Complete Genome Sequence of Enterobacter Phage vB_EcRAM-01, a New Pseudotevenvirus against the Enterobacter cloacae Complex.

Here, we present the complete genome sequence of Enterobacter phage vB_EcRAM-01, isolated from waters of the Río Abajo river, in Panama City, Panama. This phage has deployed lytic activity against the Enterobacter cloacae complex, a pathogen of clinical importance in intensive care units. It belongs to the Myoviridae family and has a double-stranded DNA genome that is 178,477 bp long and contains 293 open reading frames (ORFs).

Symmetry disruption commits vault particles to disassembly.

Vaults are ubiquitous ribonucleoprotein particles involved in a diversity of cellular processes, with promising applications as nanodevices for delivery of multiple cargos. The vault shell is assembled by the symmetrical association of multiple copies of the major vault protein that, initially, generates half vaults. The pairwise, anti-parallel association of two half vaults produces whole vaults. Here, using a combination of vault recombinant reconstitution and structural techniques, we characterized the molecular determinants for the vault opening process. This process commences with a relaxation of the vault waist, causing the expansion of the inner cavity. Then, local disengagement of amino-terminal domains at the vault midsection seeds a conformational change that leads to the aperture, facilitating access to the inner cavity where cargo is hosted. These results inform a hitherto uncharacterized step of the vault cycle and will aid current engineering efforts leveraging vault for tailored cargo delivery.

Structural basis for biologically relevant mechanical stiffening of a virus capsid by cavity-creating or spacefilling mutations.

Recent studies reveal that the mechanical properties of virus particles may have been shaped by evolution to facilitate virus survival. Manipulation of the mechanical behavior of virus capsids is leading to a better understanding of viral infection, and to the development of virus-based nanoparticles with improved mechanical properties for nanotechnological applications. In the minute virus of mice (MVM), deleterious mutations around capsid pores involved in infection-related translocation events invariably increased local mechanical stiffness and interfered with pore-associated dynamics. To provide atomic-resolution insights into biologically relevant changes in virus capsid mechanics, we have determined by X-ray crystallography the structural effects of deleterious, mechanically stiffening mutations around the capsid pores. Data show that the cavity-creating N170A mutation at the pore wall does not induce any dramatic structural change around the pores, but instead generates subtle rearrangements that propagate throughout the capsid, resulting in a more compact, less flexible structure. Analysis of the spacefilling L172W mutation revealed the same relationship between increased stiffness and compacted capsid structure. Implications for understanding connections between virus mechanics, structure, dynamics and infectivity, and for engineering modified virus-based nanoparticles, are discussed.

Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation.

The association-dissociation of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) with eIF4G is a key control step in eukaryotic translation. The paradigm on the eIF4E-eIF4G interaction states that eIF4G binds to the dorsal surface of eIF4E through a single canonical alpha-helical motif, while metazoan eIF4E-binding proteins (m4E-BPs) advantageously compete against eIF4G via bimodal interactions involving this canonical motif and a second noncanonical motif of the eIF4E surface. Metazoan eIF4Gs share this extended binding interface with m4E-BPs, with significant implications on the understanding of translation regulation and the design of therapeutic molecules. Here we show the high-resolution structure of melon (Cucumis melo) eIF4E in complex with a melon eIF4G peptide and propose the first eIF4E-eIF4G structural model for plants. Our structural data together with functional analyses demonstrate that plant eIF4G binds to eIF4E through both the canonical and noncanonical motifs, similarly to metazoan eIF4E-eIF4G complexes. As in the case of metazoan eIF4E-eIF4G, this may have very important practical implications, as plant eIF4E-eIF4G is also involved in a significant number of plant diseases. In light of our results, a universal eukaryotic bipartite mode of binding to eIF4E is proposed.

Analysis of the interacting partners eIF4F and 3'-CITE required for Melon necrotic spot virus cap-independent translation.

