De novo phasing with X-ray laser reveals mosquito larvicide BinAB structure.

BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally to toxic oligomeric pores. The small size of the crystals-50 unit cells per edge, on average-has impeded structural characterization by conventional means. Here we report the structure of Lysinibacillus sphaericus BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser. The structure reveals tyrosine- and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears to be responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation.

Diversity-generating retroelements: natural variation, classification and evolution inferred from a large-scale genomic survey.

Diversity-generating retroelements (DGRs) are novel genetic elements that use reverse transcription to generate vast numbers of sequence variants in specific target genes. Here, we present a detailed comparative bioinformatic analysis that depicts the landscape of DGR sequences in nature as represented by data in GenBank. Over 350 unique DGRs are identified, which together form a curated reference set of putatively functional DGRs. We classify target genes, variable repeats and DGR cassette architectures, and identify two new accessory genes. The great variability of target genes implies roles of DGRs in many undiscovered biological processes. There is much evidence for horizontal transfers of DGRs, and we identify lineages of DGRs that appear to have specialized properties. Because GenBank contains data from only 10% of described species, the compilation may not be wholly representative of DGRs present in nature. Indeed, many DGR subtypes are present only once in the set and DGRs of the candidate phylum radiation bacteria, and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea archaea, are exceptionally diverse in sequence, with little information available about functions of their target genes. Nonetheless, this study provides a detailed framework for classifying and studying DGRs as they are uncovered and studied in the future.

Protein crystal structure obtained at 2.9 Å resolution from injecting bacterial cells into an X-ray free-electron laser beam.

It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼ 5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.

Surface display of a massively variable lipoprotein by a Legionella diversity-generating retroelement.

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by facilitating localized DNA sequence evolution through a specialized error-prone reverse transcription process. We characterized a DGR in Legionella pneumophila, an opportunistic human pathogen that causes Legionnaires disease. The L. pneumophila DGR is found within a horizontally acquired genomic island, and it can theoretically generate 10(26) unique nucleotide sequences in its target gene, legionella determinent target A (ldtA), creating a repertoire of 10(19) distinct proteins. Expression of the L. pneumophila DGR resulted in transfer of DNA sequence information from a template repeat to a variable repeat (VR) accompanied by adenine-specific mutagenesis of progeny VRs at the 3'end of ldtA. ldtA encodes a twin-arginine translocated lipoprotein that is anchored in the outer leaflet of the outer membrane, with its C-terminal variable region surface exposed. Related DGRs were identified in L. pneumophila clinical isolates that encode unique target proteins with homologous VRs, demonstrating the adaptability of DGR components. This work characterizes a DGR that diversifies a bacterial protein and confirms the hypothesis that DGR-mediated mutagenic homing occurs through a conserved mechanism. Comparative bioinformatics predicts that surface display of massively variable proteins is a defining feature of a subset of bacterial DGRs.

Three-dimensional structure of tropism-switching Bordetella bacteriophage.

Bacteriophage BPP-1, which infects Bordetella species, can switch its specificity by mutations to the ligand-binding surface of its major tropism-determinant protein, Mtd. This targeted mutagenesis results from the activity of a phage-encoded diversity-generating retroelement. Purified Mtd binds its receptor with low affinity, yet BPP-1 binding and infection of Bordettella cells are efficient because of high-avidity binding between phage-associated Mtd and its receptor. Here, using an integrative approach of three-dimensional (3D) structural analyses of the entire phage by cryo-electron tomography and single-prticle cryo-electron microscopy, we provide direct localization of Mtd in the phage and the structural basis of the high-avidity binding of the BPP-1 phage. Our structure shows that each BPP-1 particle has a T = 7 icosahedral head and an unusual tail apparatus consisting of a short central tail "hub," six short tail spikes, and six extended tail fibers. Subtomographic averaging of the tail fiber maps revealed a two-lobed globular structure at the distal end of each long tail fiber. Tomographic reconstructions of immuno-gold-labeled BPP-1 directly localized Mtd to these globular structures. Finally, our icosahedral reconstruction of the BPP-1 head at 7A resolution reveals an HK97-like major capsid protein stabilized by a smaller cementing protein. Our structure represents a unique bacteriophage reconstruction with its tail fibers and ligand-binding domains shown in relation to its tail apparatus. The localization of Mtd at the distal ends of the six tail fibers explains the high avidity binding of Mtd molecules to cell surfaces for initiation of infection.

Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure.

Amyloid or amyloid-like fibrils are elongated, insoluble protein aggregates, formed in vivo in association with neurodegenerative diseases or in vitro from soluble native proteins, respectively. The underlying structure of the fibrillar or 'cross-beta' state has presented long-standing, fundamental puzzles of protein structure. These include whether fibril-forming proteins have two structurally distinct stable states, native and fibrillar, and whether all or only part of the native protein refolds as it converts to the fibrillar state. Here we show that a designed amyloid-like fibril of the well-characterized enzyme RNase A contains native-like molecules capable of enzymatic activity. In addition, these functional molecular units are formed from a core RNase A domain and a swapped complementary domain. These findings are consistent with the zipper-spine model in which a cross-beta spine is decorated with three-dimensional domain-swapped functional units, retaining native-like structure.

Zinc binding drives sheet formation by the SAM domain of diacylglycerol kinase δ.

The diacylglycerol kinase (DGK) family of enzymes plays critical roles in lipid signaling pathways by converting diacylglycerol to phosphatidic acid, thereby downregulating signaling by the former and upregulating signaling by the latter second messenger. Ten DGK family isozymes have been identified to date, which possess different interaction motifs imparting distinct temporal and spatial control of DGK activity to each isozyme. Two DGK family members, δ and η, contain a sterile alpha motif (SAM) domain. The SAM domain of DGKδ1 forms helical polymers that are important for retaining the enzyme in cytoplasmic puncta, thereby inhibiting activity at the plasma membrane until pathway activation. Because zinc was found to be important for stabilizing the similar SAM polymers of the scaffolding protein Shank-3, we investigated the potential role of zinc in DGKδ SAM domain (DGKδSAM) assembly. We find that DGKδSAM binds zinc at multiple sites, driving the organization of the DGKδSAM into large sheets of polymers. Moreover, a mutant DGKδ containing a SAM domain refractory to zinc binding diminishes the formation of cytoplasmic puncta, shows partially impaired regulation of transport to the plasma membrane, and lacks the ability to inhibit the formation of CopII coated vesicles. These results suggest that zinc may play an important role in the assembly and physiology of the DGKδ isozyme.

Bordetella Bsp22 forms a filamentous type III secretion system tip complex and is immunoprotective in vitro and in vivo.

Type III secretion system (T3SS) tip complexes serve as adaptors that bridge the T3SS needle and the pore-forming translocation apparatus. In this report we demonstrate that Bsp22, the most abundantly secreted substrate of the Bordetella T3SS, self-polymerizes to form the Bordetella bronchiseptica tip complex. Bsp22 is required for both T3SS-mediated cytotoxicity against eukaryotic cells and haemoglobin release from erythrocytes. Bacterial two-hybrid analysis and protein pull-down assays demonstrated the ability of Bsp22 to associate with itself and to bind BopD, a component of the Bordetella translocation pore. Immunoblot and cross-linking analysis of secreted proteins or purified Bsp22 showed extensive multimerization which was shown by transmission electron microscopy to lead to the formation of variable length flexible filaments. Immunoelectron microscopy revealed Bsp22 filaments on the surface of bacterial cells. Given its required role in secretion and cell-surface exposure, we tested the protective effects of antibodies against Bsp22 in vitro and in vivo. Polyclonal antisera against Bsp22 fully protected epithelial cells from T3SS-dependent killing and immunization with Bsp22 protected mice against Bordetella infection. Of the approximately 30 genes which encode the Bordetella T3SS apparatus, bsp22 is the only one without characterized orthologues in other well-characterized T3SS loci. A maximum likelihood phylogenetic analysis indicated that Bsp22 defines a new subfamily of T3SS tip complex proteins. Given its immunogenic and immunoprotective properties and high degree of conservation among Bordetella species, Bsp22 and its homologues may prove useful for diagnostics and next-generation subunit vaccines.

