The mechanism of vault opening from the high resolution structure of the N-terminal repeats of MVP

Vaults are ubiquitous ribonucleoprotein complexes involved in a diversity of cellular processes, including multidrug resistance, transport mechanisms and signal transmission. The vault particle shows a barrel-shaped structure organized in two identical moieties, each consisting of 39 copies of the major vault protein MVP. Earlier data indicated that vault halves can dissociate at acidic pH. The crystal structure of the vault particle solved at 8 Å resolution, together with the 2.1-Å structure of the seven N-terminal domains (R1–R7) of MVP, reveal the interactions governing vault association and provide an explanation for a reversible dissociation induced by low pH. The structural comparison with the recently published 3.5 Å model shows major discrepancies, both in the main chain tracing and in the side chain assignment of the two terminal domains R1 and R2

A non-canonical di-acidic signal at the C-terminus of Kv1.3 determines anterograde trafficking and surface expression

Impairment of Kv1.3 expression at the cell membrane in leukocytes and sensory neuron contributes to the pathophysiology of autoimmune diseases and sensory syndromes. Molecular mechanisms underlying Kv1.3 channel trafficking to the plasma membrane remain elusive. We report a novel non-canonical di-acidic signal (E483/484) at the C-terminus of Kv1.3 essential for anterograde transport and surface expression. Notably, homologous motifs are conserved in neuronal Kv1 and Shaker channels. Biochemical analysis revealed interactions with the Sec24 subunit of the coat protein complex II. Disruption of this complex retains the channel at the endoplasmic reticulum. A molecular model of the Kv1.3-Sec24a complex suggests salt-bridges between the di-acidic E483/484 motif in Kv1.3 and the di-basic R750/752 sequence in Sec24. These findings identify a previously unrecognized motif of Kv channels essential for their expression on the cell surface. Our results contribute to our understanding of how Kv1 channels target to the cell membrane, and provide new therapeutic strategies for the treatment of pathological conditions

A Multi-Step Process of Viral Adaptation to a Mutagenic Nucleoside Analogue by Modulation of Transition Types Leads to Extinction-Escape

Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin (1-β-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is mediated by amino acid substitutions in the viral polymerase that either increase the general template copying fidelity of the enzyme or decrease the incorporation of ribavirin into RNA. Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions (M296I, P44S and P169S) in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin —by avoiding the biased repertoire of transition mutations produced by this purine analogue—and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure

On the effect of carbonate on barite growth at elevated temperatures

The effect of carbonate on the growth of barite {001} surfaces from aqueous solutions supersaturated with respect to barite (Ω;barite ∼ 12) was studied by hydrothermal atomic force microscopy (HAFM) and Raman spectroscopy at temperatures ranging from 25 to 70 °C. The experiments showed that the effects of carbonate depend on the specific location of growth. For mono-layers growing on pristine barite, the carbonate-additive promotes growth and the spreading rate of two-dimensional islands increases with temperature. However, growth is inhibited in layers growing on surfaces, which grew in carbonate-containing solution. The threshold carbonate concentration necessary to completely inhibit growth is inversely correlated with temperature. Raman spectroscopy revealed the presence of carbonate within crystals, which grew in carbonate-containing solution. Judging by these findings, incorporation of carbonate into the structure of growing barite as a thermally activated process likely is a controlling factor, which inhibits barite growth. Thus the study shows that additives can exert opposing effects on growth not only depending on additive concentration but also depending on the specific growth location. The implication of this work, therefore, is that bimodal effects of additives on crystal growth occur more frequently than generally recognized. The insights into the mechanisms of such bimodal effects of additives can significantly contribute to the understanding and predictability of the kinetics of macro-scale processes such as barite scale formation or the behavior of barium sulfate in CO2-sequestration fluids.Peer Reviewe

