Showing papers in "Nature Structural & Molecular Biology in 2004"
TL;DR: It is highlighted that there are a variety of enzymes with different specificities, suggesting that individual nucleases act on distinct subpopulations of transcripts within the cell and multiple mechanisms by which mRNA degradation could be regulated.
Abstract: The degradation of eukaryotic mRNAs plays important roles in the modulation of gene expression, quality control of mRNA biogenesis and antiviral defenses. In the past five years, many of the enzymes involved in this process have been identified and mechanisms that modulate their activities have begun to be identified. In this review, we describe the enzymes of mRNA degradation and their properties. We highlight that there are a variety of enzymes with different specificities, suggesting that individual nucleases act on distinct subpopulations of transcripts within the cell. In several cases, translation factors that bind mRNA inhibit these nucleases. In addition, recent work has begun to identify distinct mRNP complexes that recruit the nucleases to transcripts through different mRNA-interacting proteins. These properties and complexes suggest multiple mechanisms by which mRNA degradation could be regulated.
835 citations
TL;DR: This work has shown that the ATP-switch model provides an exquisitely regulated switch that induces conformational changes in the TMDs to mediate membrane transport in ABC transporters.
Abstract: ABC transporters mediate active translocation of a diverse range of molecules across all cell membranes. They comprise two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Recent biochemical, structural and genetic studies have led to the ATP-switch model in which ATP binding and ATP hydrolysis, respectively, induce formation and dissociation of an NBD dimer. This provides an exquisitely regulated switch that induces conformational changes in the TMDs to mediate membrane transport.
696 citations
TL;DR: The major polyphenol in green tea, (−)-epigallocatechin-3-gallate (EGCG), has been shown to prevent carcinogenesis and a receptor that mediates the anticancer activity of EGCG is identified.
Abstract: The major polyphenol in green tea, (−)-epigallocatechin-3-gallate (EGCG), has been shown to prevent carcinogenesis We have identified a receptor that mediates the anticancer activity of EGCG Expression of the metastasis-associated 67-kDa laminin receptor confers EGCG responsiveness to cancer cells at physiologically relevant concentrations Experiments using surface plasmon resonance demonstrate binding of EGCG to the 67-kDa laminin receptor with a nanomolar K d value
638 citations
TL;DR: It is reported here that minus-strand deamination occurred over the length of the virus genome, preferentially at CCCA sequences, with a graded frequency in the 5′→3′ direction.
Abstract: Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome
607 citations
TL;DR: The first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, is described, a six-bladed β-propeller with a unique active site lid that is also involved in HDL binding.
Abstract: Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, 'good cholesterol') and are involved in the prevention of atherosclerosis. We describe the first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, at a resolution of 2.2 A. PON1 is a six-bladed beta-propeller with a unique active site lid that is also involved in HDL binding. The three-dimensional structure and directed evolution studies permit a detailed description of PON1's active site and catalytic mechanism, which are reminiscent of secreted phospholipase A2, and of the routes by which PON family members diverged toward different substrate and reaction selectivities.
588 citations
TL;DR: Kinetic analysis suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage, and product release, and the position of the scissile phosphate on the target RNA seems to be determined during RISC assembly, before the si RNA encounters its RNA target.
Abstract: The siRNA-directed ribonucleoprotein complex, RISC, catalyzes target RNA cleavage in the RNA interference pathway. Here, we show that siRNA-programmed RISC is a classical Michaelis-Menten enzyme in the presence of ATP. In the absence of ATP, the rate of multiple rounds of catalysis is limited by release of the cleaved products from the enzyme. Kinetic analysis suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage, and product release. Bases near the siRNA 5′ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3′ regions of the siRNA provide a helical geometry required for catalysis. Finally, the position of the scissile phosphate on the target RNA seems to be determined during RISC assembly, before the siRNA encounters its RNA target.
570 citations
TL;DR: X-ray structures of human MEK1 and MEK2 are presented, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 Å and 3.2 Å, and reveal a novel, noncompetitive mechanism for protein kinase inhibition.