Regulation of enzyme localization by polymerization: polymer formation by the SAM domain of diacylglycerol kinase delta1.

The diacylglycerol kinase (DGK) enzymes function as regulators of intracellular signaling by altering the levels of the second messengers, diacylglycerol and phosphatidic acid. The DGK delta and eta isozymes possess a common protein-protein interaction module known as a sterile alpha-motif (SAM) domain. In DGK delta, SAM domain self-association inhibits the translocation of DGK delta to the plasma membrane. Here we show that DGK delta SAM forms a polymer and map the polymeric interface by a genetic selection for soluble mutants. A crystal structure reveals that DGKSAM forms helical polymers through a head-to-tail interaction similar to other SAM domain polymers. Disrupting polymerization by polymer interface mutations constitutively localizes DGK delta to the plasma membrane. Thus, polymerization of DGK delta regulates the activity of the enzyme by sequestering DGK delta in an inactive cellular location. Regulation by dynamic polymerization is an emerging theme in signal transduction.

Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins.

Hfq is a nucleic acid-binding protein that functions as a global regulator of gene expression by virtue of its interactions with several small, non-coding RNA species. Originally identified as an Escherichia coli host factor required for RNA phage Qbeta replication, Hfq is now known to post-transcriptionally regulate bacterial gene expression by modulating both mRNA stability and translational activity. Recently shown to be a member of the diverse Sm protein family, Hfq adopts the OB-like fold typical of other Sm and Sm-like (Lsm) proteins, and also assembles into toroidal homo-oligomers that bind single-stranded RNA. Similarities between the structures, functions, and evolution of Sm/Lsm proteins and Hfq are continually being discovered, and we now report an additional, unexpected biophysical property that is shared by Hfq and other Sm proteins: E.coli Hfq polymerizes into well-ordered fibres whose morphologies closely resemble those found for Sm-like archaeal proteins (SmAPs). However, the hierarchical assembly of these fibres is dissimilar: whereas SmAPs polymerize into polar tubes (and striated bundles of such tubes) by head-to-tail stacking of individual homo-heptamers, helical Hfq fibres are formed by cylindrical slab-like layers that consist of 36 subunits arranged as a hexamer of Hfq homo-hexamers (i.e. protofilaments in a 6 x 6 arrangement). The different fibrillar ultrastructures formed by Hfq and SmAP are presented and examined herein, with the overall goal of elucidating another similarity amongst the diverse members of the Sm protein family.

The disassembly, reassembly and stability of CCMV protein capsids.

Efficient procedures are described for the disassembly of Cowpea Chlorotic Mottle Virus (CCMV) into its viral-RNA and capsid-protein components, the separation of the RNA and protein, and the reassembly of the purified protein into higher order nanoscale structures. These straightforward biochemical techniques result in high yield quantities of protein suitable for further biophysical studies (AFM, X-ray scattering, NMR, osmotic stress experiments, protein phase-diagram) and nanotechnology applications (protein enclosed nanoparticles, protein-lipid nanoemulsion droplets). Also discussed are solution conditions that affect the stability of the self-assembled protein structure and explicitly show that divalent cation is not required to obtain stable protein structures, while the presence of even small amounts of Ba(2+) have a significant impact on protein self-assembly. However, since high ionic strength solution conditions result in good yields of CCMV-like protein capsids, it is suggested that the highly charged cationic protein N-terminus could act as an electrostatic switch for protein self-assembly and therefore be modulated by ionic strength and salt type. It was also found that CaCl(2)/RNA precipitation methods do not yield sufficiently pure protein samples.

Crystal structures of a tetrahedral open pore ferritin from the hyperthermophilic archaeon Archaeoglobus fulgidus.

