Calicivirus VP2 forms a portal-like assembly following receptor engagement.

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly-which was not detected in undecorated virions-is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae.

A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants.

RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.

eIF4A1-dependent mRNAs employ purine-rich 5'UTR sequences to activate localised eIF4A1-unwinding through eIF4A1-multimerisation to facilitate translation.

Altered eIF4A1 activity promotes translation of highly structured, eIF4A1-dependent oncogene mRNAs at root of oncogenic translational programmes. It remains unclear how these mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that single-stranded RNA sequence motifs specifically activate eIF4A1 unwinding allowing local RNA structural rearrangement and translation of eIF4A1-dependent mRNAs in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5'UTR which specifically activate eIF4A1 unwinding of local RNA structure to facilitate translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression, and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex enabling efficient unwinding. Our data proposes a model in which AG-motifs in the 5'UTR of eIF4A1-dependent mRNAs specifically activate eIF4A1, enabling assembly of the helicase-competent multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.

Structure of UBE2K-Ub/E3/polyUb reveals mechanisms of K48-linked Ub chain extension.

Ubiquitin (Ub) chain types govern distinct biological processes. K48-linked polyUb chains target substrates for proteasomal degradation, but the mechanism of Ub chain synthesis remains elusive due to the transient nature of Ub handover. Here, we present the structure of a chemically trapped complex of the E2 UBE2K covalently linked to donor Ub and acceptor K48-linked di-Ub, primed for K48-linked Ub chain synthesis by a RING E3. The structure reveals the basis for acceptor Ub recognition by UBE2K active site residues and the C-terminal Ub-associated (UBA) domain, to impart K48-linked Ub specificity and catalysis. Furthermore, the structure unveils multiple Ub-binding surfaces on the UBA domain that allow distinct binding modes for K48- and K63-linked Ub chains. This multivalent Ub-binding feature serves to recruit UBE2K to ubiquitinated substrates to overcome weak acceptor Ub affinity and thereby promote chain elongation. These findings elucidate the mechanism of processive K48-linked polyUb chain formation by UBE2K.

Activation of a primed RING E3-E2-ubiquitin complex by non-covalent ubiquitin.

RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (Ub(B)) enhances both RING-independent and RING-dependent UbcH5B-catalyzed donor Ub (Ub(D)) transfer, but with a more prominent effect in RING-dependent transfer. Ub(B) enhances RING E3s' affinities for UbcH5B-Ub, and RING E3-UbcH5B-Ub complex improves Ub(B)'s affinity for UbcH5B. A comparison of the crystal structures of a RING E3, RNF38, bound to UbcH5B-Ub in the absence and presence of Ub(B), together with molecular dynamics simulation and biochemical analyses, suggests Ub(B) restricts the flexibility of UbcH5B's α1 and α1ß1 loop. Ub(B) supports E3 function by stabilizing the RING E3-UbcH5B-Ub complex, thereby improving the catalytic efficiency of Ub transfer. Thus, Ub(B) serves as an allosteric activator of RING E3-mediated Ub transfer.

Crystal structures of WrbA, a spurious target of the salicylidene acylhydrazide inhibitors of type III secretion in Gram-negative pathogens, and verification of improved specificity of next-generation compounds.

The enterohemorrhagic Escherichia coli pathotype is responsible for severe and dangerous infections in humans. Establishment of the infection requires colonization of the gastro-intestinal tract, which is dependent on the Type III Secretion System. The Type III Secretion System (T3SS) allows attachment of the pathogen to the mammalian host cell and cytoskeletal rearrangements within the host cell. Blocking the functionality of the T3SS is likely to reduce colonization and therefore limit the disease. This route offers an alternative to antibiotics, and problems with the development of antibiotics resistance. Salicylidene acylhydrazides have been shown to have an inhibitory effect on the T3SS in several pathogens. However, the main target of these compounds is still unclear. Past work has identified a number of putative protein targets of these compounds, one of which being WrbA. Whilst WrbA is considered an off-target interaction, this study presents the effect of the salicylidne acylhydrazide compounds on the activity of WrbA, along with crystal structures of WrbA from Yersinia pseudotuberculosis and Salmonella serovar Typhimurium; the latter also containing parts of the compound in the structure. We also present data showing that the original compounds were unstable in acidic conditions, and that later compounds showed improved stability.

