyzes the NAD-dependent redox reaction between acetoin and 2,3-butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l-BDH with a bound inhibitor at 2.0 Å. Comparison with the inhibitor binding mode of meso-BDH highlights the role of a hydrogen-bond from a conserved Trp residue¹⁹². Site-directed mutagenesis of three active site residues of meso-BDH, including Trp¹⁹⁰, which corresponds to Trp¹⁹² of l-BDH, converted its stereospecificity to that of l-BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.     

Structural basis for substrate recognition by Erwinia chrysanthemi GH30 glucuronoxylanase

Xylanase A from the phytopathogenic bacterium Erwinia chrysanthemi is classified as a glycoside hydrolase family 30 enzyme (previously in family 5) and is specialized for degradation of glucuronoxylan. The recombinant enzyme was crystallized with the aldotetraouronic acid β-D-xylopyranosyl-(1→4)-[4-O-methyl-α-D-glucuronosyl-(1→2)]-β-D-xylopyranosyl-(1→4)-D-xylose as a ligand. The crystal structure of the enzyme-ligand complex was solved at 1.39 ? resolution. The ligand xylotriose moiety occupies subsites -1, -2 and -3, whereas the methyl glucuronic acid residue attached to the middle xylopyranosyl residue of xylotriose is bound to the enzyme through hydrogen bonds to five amino acids and by the ionic interaction of the methyl glucuronic acid carboxylate with the positively charged guanidinium group of Arg293. The interaction of the enzyme with the methyl glucuronic acid residue appears to be indispensable for proper distortion of the xylan chain and its effective hydrolysis. Such a distortion does not occur with linear β-1,4-xylooligosaccharides, which are hydrolyzed by the enzyme at a negligible rate. DATABASE: Structural and experimental data are available in the Protein Data Bank database under accession number 2y24 [45].     

Continuous enzymatic assay for histone lysine methyltransferases

We describe a continuous peptide methylation assay using the Neurospora crassa Dim-5 histone H3 lysine 9 (H3K9) methyltransferase as a model system. The assay uses streptavidin FlashPlates coated with target peptide. Since no washing and pipeting steps were required after the addition of the enzyme/S-adenosyl-L-methionine (AdoMet) mixture to the microplate, a continuous readout of the reaction progress was possible. We show that this assay is highly reproducible (with errors in the order of +/- 3%). The continuous assay is well suited for the simultaneous analysis of up to 384 samples, thus allowing for a rapid screening of methylation rates of different substrates under different conditions or in the presence of inhibitors.     

Histone lysine demethylases and their impact on epigenetics

Methylation marks on the lysine residues of histone proteins are thought to contribute to epigenetic phenomena in part because of their apparent irreversibility. Will this view change with the recent discovery of histone lysine demethylases that reversibly remove methyl marks?     

Molecular basis for target RNA recognition and cleavage by human RISC

The RNA-Induced Silencing Complex (RISC) is a ribonucleoprotein particle composed of a single-stranded short interfering RNA (siRNA) and an endonucleolytically active Argonaute protein, capable of cleaving mRNAs complementary to the siRNA. The mechanism by which RISC cleaves a target RNA is well understood, however it remains enigmatic how RISC finds its target RNA. Here, we show, both in vitro and in vivo, that the accessibility of the target site correlates directly with the efficiency of cleavage, demonstrating that RISC is unable to unfold structured RNA. In the course of target recognition, RISC transiently contacts single-stranded RNA nonspecifically and promotes siRNA-target RNA annealing. Furthermore, the 5' part of the siRNA within RISC creates a thermodynamic threshold that determines the stable association of RISC and the target RNA. We therefore provide mechanistic insights by revealing features of RISC and target RNAs that are crucial to achieve efficiency and specificity in RNA interference.     

Molecular basis for substrate selectivity and specificity by an LPS biosynthetic enzyme

Diversity in the polysaccharide component of lipopolysaccharide (LPS) contributes to the persistence and pathogenesis of Gram-negative bacteria. The Nudix hydrolase GDP-mannose mannosyl hydrolase (Gmm) contributes to this diversity by regulating the concentration of mannose in LPS biosynthetic pathways. Here, we present seven high-resolution crystal structures of Gmm from the enteropathogenic E. coli strain O128: the structure of the apo enzyme, the cocrystal structure of Gmm bound to the product Mg2+-GDP, two cocrystal structures of precatalytic and turnover complexes of Gmm-Ca2+-GDP-alpha-d-mannose, and three cocrystal structures of an inactive mutant (His-124 --> Leu) Gmm bound to substrates GDP-alpha-d-mannose, GDP-alpha-d-glucose, and GDP-beta-l-fucose. These crystal structures help explain the molecular basis for substrate specificity and promiscuity and provide a structural framework for reconciling previously determined kinetic parameters. Unexpectedly, these structures reveal concerted changes in the enzyme structure that result in the formation of a catalytically competent active site only in the presence of the substrate/product. These structural views of the enzyme may provide a rationale for the design of inhibitors that target the biosynthesis of LPS by pathogenic bacteria.     

Structural basis for substrate recognition by the editing domain of isoleucyl-tRNA synthetase

In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNA(Ile), in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 A resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120 degrees and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180 degrees . The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T.thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.     

Molecular basis for the recognition of snurportin 1 by importin beta

The nuclear import of uridine-rich ribonucleoproteins is mediated by the transport adaptor snurportin 1 (SNP1). Similar to importin alpha, SNP1 uses an N-terminal importin beta binding (sIBB) domain to recruit the receptor importin beta and gain access to the nucleus. In this study, we demonstrate that the sIBB domain has a bipartite nature, which contains two distinct binding determinants for importin beta. The first determinant spans residues 25-65 and includes the previously identified importin alpha IBB (alphaIBB) region of homology. The second binding determinant encompasses residues 1-24 and resembles region 1011-1035 of the nucleoporin 153 (Nup153). The two binding determinants synergize within the sIBB domain to confer a low nanomolar binding affinity for importin beta (K(d) approximately 2 nm) in an interaction that, in vitro, is displaced by RanGTP. We propose that in vivo the synergy of Nup153 and nuclear RanGTP promotes translocation of uridine-rich ribonucleoproteins into the nucleus.