Viruses and viral proteins

For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes

Snapshots of a Non-Canonical RdRP in Action

RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication and transcription. The closed “right hand” architecture of RdRPs encircles seven conserved structural motifs (A to G) that regulate the polymerization activity. The four palm motifs, arranged in the sequential order A to D, are common to all known template dependent polynucleotide polymerases, with motifs A and C containing the catalytic aspartic acid residues. Exceptions to this design have been reported in members of the Permutotetraviridae and Birnaviridae families of positive single stranded (+ss) and double-stranded (ds) RNA viruses, respectively. In these enzymes, motif C is located upstream of motif A, displaying a permuted C–A–B–D connectivity. Here we study the details of the replication elongation process in the non-canonical RdRP of the Thosea asigna virus (TaV), an insect virus from the Permutatetraviridae family. We report the X-ray structures of three replicative complexes of the TaV polymerase obtained with an RNA template-primer in the absence and in the presence of incoming rNTPs. The structures captured different replication events and allowed to define the critical interactions involved in: (i) the positioning of the acceptor base of the template strand, (ii) the positioning of the 3’-OH group of the primer nucleotide during RNA replication and (iii) the recognition and positioning of the incoming nucleotide. Structural comparisons unveiled a closure of the active site on the RNA template-primer binding, before rNTP entry. This conformational rearrangement that also includes the repositioning of the motif A aspartate for the catalytic reaction to take place is maintained on rNTP and metal ion binding and after nucleotide incorporation, before translocation

Pauson-Khand reaction of internal dissymmetric trifluoromethyl alkynes. influence of the alkene on the regioselectivity

Abstract: The scope of the Pauson-Khand reaction (PKR) of internal trifluoromethyl alkynes, previously described with norbornadiene, is expanded to norbornene and ethylene. A thorough structural analysis of the resulting PK adducts has been carried out to unveil that α-trifluoromethylcyclopentenones are preferred in all cases, independently of the electronic properties of the alkyne. The regioselectivity observed with norbornadiene and ethylene is higher than in the case of norbornene

Pauson–Khand Adducts of <i>N</i>‑Boc-propargylamine: A New Approach to 4,5-Disubstituted Cyclopentenones

A new approach to the synthesis of 4,5-disubstituted cyclopentenones is described. The strategy is based on the Pauson–Khand (PK) reaction of norbornadiene and <i>N</i>-Boc-propargylamine as an alkyne with a masked leaving group, which can be eliminated at will. This approach to the synthesis of 4,5-disubstituted cyclopentenones overcomes the problem of using the alkylation to introduce the α side chain. As an example, prostane 13-<i>epi</i>-12-oxo-phytodienoic acid (13-<i>epi</i>-12-oxo-PDA) methyl ester was synthesized

Caveolin interaction governs Kv1.3 lipid raft targeting

The spatial localization of ion channels at the cell surface is crucial for their functional role. Many channels localize in lipid raft microdomains, which are enriched in cholesterol and sphingolipids. Caveolae, specific lipid rafts which concentrate caveolins, harbor signaling molecules and their targets becoming signaling platforms crucial in cell physiology. However, the molecular mechanisms involved in such spatial localization are under debate. Kv1.3 localizes in lipid rafts and participates in the immunological response. We sought to elucidate the mechanisms of Kv1.3 surface targeting, which govern leukocyte physiology. Kv1 channels share a putative caveolin-binding domain located at the intracellular N-terminal of the channel. This motif, lying close to the S1 transmembrane segment, is situated near the T1 tetramerization domain and the determinants involved in the Kvβ subunit association. The highly hydrophobic domain (FQRQVWLLF) interacts with caveolin 1 targeting Kv1.3 to caveolar rafts. However, subtle variations of this cluster, putative ancillary associations and different structural conformations can impair the caveolin recognition, thereby altering channel's spatial localization. Our results identify a caveolin-binding domain in Kv1 channels and highlight the mechanisms that govern the regulation of channel surface localization during cellular processes