Abstract: MEK1 and MEK2 are closely related, dual-specificity tyrosine/threonine protein kinases found in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway. Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK1 results in cellular transformation. Here we present the X-ray structures of human MEK1 and MEK2, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 A and 3.2 A, respectively. The structures reveal that MEK1 and MEK2 each have a unique inhibitor-binding pocket adjacent to the MgATP-binding site. The presence of the potent inhibitor induces several conformational changes in the unphosphorylated MEK1 and MEK2 enzymes that lock them into a closed but catalytically inactive species. Thus, the structures reported here reveal a novel, noncompetitive mechanism for protein kinase inhibition.
566 citations
TL;DR: Models for the specific assembly of the RNA polymerase II transcription machinery at a promoter, conformational changes that occur during initiation of transcription, and the mechanism of initiation are discussed in light of recent developments.
Abstract: Advances in structure determination of the bacterial and eukaryotic transcription machinery have led to a marked increase in the understanding of the mechanism of transcription. Models for the specific assembly of the RNA polymerase II transcription machinery at a promoter, conformational changes that occur during initiation of transcription, and the mechanism of initiation are discussed in light of recent developments.
563 citations
TL;DR: The spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin, and it is speculated that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions.
Abstract: Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.
557 citations
TL;DR: It is reported that three representatives of this riboswitch class bind adenine with values for apparent dissociation constant (apparent Kd) that are several orders of magnitude lower than those for binding guanine.
Abstract: A class of riboswitches that recognizes guanine and discriminates against other purine analogs was recently identified. RNAs that carry the consensus sequence and structural features of guanine riboswitches are located in the 5′ untranslated region (UTR) of numerous prokaryotic genes, where they control the expression of proteins involved in purine salvage and biosynthesis. We report that three representatives of this riboswitch class bind adenine with values for apparent dissociation constant (apparent Kd) that are several orders of magnitude lower than those for binding guanine. Because preference for adenine is attributable to a single nucleotide substitution, the RNA most likely recognizes its ligand by forming a Watson-Crick base pair. In addition, the adenine riboswitch associated with the ydhL gene of Bacillus subtilis functions as a genetic 'on' switch, wherein adenine binding causes a structural rearrangement that precludes formation of an intrinsic transcription terminator stem.
557 citations
TL;DR: Structural and mutagenesis data explain how both receptors bind high-mannose oligosaccharides on enveloped viruses and why only DC-SIGN binds blood group antigens, including those present on microorganisms.
Abstract: Both the dendritic cell receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR bind human immunodeficiency virus and enhance infection. However, biochemical and structural comparison of these receptors now reveals that they have very different physiological functions. By screening an extensive glycan array, we demonstrated that DC-SIGN and DC-SIGNR have distinct ligand-binding properties. Our structural and mutagenesis data explain how both receptors bind high-mannose oligosaccharides on enveloped viruses and why only DC-SIGN binds blood group antigens, including those present on microorganisms. DC-SIGN mediates endocytosis, trafficking as a recycling receptor and releasing ligand at endosomal pH, whereas DC-SIGNR does not release ligand at low pH or mediate endocytosis. Thus, whereas DC-SIGN has dual ligand-binding properties and functions both in adhesion and in endocytosis of pathogens, DC-SIGNR binds a restricted set of ligands and has only the properties of an adhesion receptor.
TL;DR: Cryo-EM and scanning transmission EM are used to determine that an average complete immature HIV particle contains ∼5,000 structural (Gag) proteins, more than twice the number from previous estimates.
Abstract: The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for virus particle assembly Initially, Gag forms an approximately spherical shell underlying the membrane of the immature particle After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome This mature shell contains 1,000-1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm By contrast, little is known about the structure of the immature virus We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete immature HIV particle contains approximately 5,000 structural (Gag) proteins, more than twice the number from previous estimates In the immature virus, Gag forms a hexameric lattice with a spacing of 80 nm Thus, less than half of the CA proteins form the mature core
TL;DR: The structure shows a novel lipid II–binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds, which provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics.
Abstract: The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.
TL;DR: The data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu–GTP–aa-tRNA toward peptidyl-t RNA during selection on the ribosome, and propose a mechanistic model of initial selection and proofreading.
Abstract: Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu–GTP–aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.
TL;DR: It is proposed that dense intragenic DNA methylation in mammalian cells initiates formation of a chromatin structure that reduces the efficiency of Pol II elongation.
