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Clostridioides difficile is one of the most common causes of antibiotic-associated diarrhea in developed countries. As key virulence factors of C. difficile , toxin A (TcdA) and toxin B (TcdB) act by glucosylating and inactivating Rho and Ras family small GTPases in host cells, which leads to actin cytoskeleton disruption, cell rounding, and ultimately cell death. Here we present the co-crystal structure of the glucosyltransferase domain (GTD) of TcdA in complex with its substrate human RhoA at 2.60-angstrom resolution. This structure reveals that TcdA GTD grips RhoA mainly through its switch I and switch II regions, which is complemented by interactions involving RhoA’s pre-switch I region. Comprehensive structural comparisons between the TcdA GTD–RhoA complex and the structures of TcdB GTD in complex with Cdc42 and R-Ras reveal both the conserved and divergent features of these two toxins in terms of substrate recognition. Taken together, these findings establish the structural basis for TcdA recognition of small GTPases and advance our understanding of the substrates selectivity of large clostridial toxins.

Mitochondrial calcium uptake is critical for regulating ATP production, intracellular calcium signalling, and cell death. This uptake is mediated by a highly selective calcium channel called the mitochondrial calcium uniporter (MCU). Here, we determined the structures of the pore-forming MCU proteins from two fungi by X-ray crystallography and single-particle cryo-electron microscopy. The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent nuclear magnetic resonance structure of Caenorhabditis elegans MCU. We observed a dimer-of-dimer architecture across species and chemical environments, which was corroborated by biochemical experiments. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results reveal a new ion channel architecture, provide insights into calcium coordination, selectivity and conduction, and establish a structural framework for understanding the mechanism of mitochondrial calcium uniporter function. X-ray and cryo-electron microscopy structures of fungal mitochondrial calcium uniporter proteins reveal a tetrameric architecture and shed light on the function of the channel.

The X-ray crystal structure is presented of a seven-transmembrane eukaryotic SWEET glucose transporter, revealing the link between seven-transmembrane eukaryotic SWEETs and their three-transmembrane bacterial homologues and providing insight into eukaryotic sugar transport mechanisms. Structure of a SWEET sugar transporter SWEET sugar transporters are involved in various processes in plants and in glucose transport in animals. The authors report the first X-ray crystal structure of a eukaryotic SWEET glucose transporter, a vacuolar glucose transporter from rice. The structure (of the inward-open state) shows that this transporter forms homomeric trimers. It contains seven transmembrane helices — in contrast to the three helices reported for bacterial homologues — suggesting a molecular basis for understanding functional cross-talk and coupling of SWEET transporters. Eukaryotes rely on efficient distribution of energy and carbon skeletons between organs in the form of sugars. Glucose in animals and sucrose in plants serve as the dominant distribution forms. Cellular sugar uptake and release require vesicular and/or plasma membrane transport proteins. Humans and plants use proteins from three superfamilies for sugar translocation: the major facilitator superfamily (MFS), the sodium solute symporter family (SSF; only in the animal kingdom), and SWEETs 1 , 2 , 3 , 4 , 5 . SWEETs carry mono- and disaccharides 6 across vacuolar or plasma membranes. Plant SWEETs play key roles in sugar translocation between compartments, cells, and organs, notably in nectar secretion 7 , phloem loading for long distance translocation 8 , pollen nutrition 9 , and seed filling 10 . Plant SWEETs cause pathogen susceptibility possibly by sugar leakage from infected cells 3 , 11 , 12 . The vacuolar Arabidopsis thaliana AtSWEET2 sequesters sugars in root vacuoles; loss-of-function mutants show increased susceptibility to Pythium infection 13 . Here we show that its orthologue, the vacuolar glucose transporter OsSWEET2b from rice ( Oryza sativa ), consists of an asymmetrical pair of triple-helix bundles, connected by an inversion linker transmembrane helix (TM4) to create the translocation pathway. Structural and biochemical analyses show OsSWEET2b in an apparent inward (cytosolic) open state forming homomeric trimers. TM4 tightly interacts with the first triple-helix bundle within a protomer and mediates key contacts among protomers. Structure-guided mutagenesis of the close paralogue SWEET1 from Arabidopsis identified key residues in substrate translocation and protomer crosstalk. Insights into the structure–function relationship of SWEETs are valuable for understanding the transport mechanism of eukaryotic SWEETs and may be useful for engineering sugar flux.