We have shown previously that the translation of Melon necrotic spot virus (MNSV, family Tombusviridae, genus Carmovirus) RNAs is controlled by a 3'-cap-independent translation enhancer (CITE), which is genetically and functionally dependent on the eukaryotic translation initiation factor (eIF) 4E. Here, we describe structural and functional analyses of the MNSV-Mα5 3'-CITE and its translation initiation factor partner. We first mapped the minimal 3'-CITE (Ma5TE) to a 45-nucleotide sequence, which consists of a stem-loop structure with two internal loops, similar to other I-shaped 3'-CITEs. UV crosslinking, followed by gel retardation assays, indicated that Ma5TE interacts in vitro with the complex formed by eIF4E + eIF4G980-1159 (eIF4Fp20 ), but not with each subunit alone or with eIF4E + eIF4G1003-1092 , suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G980-1159 , or through a double interaction with eIF4E and eIF4G980-1159 . Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G1003-1092 and cap-independent translation. We also developed an in vivo system to test the effect of mutations in eIF4E in Ma5TE-driven cap-independent translation, showing that conserved amino acids in a positively charged RNA-binding motif around amino acid position 228, implicated in eIF4E-eIF4G binding or belonging to the cap-recognition pocket, are essential for cap-independent translation controlled by Ma5TE, and thus for the multiplication of MNSV.

A novel benzonitrile analogue inhibits rhinovirus replication.

OBJECTIVES: To study the characteristics and the mode of action of the anti-rhinovirus compound 4-[1-hydroxy-2-(4,5-dimethoxy-2-nitrophenyl)ethyl]benzonitrile (LPCRW_0005). METHODS: The antiviral activity of LPCRW_0005 was evaluated in a cytopathic effect reduction assay against a panel of human rhinovirus (HRV) strains. To unravel its precise molecular mechanism of action, a time-of-drug-addition study, resistance selection and thermostability assays were performed. The crystal structure of the HRV14/LPCRW_0005 complex was elucidated as well. RESULTS: LPCRW_0005 proved to be a selective inhibitor of the replication of HRV14 (EC(50) of 2 ±â€Š1 µM). Time-of-drug-addition studies revealed that LPCRW_0005 interferes with the earliest stages of virus replication. Phenotypic drug-resistant virus variants were obtained (≥30-fold decrease in susceptibility to the inhibitory effect of LPCRW_0005), which carried either an A150T or A150V amino acid substitution in the VP1 capsid protein. The link between the mutant genotype and drug-resistant phenotype was confirmed by reverse genetics. Cross-resistance studies and thermostability assays revealed that LPCRW_0005 has a similar mechanism of action to the capsid binder pleconaril. Elucidation of the crystal structure of the HRV14/LPCRW_0005 complex revealed the existence of multiple hydrophobic and polar interactions between the VP1 pocket and LPCRW_0005. CONCLUSIONS: LPCRW_0005 is a novel inhibitor of HRV14 replication that acts as a capsid binder. The compound has a chemical structure that is markedly smaller than that of other capsid binders. Structural studies show that LPCRW_0005, in contrast to pleconaril, leaves the toe end of the pocket in VP1 empty. This suggests that extended analogues of LPCRW_0005 that fill the full cavity could be more potent inhibitors of rhinovirus replication.

Uncoating of common cold virus is preceded by RNA switching as determined by X-ray and cryo-EM analyses of the subviral A-particle.

During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNA-protein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp(38) of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress.

Human-like eukaryotic translation initiation factor 3 from Neurospora crassa.

Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13(th) subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells.

Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation.

The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome.

New features of vault architecture and dynamics revealed by novel refinement using the deformable elastic network approach.

The vault particle, with a molecular weight of about 10 MDa, is the largest ribonucleoprotein that has been described. The X-ray structure of intact rat vault has been solved at a resolution of 3.5 Å [Tanaka et al. (2009), Science, 323, 384-388], showing an overall barrel-shaped architecture organized into two identical moieties, each consisting of 39 copies of the major vault protein (MVP). The model deposited in the PDB includes 39 MVP copies (half a vault) in the crystal asymmetric unit. A 2.1 Å resolution structure of the seven N-terminal repeats (R1-7) of MVP has also been determined [Querol-Audí et al. (2009), EMBO J. 28, 3450-3457], revealing important discrepancies with respect to the MVP models for repeats R1 and R2. Here, the re-refinement of the vault structure by incorporating the high-resolution information available for the R1-7 domains, using the deformable elastic network (DEN) approach and maintaining strict 39-fold noncrystallographic symmetry is reported. The new refinement indicates that at the resolution presently available the MVP shell can be described well as only one independent subunit organized with perfect D39 molecular symmetry. This refinement reveals that significant rearrangements occur in the N-terminus of MVP during the closing of the two vault halves and that the 39-fold symmetry breaks in the cap region. These results reflect the highly dynamic nature of the vault structure and represent a necessary step towards a better understanding of the biology and regulation of this particle.