Abstract: Transcriptional silencing in mammals is often associated with promoter methylation. However, a considerable number of genomic methylated CpGs exist in transposable elements, which are frequently found in intronic regions. To determine whether intragenic methylation influences transcription efficiency, we used the Cre/loxP-based system, RMCE, to introduce a transgene, methylated exclusively in a region downstream of the promoter, into a specific genomic site. This methylation pattern was maintained in vivo, and yielded a clear decrease in transgene expression relative to an unmethylated control. Notably, RNA polymerase II (Pol II) was depleted exclusively in the methylated region, as was histone H3 di- and trimethylated on Lys4 and acetylated on Lys9 and Lys14. As the methylated region adopts a closed chromatin structure in vivo, we propose that dense intragenic DNA methylation in mammalian cells initiates formation of a chromatin structure that reduces the efficiency of Pol II elongation.
TL;DR: A comparative and quantitative study of activity, structure and dynamics revealed a close link between protein dynamics and catalytic turnover in hyperthermophilic and mesophilic homologs of adenylate kinase.
Abstract: A fundamental question is how enzymes can accelerate chemical reactions. Catalysis is not only defined by actual chemical steps, but also by enzyme structure and dynamics. To investigate the role of protein dynamics in enzymatic turnover, we measured residue-specific protein dynamics in hyperthermophilic and mesophilic homologs of adenylate kinase during catalysis. A dynamic process, the opening of the nucleotide-binding lids, was found to be rate-limiting for both enzymes as measured by NMR relaxation. Moreover, we found that the reduced catalytic activity of the hyperthermophilic enzyme at ambient temperatures is caused solely by a slower lid-opening rate. This comparative and quantitative study of activity, structure and dynamics revealed a close link between protein dynamics and catalytic turnover.
TL;DR: The crystal structure of the N-terminal half of human POT1 (hPOT1) protein bound to a telomeric single-stranded DNA (ssDNA) decamer, TTAGGGTTAG, is reported, providing an atomic-resolution model for chromosome end-capping.
Abstract: The POT1 (protection of telomeres 1) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryotes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere length in human cells. Here, we report the crystal structure at a resolution of 1.73 A of the N-terminal half of human POT1 (hPOT1) protein bound to a telomeric single-stranded DNA (ssDNA) decamer, TTAGGGTTAG, the minimum tight-binding sequence indicated by in vitro binding assays. The structure reveals that hPOT1 contains two oligonucleotide/ oligosaccharide-binding (OB) folds; the N-terminal OB fold binds the first six nucleotides, resembling the structure of the S. pombe Pot1pN-ssDNA complex, whereas the second OB fold binds and protects the 3' end of the ssDNA. These results provide an atomic-resolution model for chromosome end-capping.
TL;DR: It is shown that allosteric inhibitors prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop, thereby exploiting a general mechanism used by tyrosine phosphatases.
Abstract: Obesity and type II diabetes are closely linked metabolic syndromes that afflict >100 million people worldwide. Although protein tyrosine phosphatase 1B (PTP1B) has emerged as a promising target for the treatment of both syndromes, the discovery of pharmaceutically acceptable inhibitors that bind at the active site remains a substantial challenge. Here we describe the discovery of an allosteric site in PTP1B. Crystal structures of PTP1B in complex with allosteric inhibitors reveal a novel site located approximately 20 A from the catalytic site. We show that allosteric inhibitors prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop, thereby exploiting a general mechanism used by tyrosine phosphatases. Notably, these inhibitors exhibit selectivity for PTP1B and enhance insulin signaling in cells. Allosteric inhibition is a promising strategy for targeting PTP1B and constitutes a mechanism that may be applicable to other tyrosine phosphatases.
TL;DR: This work has studied the entry mechanisms of influenza viruses by tracking the interaction of single viruses with cellular endocytic structures in real time using fluorescence microscopy, and shows that influenza can exploit clathrin-mediated and clathin- and caveolin-independent endocytical pathways in parallel, both pathways leading to viral fusion with similar efficiency.
