The ABC toxin complex from Yersinia entomophaga can package three different cytotoxic components expressed from distinct genetic loci in an unfolded state: the structures of both shell and cargo.

Bacterial ABC toxin complexes (Tcs) comprise three core proteins: TcA, TcB and TcC. The TcA protein forms a pentameric assembly that attaches to the surface of target cells and penetrates the cell membrane. The TcB and TcC proteins assemble as a heterodimeric TcB-TcC subcomplex that makes a hollow shell. This TcB-TcC subcomplex self-cleaves and encapsulates within the shell a cytotoxic `cargo' encoded by the C-terminal region of the TcC protein. Here, we describe the structure of a previously uncharacterized TcC protein from Yersinia entomophaga, encoded by a gene at a distant genomic location from the genes encoding the rest of the toxin complex, in complex with the TcB protein. When encapsulated within the TcB-TcC shell, the C-terminal toxin adopts an unfolded and disordered state, with limited areas of local order stabilized by the chaperone-like inner surface of the shell. We also determined the structure of the toxin cargo alone and show that when not encapsulated within the shell, it adopts an ADP-ribosyltransferase fold most similar to the catalytic domain of the SpvB toxin from Salmonella typhimurium. Our structural analysis points to a likely mechanism whereby the toxin acts directly on actin, modifying it in a way that prevents normal polymerization.

Teneurin Structures Are Composed of Ancient Bacterial Protein Domains.

Pioneering bioinformatic analysis using sequence data revealed that teneurins evolved from bacterial tyrosine-aspartate (YD)-repeat protein precursors. Here, we discuss how structures of the C-terminal domain of teneurins, determined using X-ray crystallography and electron microscopy, support the earlier findings on the proteins' ancestry. This chapter describes the structure of the teneurin scaffold with reference to a large family of teneurin-like proteins that are widespread in modern prokaryotes. The central scaffold of modern eukaryotic teneurins is decorated by additional domains typically found in bacteria, which are re-purposed in eukaryotes to generate highly multifunctional receptors. We discuss how alternative splicing contributed to further diversifying teneurin structure and thereby function. This chapter traces the evolution of teneurins from a structural point of view and presents the state-of-the-art of how teneurin function is encoded by its specific structural features.

Cryo-EM structures of the pore-forming A subunit from the Yersinia entomophaga ABC toxin.

ABC toxins are pore-forming virulence factors produced by pathogenic bacteria. YenTcA is the pore-forming and membrane binding A subunit of the ABC toxin YenTc, produced by the insect pathogen Yersinia entomophaga. Here we present cryo-EM structures of YenTcA, purified from the native source. The soluble pre-pore structure, determined at an average resolution of 4.4 Å, reveals a pentameric assembly that in contrast to other characterised ABC toxins is formed by two TcA-like proteins (YenA1 and YenA2) and decorated by two endochitinases (Chi1 and Chi2). We also identify conformational changes that accompany membrane pore formation by visualising YenTcA inserted into liposomes. A clear outward rotation of the Chi1 subunits allows for access of the protruding translocation pore to the membrane. Our results highlight structural and functional diversity within the ABC toxin subfamily, explaining how different ABC toxins are capable of recognising diverse hosts.

Crystal structure of PhnF, a GntR-family transcriptional regulator of phosphate transport in Mycobacterium smegmatis.

Bacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains.

The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device.

The ABC toxin complexes produced by certain bacteria are of interest owing to their potent insecticidal activity and potential role in human disease. These complexes comprise at least three proteins (A, B and C), which must assemble to be fully toxic. The carboxy-terminal region of the C protein is the main cytotoxic component, and is poorly conserved between different toxin complexes. A general model of action has been proposed, in which the toxin complex binds to the cell surface via the A protein, is endocytosed, and subsequently forms a pH-triggered channel, allowing the translocation of C into the cytoplasm, where it can cause cytoskeletal disruption in both insect and mammalian cells. Toxin complexes have been visualized using single-particle electron microscopy, but no high-resolution structures of the components are available, and the role of the B protein in the mechanism of toxicity remains unknown. Here we report the three-dimensional structure of the complex formed between the B and C proteins, determined to 2.5 Å by X-ray crystallography. These proteins assemble to form an unprecedented, large hollow structure that encapsulates and sequesters the cytotoxic, C-terminal region of the C protein like the shell of an egg. The shell is decorated on one end by a ß-propeller domain, which mediates attachment of the B-C heterodimer to the A protein in the native complex. The structure reveals how C auto-proteolyses when folded in complex with B. The C protein is the first example, to our knowledge, of a structure that contains rearrangement hotspot (RHS) repeats, and illustrates a marked structural architecture that is probably conserved across both this widely distributed bacterial protein family and the related eukaryotic tyrosine-aspartate (YD)-repeat-containing protein family, which includes the teneurins. The structure provides the first clues about the function of these protein repeat families, and suggests a generic mechanism for protein encapsulation and delivery.