Architecture of human translation initiation factor 3.

Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans.

Role of motif B loop in allosteric regulation of RNA-dependent RNA polymerization activity.

Increasing amounts of data show that conformational dynamics are essential for protein function. Unveiling the mechanisms by which this flexibility affects the activity of a given enzyme and how it is controlled by other effectors opens the door to the design of a new generation of highly specific drugs. Viral RNA-dependent RNA polymerases (RdRPs) are not an exception. These enzymes, essential for the multiplication of all RNA viruses, catalyze the formation of phosphodiester bonds between ribonucleotides in an RNA-template-dependent fashion. Inhibition of RdRP activity will prevent genome replication and virus multiplication. Thus, RdRPs, like the reverse transcriptase of retroviruses, are validated targets for the development of antiviral therapeutics. X-ray crystallography of RdRPs trapped in multiple steps throughout the catalytic process, together with NMR data and molecular dynamics simulations, have shown that all polymerase regions contributing to conserved motifs required for substrate binding, catalysis and product release are highly flexible and some of them are predicted to display correlated motions. All these dynamic elements can be modulated by external effectors, which appear as useful tools for the development of effective allosteric inhibitors that block or disturb the flexibility of these enzymes, ultimately impeding their function. Among all movements observed, motif B, and the B-loop at its N-terminus in particular, appears as a new potential druggable site.

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability.

Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with sliding clamps are key processes in biology, which are incompletely understood from a mechanistic point of view. We have used an integrative computational and experimental approach to define the assemblies of FEN1 with double-flap DNA substrates and either proliferating cell nuclear antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models of these two ternary complexes were developed and refined through extensive molecular dynamics simulations to expose their conformational dynamics. Clustering analysis revealed the most dominant conformations accessible to the complexes. The cluster centroids were subsequently used in conjunction with single-particle electron microscopy data to obtain a 3D EM reconstruction of the human 9-1-1/FEN1/DNA assembly at 18-Å resolution. Comparing the structures of the complexes revealed key differences in the orientation and interactions of FEN1 and double-flap DNA with the two clamps that are consistent with their respective functions in providing inherent flexibility for lagging strand DNA replication or inherent stability for DNA repair.

Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3).

Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.

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 A resolution, together with the 2.1-A 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 A model shows major discrepancies, both in the main chain tracing and in the side chain assignment of the two terminal domains R1 and R2.

Structural and mechanistic insights into the association of PKCalpha-C2 domain to PtdIns(4,5)P2.

C2 domains are widely-spread protein signaling motifs that in classical PKCs act as Ca(2+)-binding modules. However, the molecular mechanisms of their targeting process at the plasma membrane remain poorly understood. Here, the crystal structure of PKCalpha-C2 domain in complex with Ca(2+), 1,2-dihexanoyl-sn-glycero-3-[phospho-L-serine] (PtdSer), and 1,2-diayl-sn-glycero-3-[phosphoinositol-4,5-bisphosphate] [PtdIns(4,5)P(2)] shows that PtdSer binds specifically to the calcium-binding region, whereas PtdIns(4,5)P(2) occupies the concave surface of strands beta3 and beta4. Strikingly, the structure reveals a PtdIns(4,5)P(2)-C2 domain-binding mode in which the aromatic residues Tyr-195 and Trp-245 establish direct interactions with the phosphate moieties of the inositol ring. Mutations that abrogate Tyr-195 and Trp-245 recognition of PtdIns(4,5)P(2) severely impaired the ability of PKCalpha to localize to the plasma membrane. Notably, these residues are highly conserved among C2 domains of topology I, and a general mechanism of C2 domain-membrane docking mediated by PtdIns(4,5)P(2) is presented.

Minor group human rhinovirus-receptor interactions: geometry of multimodular attachment and basis of recognition.