Ferritins are known as important iron storage/detoxification proteins and are widely found in living organisms. This report details the 2.1 A resolution native and 2.7 A resolution iron bound structures of the ferritin from the hyperthermophilic Archaeon Archaeoglobus fulgidus, and represents the first structure of a ferritin from an archaeon, or a hyperthermophilic organism. The A. fulgidus ferritin (AfFtn) monomer has a high degree of structural similarity with archetypal ferritins from E. coli and humans, but the AfFtn quaternary structure is novel; 24 subunits assemble into a shell having tetrahedral (2-3) rather than the canonical octahedral (4-3-2) symmetry of archetypal ferritins. The difference in assembly opens four large (approximately 45 A) pores in the AfFtn shell. Two nonconservative amino acid substitutions may be critical for stabilizing the tetrahedral form.

Isolation and characterization of mitochondrial ribosomes and ribosomal subunits from Leishmania tarentolae.

We have analyzed Leishmania tarentolae mitochondrial ribonucleoprotein (RNP) complexes using the 9S small subunit (SSU) rRNA and the 12S large subunit (LSU) rRNA as markers, and have identified a 50S RNP particle as the putative mitochondrial monosome, a 40S particle as the putative LSU and a 30S particle as the putative SSU. These assignments are supported by morphological analysis by cryo-electron microscopy and proteomics analyses by mass spectrometry. The presence of additional rRNA-containing particles complicated the analysis and most likely was the basis for previous difficulties in identification of these ribosomes; thus, in addition to the monosomes and their subunits, there are abundant stable 45S particles (SSU(*)) containing only 9S rRNA, which may represent homodimers of the SSU or SSU associated with additional proteins, and variable minor amounts of 65S and 70S particles, which represent homodimers of the LSU and SSU(*), respectively. These additional rRNA particles might be due to the lengthy mitochondrial isolation and ribosome isolation procedures or may be present in vivo and play yet undetermined roles.

The oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs).

Intron splicing is a prime example of the many types of RNA processing catalyzed by small nuclear ribonucleoprotein (snRNP) complexes. Sm proteins form the cores of most snRNPs, and thus to learn principles of snRNP assembly we characterized the oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs) from Pyrobaculum aerophilum (Pae) and Methanobacterium thermautotrophicum (Mth). Ultracentrifugation shows that Mth SmAP1 is exclusively heptameric in solution, whereas Pae SmAP1 forms either disulfide-bonded 14-mers or sub-heptameric states (depending on the redox potential). By electron microscopy, we show that Pae and Mth SmAP1 polymerize into bundles of well ordered fibers that probably form by head-to-tail stacking of heptamers. The crystallographic results reported here corroborate these findings by showing heptamers and 14-mers of both Mth and Pae SmAP1 in four new crystal forms. The 1.9 A-resolution structure of Mth SmAP1 bound to uridine-5'-monophosphate (UMP) reveals conserved ligand-binding sites. The likely RNA binding site in Mth agrees with that determined for Archaeoglobus fulgidus (Afu) SmAP1. Finally, we found that both Pae and Mth SmAP1 gel-shift negatively supercoiled DNA. These results distinguish SmAPs from eukaryotic Sm proteins and suggest that SmAPs have a generic single-stranded nucleic acid-binding activity.

Efficient purification of bromoviruses by ultrafiltration.

Ultrafiltration using polyethersulfone-membranes was evaluated as an efficient and preferred method for purifying Cowpea Chlorotic Mottle Virus (CCMV). Cesium chloride (CsCl) ultracentrifugation and ultrafiltration protocols are described, and comparative UV-spectroscopic and electron micrograph results are presented. CCMV purified by ultrafiltration are shown to be equivalent to CCMV purified by ultracentrifugation, while reducing purification time by two days and avoiding the need for expensive capital overheads such as ultracentrifuges, rotors and toxic CsCl chemical waste.

A human sterile alpha motif domain polymerizome.