Abstract: Most viruses enter cells via receptor-mediated endocytosis. However, the entry mechanisms used by many of them remain unclear. Also largely unknown is the way in which viruses are targeted to cellular endocytic machinery. We have studied the entry mechanisms of influenza viruses by tracking the interaction of single viruses with cellular endocytic structures in real time using fluorescence microscopy. Our results show that influenza can exploit clathrin-mediated and clathrin- and caveolin-independent endocytic pathways in parallel, both pathways leading to viral fusion with similar efficiency. Remarkably, viruses taking the clathrin-mediated pathway enter cells via the de novo formation of clathrin-coated pits (CCPs) at viral-binding sites. CCP formation at these sites is much faster than elsewhere on the cell surface, suggesting a virus-induced CCP formation mechanism that may be commonly exploited by many other types of viruses.
TL;DR: The initiation site is identified as a novel component of the targeting signal, which is required to engage the proteasome unfolding machinery efficiently and accelerate proteolysis of tightly folded proteins.
Abstract: The proteasome is the main ATP-dependent protease in eukaryotic cells and controls the concentration of many regulatory proteins in the cytosol and nucleus. Proteins are targeted to the proteasome by the covalent attachment of polyubiquitin chains. The ubiquitin modification serves as the proteasome recognition element but by itself is not sufficient for efficient degradation of folded proteins. We report that proteolysis of tightly folded proteins is accelerated greatly when an unstructured region is attached to the substrate. The unstructured region serves as the initiation site for degradation and is hydrolyzed first, after which the rest of the protein is digested sequentially. These results identify the initiation site as a novel component of the targeting signal, which is required to engage the proteasome unfolding machinery efficiently. The proteasome degrades a substrate by first binding to its ubiquitin modification and then initiating unfolding at an unstructured region.
TL;DR: The role of Dicer has previously been established in RNAi as the originator of 22-mers characteristic of silencing phenomena and a related RNAse III enzyme, Drosha, has surfaced as another component of the RNAi pathway.
Abstract: Our understanding of RNA interference has been enhanced by new data concerning RNase III molecules. The role of Dicer has previously been established in RNAi as the originator of 22-mers characteristic of silencing phenomena. Recently, a related RNAse III enzyme, Drosha, has surfaced as another component of the RNAi pathway. In addition to biochemistry, protein structures have proven to be helpful in deciphering the enzymology of RNase III molecules.
TL;DR: This work incorporated double-labeled F0F1-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis.
Abstract: Synthesis of ATP from ADP and phosphate, catalyzed by F0F1-ATP synthases, is the most abundant physiological reaction in almost any cell. F0F1-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F0F1-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The γ subunit rotates stepwise during proton transport–powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.
TL;DR: This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation.
Abstract: The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. However, the molecular events that regulate p300 HAT activity are poorly understood. We evaluated autoacetylation of the p300 HAT protein domain to determine its function. Using expressed protein ligation, the p300 HAT protein domain was generated in hypoacetylated form and found to have reduced catalytic activity. This basal catalytic rate was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif. This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation. We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide variety of signaling and transcriptional events.
TL;DR: A new and testable model for how cells implement the histone code by modulating nucleosome dynamics is proposed.
Abstract: Post-translational modifications of the histone tails are correlated with distinct chromatin states that regulate access to DNA Recent proteomic analyses have revealed several new modifications in the globular nucleosome core, many of which lie at the histone-DNA interface We interpret these modifications in light of previously published data and propose a new and testable model for how cells implement the histone code by modulating nucleosome dynamics
TL;DR: It is reported that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways, one involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylated and activation of IRF-3.
Abstract: Double-stranded RNA (dsRNA), a frequent byproduct of virus infection, is recognized by Toll-like receptor 3 (TLR3) to mediate innate immune response to virus infection. TLR3 signaling activates the transcription factor IRF-3 by its Ser/Thr phosphorylation, accompanied by its dimerization and nuclear translocation. It has been reported that the Ser/Thr kinase TBK-1 is essential for TLR3-mediated activation and phosphorylation of IRF-3. Here we report that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways. One involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells. Thus, the PI3K-Akt pathway plays an essential role in TLR3-mediated gene induction.
TL;DR: It is proposed that syt-PIP2 interactions are involved in exocytosis by facilitating the close apposition of the vesicle and target membrane on rapid time scales in response to Ca2+.
