Crystal structure of the Leishmania major phosphodiesterase LmjPDEB1 and insight into the design of the parasite-selective inhibitors

Human leishmaniasis is a major public health problem in many countries, but chemotherapy is in an unsatisfactory state. Leishmania major phosphodiesterases (LmjPDEs) have been shown to play important roles in cell proliferation and apoptosis of the parasite. Thus LmjPDE inhibitors may potentially represent a novel class of drugs for the treatment of leishmaniasis. Reported here are the kinetic characterization of the LmjPDEB1 catalytic domain and its crystal structure as a complex with 3-isobutyl-1-methylxanthine (IBMX) at 1.55 A resolution. The structure of LmjPDEB1 is similar to that of human PDEs. IBMX stacks against the conserved phenylalanine and forms a hydrogen bond with the invariant glutamine, in a pattern common to most inhibitors bound to human PDEs. However, an extensive structural comparison reveals subtle, but significant differences between the active sites of LmjPDEB1 and human PDEs. In addition, a pocket next to the inhibitor binding site is found to be unique to LmjPDEB1. This pocket is isolated by two gating residues in human PDE families, but constitutes a natural expansion of the inhibitor binding pocket in LmjPDEB1. The structure particularity might be useful for the development of parasite-selective inhibitors for the treatment of leishmaniasis.

Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity

Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 A) and in complex with acarbose (to 1.9 A). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.

The crystal structure of the platelet activator aggretin reveals a novel (alphabeta)2 dimeric structure

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.

Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions

Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5' end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-A resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.

The Age of the Universe and its Measurement

Current wisdom in cosmology has it that the Universe is about 13.8 billions year old. Statements about the age of the Universe are not just difficult to confirm, but also carry a lot of presuppositions. The aim of this talk is to make explicit these presuppositions, to discuss their significance and to trace the implications for an emipirical investigation of the age of the Universe.

In search of the internal structure of the processes underlying interval timing in the sub-second and the second range: A confirmatory factor analysis approach

One of the earliest accounts of duration perception by Karl von Vierordt implied a common process underlying the timing of intervals in the sub-second and the second range. To date, there are two major explanatory approaches for the timing of brief intervals: the Common Timing Hypothesis and the Distinct Timing Hypothesis. While the common timing hypothesis also proceeds from a unitary timing process, the distinct timing hypothesis suggests two dissociable, independent mechanisms for the timing of intervals in the sub-second and the second range, respectively. In the present paper, we introduce confirmatory factor analysis (CFA) to elucidate the internal structure of interval timing in the sub-second and the second range. Our results indicate that the assumption of two mechanisms underlying the processing of intervals in the second and the sub-second range might be more appropriate than the assumption of a unitary timing mechanism. In contrast to the basic assumption of the distinct timing hypothesis, however, these two timing mechanisms are closely associated with each other and share 77% of common variance. This finding suggests either a strong functional relationship between the two timing mechanisms or a hierarchically organized internal structure. Findings are discussed in the light of existing psychophysical and neurophysiological data.

Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights

Sodium-proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium-proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium-proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium-proton antiport in NhaA.

The structure of the debate on EU integration issues. A comparative analysis of party communication in the EP 2014 elections

The results of Eurosceptic parties in the recent European parliament election provide further evidence that the “permissive consensus” on European integration blurred. This paper focuses on the structure of the debate on EU integration issues. Which EU integration issues and positions do parties put forward? Can the debate on EU integration issues be subsumed in one or several dimensions? Do they reflect national political conflicts such as the left-right and the ‘new politics’/cultural divide? Or do they form one unique or several EU-specific dimensions, e.g. national sovereignty versus integration? In order to address these questions, this paper departs from the assumption that debate on European integration is multidimensional in its nature and therefore entails a multitude of issue areas. In other words, it does not look at how socio-economic and cultural issues are related to European integration but focuses on its components, i.e. particular EU-specific policies such as EU-wide employment, environment, immigration and monetary policy. The paper departs from the cleavage theory on political di-visions and different approaches transferring them to EU politics. Two points should be noted; first, this paper does not compare the debate on European integration issues between the national level and the EU level, but whether domestic divisions are reflected at the EU level. Second, it is not concerned with the general ideo-logical profile of political parties on EU integration issues, but on EU issues that parties communicated through press releases. By doing this, the paper is concerned with the salient EU issues that parties touch upon.

The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein

The polypeptide composition of the U7 small nuclear ribonucleoprotein (snRNP) involved in histone messenger RNA (mRNA) 3' end formation has recently been elucidated. In contrast to spliceosomal snRNPs, which contain a ring-shaped assembly of seven so-called Sm proteins, in the U7 snRNP the Sm proteins D1 and D2 are replaced by U7-specific Sm-like proteins, Lsm10 and Lsm11. This polypeptide composition and the unusual structure of Lsm11, which plays a role in histone RNA processing, represent new themes in the biology of Sm/Lsm proteins. Moreover this structure has important consequences for snRNP assembly that is mediated by two complexes containing the PRMT5 methyltransferase and the SMN (survival of motor neurons) protein, respectively. Finally, the ability to alter this polypeptide composition by a small mutation in U7 snRNA forms the basis for using modified U7 snRNA derivatives to alter specific pre-mRNA splicing events, thereby opening up a new way for antisense gene therapy.

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.