X-ray structures of human rhinovirus 2 (HRV2) in complex with soluble very-low-density lipoprotein receptors encompassing modules 1, 2, and 3 (V123) and five V3 modules arranged in tandem (V33333) demonstrates multi-modular binding around the virion's five-fold axes. Occupancy was 60% for V123 and 100% for V33333 explaining the high-avidity of the interaction. Surface potentials of 3D-models of all minor group HRVs and K-type major group HRVs were compared; hydrophobic interactions between a conserved lysine in the viruses and a tryptophan in the receptor modules together with coulombic attraction via diffuse opposite surface potentials determine minor group HRV receptor specificity.

Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases.

TREX1 is the major exonuclease in mammalian cells, exhibiting the highest level of activity with a 3'-->5' activity. This exonuclease is responsible in humans for Aicardi-Goutières syndrome and for an autosomal dominant retinal vasculopathy with cerebral leukodystrophy. In addition, this enzyme is associated with systemic lupus erythematosus. TREX1 belongs to the exonuclease DEDDh family, whose members display low levels of sequence identity, while possessing a common fold and active site organization. For these exonucleases, a catalytic mechanism has been proposed that involves two divalent metal ions bound to the DEDD motif. Here we studied the interaction of TREX1 with the monovalent cations lithium and sodium. We demonstrate that these metals inhibit the exonucleolytic activity of TREX1, as measured by the classical gel method, as well as by a new technique developed for monitoring the real-time exonuclease reaction. The X-ray structures of the enzyme in complex with these two cations and with a nucleotide, a product of the exonuclease reaction, were determined at 2.1 A and 2.3 A, respectively. A comparison with the structures of the active complexes (in the presence of magnesium or manganese) explains that the inhibition mechanism is caused by the noncatalytic metals competing with distinct affinities for the two metal-binding sites and inducing subtle rearrangements in active centers. Our analysis also reveals that a histidine residue (His124), highly conserved in the DEDDh family, is involved in the activity of TREX1, as confirmed by mutational studies. Our results shed further light on the mechanism of activity of the DEDEh family of exonucleases.

Activation mechanism of a noncanonical RNA-dependent RNA polymerase.

Two lineages of viral RNA-dependent RNA polymerases (RDRPs) differing in the organization (canonical vs. noncanonical) of the palm subdomain have been identified. Phylogenetic analyses indicate that both lineages diverged at a very early stage of the evolution of the enzyme [Gorbalenya AE, Pringle FM, Zeddam JL, Luke BT, Cameron CE, Kalmakoff J, Hanzlik TN, Gordon KH, Ward VK (2002) J Mol Biol 324:47-62]. Here, we report the x-ray structure of a noncanonical birnaviral RDRP, named VP1, in its free form, bound to Mg(2+) ions, and bound to a peptide representing the polymerase-binding motif of the regulatory viral protein VP3. The structure of VP1 reveals that the noncanonical connectivity of the palm subdomain maintains the geometry of the catalytic residues found in canonical polymerases but results in a partial blocking of the active site cavity. The VP1-VP3 peptide complex shows a mode of polymerase activation in which VP3 binding promotes a conformational change that removes the steric blockade of the VP1 active site, facilitating the accommodation of the template and incoming nucleotides for catalysis. The striking structural similarities between birnavirus (dsRNA) and the positive-stranded RNA picornavirus and calicivirus RDRPs provide evidence supporting the existence of functional and evolutionary relationships between these two virus groups.

Biosynthesis of isoprenoids in plants: structure of the 2C-methyl-D-erithrytol 2,4-cyclodiphosphate synthase from Arabidopsis thaliana. Comparison with the bacterial enzymes.

The X-ray crystal structure of the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) from Arabidopsis thaliana has been solved at 2.3 A resolution in complex with a cytidine-5-monophosphate (CMP) molecule. This is the first structure determined of an MCS enzyme from a plant. Major differences between the A. thaliana and bacterial MCS structures are found in the large molecular cavity that forms between subunits and involve residues that are highly conserved among plants. In some bacterial enzymes, the corresponding cavity has been shown to be an isoprenoid diphosphate-like binding pocket, with a proposed feedback-regulatory role. Instead, in the structure from A. thaliana the cavity is unsuited for binding a diphosphate moiety, which suggests a different regulatory mechanism of MCS enzymes between bacteria and plants.