Abstract: Synaptotagmin-1 (syt), the putative Ca2+ sensor for exocytosis, is anchored to the membrane of secretory organelles. Its cytoplasmic domain is composed of two Ca2+-sensing modules, C2A and C2B. Syt binds phosphatidylinositol 4,5-bisphosphate (PIP2), a plasma membrane lipid with an essential role in exocytosis and endocytosis. We resolved two modes of PIP2 binding that are mediated by distinct surfaces on the C2B domain of syt. A novel Ca2+-independent mode of binding predisposes syt to penetrate PIP2-harboring target membranes in response to Ca2+ with submillisecond kinetics. Thus, PIP2 increases the speed of response of syt and steers its membrane-penetration activity toward the plasma membrane. We propose that syt-PIP2 interactions are involved in exocytosis by facilitating the close apposition of the vesicle and target membrane on rapid time scales in response to Ca2+.
TL;DR: Direct detection of a dynamic equilibrium conformational transition in nucleosomes that greatly increases the distance between the end of the nucleosomal DNA and the histone core is reported and the equilibrium constant for this transition under physiological conditions is quantified.
Abstract: DNA wrapped in nucleosomes is sterically occluded, creating obstacles for polymerase, regulatory, remodeling, repair and recombination complexes, which require access to the wrapped DNA. How such complexes recognize and gain access to their DNA target sites is not known. Here we report the direct detection of a dynamic equilibrium conformational transition in nucleosomes that greatly increases the distance between the end of the nucleosomal DNA and the histone core. We quantified the equilibrium constant for this transition under physiological conditions. As predicted by these findings, addition of LexA protein to nucleosomes containing the LexA target site drives this conformational equilibrium toward the unwrapped, accessible state, simultaneously allowing stable LexA binding. This inherent property of nucleosomes allows any protein, whether an energy-dependent machine or a passive binder, to gain access even to buried stretches of nucleosomal DNA.
TL;DR: It is proposed that the PAZ domain contributes to the specific recognition of siRNAs by providing a binding pocket for their characteristic two-nucleotide 3′ overhangs.
Abstract: We describe the solution structures of the Argonaute2 PAZ domain bound to RNA and DNA oligonucleotides. The structures reveal a unique mode of single-stranded nucleic acid binding in which the two 3'-terminal nucleotides are buried in a hydrophobic cleft. We propose that the PAZ domain contributes to the specific recognition of siRNAs by providing a binding pocket for their characteristic two-nucleotide 3' overhangs.
TL;DR: A notably close contact interface, comprised of extensive active site and exosite interactions, explains, in molecular detail, the basis of the antithrombotic properties of therapeutic heparin.
Abstract: The maintenance of normal blood flow depends completely on the inhibition of thrombin by antithrombin, a member of the serpin family. Antithrombin circulates at a high concentration, but only becomes capable of efficient thrombin inhibition on interaction with heparin or related glycosaminoglycans. The anticoagulant properties of therapeutic heparin are mediated by its interaction with antithrombin, although the structural basis for this interaction is unclear. Here we present the crystal structure at a resolution of 2.5 A of the ternary complex between antithrombin, thrombin and a heparin mimetic (SR123781). The structure reveals a template mechanism with antithrombin and thrombin bound to the same heparin chain. A notably close contact interface, comprised of extensive active site and exosite interactions, explains, in molecular detail, the basis of the antithrombotic properties of therapeutic heparin.
TL;DR: The crystal structure of Ca2+-free wild-type PAD4 shows that the polypeptide chain adopts an elongated fold in which the N-terminal domain forms two immunoglobulin-like subdomains, and the C-terminals forms an α/β propeller structure, which indicates that Ca2-binding induces conformational changes that generate the active site cleft.
Abstract: Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. Its gene is a susceptibility locus for rheumatoid arthritis. Here we present the crystal structure of Ca(2+)-free wild-type PAD4, which shows that the polypeptide chain adopts an elongated fold in which the N-terminal domain forms two immunoglobulin-like subdomains, and the C-terminal domain forms an alpha/beta propeller structure. Five Ca(2+)-binding sites, none of which adopt an EF-hand motif, were identified in the structure of a Ca(2+)-bound inactive mutant with and without bound substrate. These structural data indicate that Ca(2+) binding induces conformational changes that generate the active site cleft. Our findings identify a novel mechanism for enzyme activation by Ca(2+) ions, and are important for understanding the mechanism of protein citrullination and for developing PAD-inhibiting drugs for the treatment of rheumatoid arthritis.