Structure of the Macrobrachium rosenbergii nodavirus: A new genus within the Nodaviridae?

Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, and thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here, we present an atomic-resolution model of the MrNV capsid protein (CP), calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional (3D) image reconstruction at 3.3 Å resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 Å resolution structure, confirming the biological relevance of the VLP structure. Our data revealed that unlike other known nodavirus structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T = 3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae: 1) an extensive network of N-terminal arms (NTAs) lines the capsid interior, forming long-range interactions to lace together asymmetric units; 2) the capsid shell is stabilised by 3 pairs of Ca2+ ions in each asymmetric unit; 3) the protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the taxonomic classification of MrNV.

A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus.

Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein-ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male.

A substrate-interacting region of Parkin directs ubiquitination of the mitochondrial GTPase Miro1.

Mutations in the gene encoding for the E3 ubiquitin ligase Parkin have been linked to early-onset Parkinson's disease. Besides many other cellular roles, Parkin is involved in clearance of damaged mitochondria via mitophagy - a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognises its substrates for ubiquitination, and no substrate interaction site in Parkin has been reported. Here, we identify a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results explain the preferential targeting and ubiquitination of Miro1 by Parkin and provide a biochemical explanation for the presence of Parkin at the mitochondrial membrane prior to activation induced by mitochondrial damage. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinson's disease.

Analysis of the human cofilin 1 structure reveals conformational changes required for actin binding.

The actin cytoskeleton is the chassis that gives a cell its shape and structure, and supplies the power for numerous dynamic processes including motility, endocytosis, intracellular transport and division. To perform these activities, the cytoskeleton undergoes constant remodelling and reorganization. One of the major actin-remodelling families are the cofilin proteins, made up of cofilin 1, cofilin 2 and actin-depolymerizing factor (ADF), which sever aged ADP-associated actin filaments to reduce filament length and provide new potential nucleation sites. Despite the significant interest in cofilin as a central node in actin-cytoskeleton dynamics, to date the only forms of cofilin for which crystal structures have been solved are from the yeast, Chromalveolata and plant kingdoms; none have previously been reported for an animal cofilin protein. Two distinct regions in animal cofilin are significantly larger than in the forms previously crystallized, suggesting that they would be uniquely organized. Therefore, it was sought to determine the structure of human cofilin 1 by X-ray crystallography to elucidate how it could interact with and regulate dynamic actin-cytoskeletal structures. Although wild-type human cofilin 1 proved to be recalcitrant, a C147A point mutant yielded crystals that diffracted to 2.8 Šresolution. These studies revealed how the actin-binding helix undergoes a conformational change that increases the number of potential hydrogen bonds available for substrate binding.

Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target.

BACKGROUND: Cucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology. FINDINGS: Cucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation. CONCLUSIONS: The implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution.

Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence-blocking compounds.

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

The structure of an orthorhombic crystal form of a 'forced reduced' thiol peroxidase reveals lattice formation aided by the presence of the affinity tag.

Thiol peroxidase (Tpx) is an atypical 2-Cys peroxiredoxin, which has been suggested to be important for cell survival and virulence in Gram-negative pathogens. The structure of a catalytically inactive version of this protein in an orthorhombic crystal form has been determined by molecular replacement. Structural alignments revealed that Tpx is conserved. Analysis of the crystal packing shows that the linker region of the affinity tag is important for formation of the crystal lattice.

Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus.

Na-FAR-1 is an unusual α-helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = ß = γ = 90°) and diffracted to 2.5 Šresolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = ß = γ = 90°) and diffracted to 3.2 Šresolution. Crystal form 2 showed signs of significant twinning.

Useable diffraction data from a multiple microdomain-containing crystal of Ascaris suum As-p18 fatty-acid-binding protein using a microfocus beamline.