Structural analysis of Chi1 Chitinase from Yen-Tc: the multisubunit insecticidal ABC toxin complex of Yersinia entomophaga.

Yersinia entomophaga MH96 is a native New Zealand soil bacterium that secretes a large ABC-type protein toxin complex, Yen-Tc, similar to those produced by nematode-associated bacteria such as Photorhabdus luminescens. Y. entomophaga displays an exceptionally virulent pathogenic phenotype in sensitive insect species, causing death within 72 h of infection. Because of this phenotype, there is intrinsic interest in the mechanism of action of Yen-Tc, and it also has the potential to function as a novel class of biopesticide. We have identified genes that encode chitinases as part of the toxin complex loci in Y. entomophaga MH96, P. luminescens, Photorhabdus asymbiotica and Xenorhabdus nematophila. Furthermore, we have shown that the secreted toxin complex from Y. entomophaga MH96 includes two chitinases as an integral part of the complex, a feature not described previously in other ABC toxins and possibly related to the severe disease caused by this bacterium. We present here the structure of the Y. entomophaga MH96 Chi1 chitinase, determined by X-ray crystallography to 1.74 Å resolution, and show that a ring of five symmetrically arranged lobes on the surface of the Yen-Tc toxin complex structure, as determined by single-particle electron microscopy, provides a good fit to the Chi1 monomer. We also confirm that the isolated chitinases display endochitinase activity, as does the complete toxin complex.

3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity.

Toxin complex (Tc) proteins are a class of bacterial protein toxins that form large, multisubunit complexes. Comprising TcA, B, and C components, they are of great interest because many exhibit potent insecticidal activity. Here we report the structure of a novel Tc, Yen-Tc, isolated from the bacterium Yersinia entomophaga MH96, which differs from the majority of bacterially derived Tcs in that it exhibits oral activity toward a broad range of insect pests, including the diamondback moth (Plutella xylostella). We have determined the structure of the Yen-Tc using single particle electron microscopy and studied its mechanism of toxicity by comparative analyses of two variants of the complex exhibiting different toxicity profiles. We show that the A subunits form the basis of a fivefold symmetric assembly that differs substantially in structure and subunit arrangement from its most well characterized homologue, the Xenorhabdus nematophila toxin XptA1. Histopathological and quantitative dose response analyses identify the B and C subunits, which map to a single, surface-accessible region of the structure, as the sole determinants of toxicity. Finally, we show that the assembled Yen-Tc has endochitinase activity and attribute this to putative chitinase subunits that decorate the surface of the TcA scaffold, an observation that may explain the oral toxicity associated with the complex.

A yeast model for target-primed (non-LTR) retrotransposition.

BACKGROUND: Target-primed (non-LTR) retrotransposons, such as the human L1 element, are mobile genetic elements found in many eukaryotic genomes. They are often present in large numbers and their retrotransposition can cause mutations and genomic rearrangements. Despite their importance, many aspects of their replication are not well understood. RESULTS: We have developed a yeast model system for studying target-primed retrotransposons. This system uses the Zorro3 element from Candida albicans. A cloned copy of Zorro3, tagged with a retrotransposition indicator gene, retrotransposes at a high frequency when introduced into an appropriate C. albicans host strain. Retrotransposed copies of the tagged element exhibit similar features to the native copies, indicating that the natural retrotransposition pathway is being used. Retrotransposition is dependent on the products of the tagged element's own genes and is highly temperature-regulated. The new assay permits the analysis of the effects of specific mutations introduced into the cloned element. CONCLUSION: This Zorro3 retrotransposition assay system complements previously available target-primed retrotransposition assays. Due to the relative simplicity of the growth, manipulation and analysis of yeast cells, the system should advance our understanding of target-primed retrotransposition.