The sterile alpha motif (SAM) domain is one of the most common protein modules found in eukaryotic genomes. Many SAM domains have been shown to form helical polymer structures suggesting that SAM modules can be used to create large protein complexes in the cell. Because many polymeric SAM domains form heterogenous and insoluble aggregates that are experimentally intractable when isolated, it is likely that many polymeric SAM domains have gone uncharacterized. We, therefore, developed a method to maintain polymeric SAM domains in a soluble form that allowed rapid screening for potential SAM polymers. SAM domains were expressed as fusions to a super-negatively charged green fluorescent protein (negGFP). The negGFP imparts three useful properties to the SAM domains: (1) the charge helps to maintain solubility; (2) the charge leads to reliable migration toward the cathode on native gels; and (3) the fluorescence emission allows visualization in crude extracts. Using the negGFP-SAM fusions, we screened a large library of human SAM domains for polymerization using a native gel screen. A selected set of hSAM domains were then purified and examined for true polymer formation by electron microscopy. In this manner, we identified a set of new potential SAM polymers: ANKS3, Atherin, BicaudalC1, Caskin1, Caskin2, Kazrin, L3MBTL3, L3MBTL4, LBP, LiprinB1, LiprinB2, SAMD8, SAMD9, and STIM2. While further characterization will be necessary to verify that the SAM domains identified here truly form polymers, our results provide a much stronger working hypothesis for a large number of proteins that was possible from sequence analysis alone.

An architectural framework that may lie at the core of the postsynaptic density.

The postsynaptic density (PSD) is a complex assembly of proteins associated with the postsynaptic membrane that organizes neurotransmitter receptors, signaling pathways, and regulatory elements within a cytoskeletal matrix. Here we show that the sterile alpha motif domain of rat Shank3/ProSAP2, a master scaffolding protein located deep within the PSD, can form large sheets composed of helical fibers stacked side by side. Zn2+, which is found in high concentrations in the PSD, binds tightly to Shank3 and may regulate assembly. Sheets of the Shank protein could form a platform for the construction of the PSD complex.

Role of the C-terminal 28 residues of beta2-microglobulin in amyloid fibril formation.

Beta2microglobulin (beta2m) is the major protein component of the fibrillar amyloid deposits isolated from patients diagnosed with dialysis-related amyloidosis (DRA). While investigating the molecular mechanism of amyloid fibril formation by beta2m, we found that the beta2m C-terminal peptide of 28 residues (cbeta2m) itself forms amyloid fibrils. When viewed by electron microscopy, cbeta2m aggregates appear as elongated unbranched fibers, the morphology typical for amyloids. Cbeta2m fibers stain with Congo red and show apple-green birefringence in polarized light, characteristic of amyloids. The observation that the beta2m C-terminal fragment readily forms amyloid fibrils implies that beta2m amyloid fibril formation proceeds via interactions of amyloid forming segments, which become exposed when the beta2m subunit is partially unfolded.

The SAM domain of polyhomeotic forms a helical polymer.

The polycomb group (PcG) proteins are important in the maintenance of stable repression patterns during development. Several PcG members contain a protein protein interaction module called a SAM domain (also known as SPM, PNT and HLH). Here we report the high-resolution structure of the SAM domain of polyhomeotic (Ph). Ph-SAM forms a helical polymer structure, providing a likely mechanism for the extension of PcG complexes. The structure of the polymer resembles that formed by the SAM domain of another transcriptional repressor, TEL. The formation of these polymer structures by SAM domains in two divergent repressors suggests a conserved mode of repression involving a higher order chromatin structure.

Arrangement of subunits and ordering of beta-strands in an amyloid sheet.

Amyloid fibrils are associated with several disease states, but their structures have yet to be fully defined. Here we use site-directed spin labeling to explain some of the specific interactions that are formed between subunits when the protein transthyretin (TTR) assembles into amyloid fibrils, which are associated with both spontaneous and familial amyloid diseases in humans. The results suggest that fibrils are formed when a major conformational change displaces the terminal beta-strand from the edge of a beta-sheet in the native structure, exposing the penultimate strand. The newly exposed strand then allows a novel beta-sheet interaction to form between the TTR subunits. This interaction and another previously identified subunit association lead to a plausible model for the specific sequence of beta-strands in one of the indefinitely repeating beta-sheets of TTR amyloid, which is formed by a head-to-head, tail-to-tail arrangement of subunits.