As-p18 is a fatty-acid-binding protein from the parasitic nematode Ascaris suum. Although it exhibits sequence similarity to mammalian intracellular fatty-acid-binding proteins, it contains features that are unique to nematodes. Crystals were obtained, but initial diffraction data analysis revealed that they were composed of a number of `microdomains'. Interpretable data could only be collected using a microfocus beamline with a beam size of 12 × 8 µm.

High-throughput identification of purification conditions leads to preliminary crystallization conditions for three inner membrane proteins.

An important factor in the crystallization, and subsequent structural determination, of integral membrane proteins is the ability to produce a stable and monodisperse solution of the protein. Obtaining the correct purification detergent to achieve this can be laborious and is often serendipitous. In this study, high-throughput methods are used to analyze the suitability of eight different detergents on the stability of 12 inner transmembrane proteins from Escherichia coli. The best results obtained from the small-scale experiments were scaled up, the aggregation state of the proteins assessed, and all monodisperse protein solutions entered into crystallization trials. This resulted in preliminary crystallization hits for three inner membrane proteins: XylH, PgpB and YjdL and this study reports the methods, purification procedures and crystallization conditions used to achieve this.

E3 ligase-inactivation rewires CBL interactome to elicit oncogenesis by hijacking RTK-CBL-CIN85 axis.

Casitas B-lineage lymphoma (CBL) is a ubiquitin ligase (E3) that becomes activated upon Tyr371-phosphorylation and targets receptor protein tyrosine kinases for ubiquitin-mediated degradation. Deregulation of CBL and its E3 activity is observed in myeloproliferative neoplasms and other cancers, including breast, colon, and prostate cancer. Here, we explore the oncogenic mechanism of E3-inactive CBL mutants identified in myeloproliferative neoplasms. We show that these mutants bind strongly to CIN85 under normal growth conditions and alter the CBL interactome. Lack of E3 activity deregulates CIN85 endosomal trafficking, leading to an altered transcriptome that amplifies signaling events to promote oncogenesis. Disruption of CBL mutant interactions with EGFR or CIN85 reduces oncogenic transformation. Given the importance of the CBL-CIN85 interaction in breast cancers, we examined the expression levels of CIN85, CBL, and the status of Tyr371-phosphorylated CBL (pCBL) in human breast cancer tissue microarrays. Interestingly, pCBL shows an inverse correlation with both CIN85 and CBL, suggesting that high expression of inactivated CBL could coordinate with CIN85 for breast cancer progression. Inhibition of the CBL-CIN85 interaction with a proline-rich peptide of CBL that binds CIN85 reduced the proliferation of MDA-MB-231 cells. Together, these results provide a rationale for exploring the potential of targeting the EGFR-CBL-CIN85 axis in CBL-inactivated mutant cancers.

Self-interaction chromatography as a tool for optimizing conditions for membrane protein crystallization.

The second virial coefficient, or B value, is a measurement of how well a protein interacts with itself in solution. These interactions can lead to protein crystallization or precipitation, depending on their strength, with a narrow range of B values (the 'crystallization slot') being known to promote crystallization. A convenient method of determining the B value is by self-interaction chromatography. This paper describes how the light-harvesting complex 1-reaction centre core complex from Allochromatium vinosum yielded single straight-edged crystals after iterative cycles of self-interaction chromatography and crystallization. This process allowed the rapid screening of small molecules and detergents as crystallization additives. Here, a description is given of how self-interaction chromatography has been utilized to improve the crystallization conditions of a membrane protein.

Expression, purification, crystallization and initial X-ray diffraction analysis of thiol peroxidase from Yersinia pseudotuberculosis.

Thiol peroxidase is an atypical 2-Cys peroxiredoxin that reduces alkyl hydroperoxides. Wild-type and C61S mutant protein have been recombinantly expressed in Escherichia coli and purified using nickel-affinity chromatography. Initial crystallization trials yielded three crystal forms in three different space groups (P2(1), P6(4) and P2(1)2(1)2(1)) both in the presence and the absence of DTT.