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Tc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigate whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, Ras-binding domain of CRAF kinase, and TEV protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to apply Tc toxins as a universal protein translocation system. Tc toxins are a major class of bacterial toxin translocation systems that inject toxic enzymes into target cells. Here the authors present functional and structural data showing that the toxic enzyme can be replaced by other small proteins and identify prerequisites required for successful translocation, which could facilitate the development of functional Tc-based protein injection devices.

A cryo-EM structure of toxin component TcdA1 embedded in lipid nanodiscs reveals details of the mechanism used by this bacterial toxin to insert into the host cell membrane. Tc toxins from pathogenic bacteria use a special syringe-like mechanism to perforate the host cell membrane and inject a deadly enzyme into the host cytosol. The molecular mechanism of this unusual injection system is poorly understood. Using electron cryomicroscopy, we determined the structure of TcdA1 from Photorhabdus luminescens embedded in lipid nanodiscs. In our structure, compared with the previous structure of TcdA1 in the prepore state, the transmembrane helices rearrange in the membrane and open the initially closed pore. However, the helices do not span the complete membrane; instead, the loops connecting the helices form the rim of the funnel. Lipid head groups reach into the space between the loops and consequently stabilize the pore conformation. The linker domain is folded and packed into a pocket formed by the other domains of the toxin, thereby considerably contributing to stabilization of the pore state.

Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets—typically comprising thousands of particles—is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200–2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE. Thorsten Wagner et al. present SPHIRE-crYOLO, a particle picking software for selecting particles from digital micrographs in cryoEM data. After training, the method automatically recognizes particles with high recall and precision, simplifying data pre-processing.

Fusobacterium nucleatum is overrepresented in the colon microbiome of colorectal cancer patients and has been associated with tumor growth enhancement and metastasis. A pivotal pathogenic factor, the autotransporter adhesin Fap2, facilitates association to cancer and immune cells via the receptors Gal-GalNAc and TIGIT, respectively, leading to deactivation of immune cells. Mechanistic details of the Fap2/TIGIT interaction remain elusive as no structural data are available. Here, we report a system to recombinantly express functional Fap2 on the Escherichia coli surface, which interacts with Gal-GalNAc on cancer cells and with purified TIGIT with submicromolar affinity. Cryo-EM structures of Fap2, alone and in complex with TIGIT, show that the elongated ~50 nm long Fap2 extracellular region binds to TIGIT on its membrane-distal tip via an extension of a β-helix domain. Moreover, by combining structure predictions, cryo-EM, docking and molecular dynamics simulations, we identified a binding pit for Gal-GalNAc on the tip of Fap2. Fusobacterium nucleatum promotes colorectal cancer via its adhesin Fap2. Here, Schöpf et al. show the molecular basis for Fap2 interaction with its receptors TIGIT on immune cells and Gal-GalNAc on tumor cells using cryo-EM and integrative modeling.

Pore-forming toxins are protein assemblies used by many organisms to disrupt the membranes of target cells. They are expressed as soluble monomers that assemble spontaneously into multimeric pores. However, owing to their complexity, the assembly processes have not been resolved in detail for any pore-forming toxin. To determine the assembly mechanism for the ring-shaped, homododecameric pore of the bacterial cytolytic toxin ClyA, we collected a diverse set of kinetic data using single-molecule spectroscopy and complementary techniques on timescales from milliseconds to hours, and from picomolar to micromolar ClyA concentrations. The entire range of experimental results can be explained quantitatively by a surprisingly simple mechanism. First, addition of the detergent n -dodecyl-β- D -maltopyranoside to the soluble monomers triggers the formation of assembly-competent toxin subunits, accompanied by the transient formation of a molten-globule-like intermediate. Then, all sterically compatible oligomers contribute to assembly, which greatly enhances the efficiency of pore formation compared with simple monomer addition. Pore-forming toxins are expressed as monomers and assemble into multimeric pores. Here, Benke et al . follow the kinetics of pore formation for the bacterial toxin ClyA with single-molecule methods and show that pore formation progresses through the assembly of oligomeric intermediates, rather than by the addition of monomers to a nascent pore.

Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is the actin ADP-ribosyltransferase TccC3, whose activity leads to the clustering of the cellular cytoskeleton and ultimately cell death. Here, we show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD + to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a mechanism of action of a bacterial toxin through specific targeting and modification of F-actin. Entomopathogenic bacteria used for pest control secrete potent Tc toxins. Here, the authors combine biochemistry, solution and solid-state NMR spectroscopy and cryo-EM to show in atomic detail how the toxin disrupts the host cell cytoskeleton and kills the target cell.

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor–toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface. Although Tc toxins are a major class of bacterial toxin translocation systems, little is known about their receptor binding. Here, the authors identify heparins/heparan sulfates and Lewis antigens as receptors for different Tc toxins, determine cryo-EM structures of three toxin-glycan complexes and propose a two-step cell adhesion mechanism for Tc toxins.

Cis prolyl peptide bonds are conserved structural elements in numerous protein families, although their formation is energetically unfavorable, intrinsically slow and often rate-limiting for folding. Here we investigate the reasons underlying the conservation of the cis proline that is diagnostic for the fold of thioredoxin-like thiol-disulfide oxidoreductases. We show that replacement of the conserved cis proline in thioredoxin by alanine can accelerate spontaneous folding to the native, thermodynamically most stable state by more than four orders of magnitude. However, the resulting trans alanine bond leads to small structural rearrangements around the active site that impair the function of thioredoxin as catalyst of electron transfer reactions by more than 100-fold. Our data provide evidence for the absence of a strong evolutionary pressure to achieve intrinsically fast folding rates, which is most likely a consequence of proline isomerases and molecular chaperones that guarantee high in vivo folding rates and yields.

Membrane glycoproteins frequently adopt different conformations when altering between active and inactive states. Here, we discover a molecular switch that exploits dynamic spatial rearrangements of N-glycans during such conformational transitions to control protein function. For the conformationally switchable cell adhesion glycoprotein α 5 β 1 integrin, we find that only the bent-closed state arranges N-glycans to nucleate the formation of up to tetrameric oligomers of the glycan-binding protein galectin-3. We propose a structural model of how these galectin-3 oligomers are built and how they clamp the bent-closed state to select it for endocytic uptake and subsequent retrograde trafficking to the Golgi for polarized distribution in cells. Our findings reveal the dynamic regulation of the glycan landscape at the cell surface to achieve oligomerization of galectin-3. Galectin-3 oligomers are thereby identified as functional decoders of defined spatial patterns of N-glycans on specifically the bent-closed conformational state of α 5 β 1 integrin and possibly other integrin family members. Specificity in interactions with otherwise common glycan structures is likely achieved through glycan context. Here, the authors show that such glycan context is generated through tunable glycan arrangements that are translated into function by galectins.

Nature Communications 6: Article number: 6198 (2015); Published: 5 February 2015; Updated: 1 February 2016 In Figure 2b of this article, the sets of arrows connecting 'I' and 'M' in the kinetic scheme are inverted. The correct version of Fig. 2 appears below. Figure 2 |