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB–antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.

Botulinum neurotoxins (BoNTs) are highly poisonous substances that are also effective medicines. Accidental BoNT poisoning often occurs through ingestion of Clostrídium botulinum-contaminated food. Here, we present the crystal structure of a BoNT in complex with a clostridial nontoxic nonhemagglutinin (NTNHA) protein at 2.7 angstroms. Biochemical and functional studies show that NTNHA provides large and multivalent binding interfaces to protect BoNT from gastrointestinal degradation. Moreover, the structure highlights key residues in BoNT that regulate complex assembly in a pH-dependent manner. Collectively, our findings define the molecular mechanisms by which NTNHA shields BoNT in the hostile gastrointestinal environment and releases it upon entry into the circulation. These results will assist in the design of small molecules for inhibiting oral BoNT intoxication and of delivery vehicles for oral administration of biologies.

The X-ray crystal structures of two bacterial homologues of the SWEET sugar transporters are solved in two conformational states, and comparison of these states suggests that transport occurs via a ‘rocker-switch’ mechanism. SemiSWEET transporters in open and occluded states There are many similarities between the sugar transporter proteins used by prokaryotes, plants and animals. This paper presents the X-ray crystal structures of two bacterial homologues of the SWEET sugar transporters that are involved in various sugar efflux processes (for example, phloem loading, pollen nutrition, and nectar secretion) in plants in and glucose transport in animals. The overall three-dimensional structures of both proteins — SemiSWEET from Vibrio sp. in an outward-facing, open state and Leptospira biflexa SemiSWEET in the occluded state — differ markedly from those of the MFS and SGLT sugar transporter superfamilies but all three transporter types feature a triple-helix bundle . Comparisons of the conformations of the open and occluded states of SemiSWEET suggest that transport occurs via a 'rocker-switch' mechanism. SWEETs and their prokaryotic homologues are monosaccharide and disaccharide transporters that are present from Archaea to plants and humans 1 , 2 , 3 . SWEETs play crucial roles in cellular sugar efflux processes: that is, in phloem loading 4 , pollen nutrition 5 and nectar secretion 6 . Their bacterial homologues, which are called SemiSWEETs, are among the smallest known transporters 1 , 3 . Here we show that SemiSWEET molecules, which consist of a triple-helix bundle, form symmetrical, parallel dimers, thereby generating the translocation pathway. Two SemiSWEET isoforms were crystallized, one in an apparently open state and one in an occluded state, indicating that SemiSWEETs and SWEETs are transporters that undergo rocking-type movements during the transport cycle. The topology of the triple-helix bundle is similar yet distinct to that of the basic building block of animal and plant major facilitator superfamily (MFS) transporters (for example, GLUTs and SUTs). This finding indicates two possibilities: that SWEETs and MFS transporters evolved from an ancestral triple-helix bundle or that the triple-helix bundle represents convergent evolution. In SemiSWEETs and SWEETs, two triple-helix bundles are arranged in a parallel configuration to produce the 6- and 6 + 1-transmembrane-helix pores, respectively. In the 12-transmembrane-helix MFS transporters, four triple-helix bundles are arranged into an alternating antiparallel configuration, resulting in a much larger 2 × 2 triple-helix bundle forming the pore. Given the similarity of SemiSWEETs and SWEETs to PQ-loop amino acid transporters and to mitochondrial pyruvate carriers (MPCs), the structures characterized here may also be relevant to other transporters in the MtN3 clan 7 , 8 , 9 . The insight gained from the structures of these transporters and from the analysis of mutations of conserved residues will improve the understanding of the transport mechanism, as well as allow comparative studies of the different superfamilies involved in sugar transport and the evolution of transporters in general.

BoNT/A1 invades motoneurons by binding to the neuronal receptor SV2. A combination of structural, biophysical and cellular analyses reveal that BoNT/A1 binding and uptake require glycosylation of SV2. Botulinum neurotoxin serotype A1 (BoNT/A1), a licensed drug widely used for medical and cosmetic applications, exerts its action by invading motoneurons. Here we report a 2.0-Å-resolution crystal structure of the BoNT/A1 receptor-binding domain in complex with its neuronal receptor, glycosylated human SV2C. We found that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan—which is conserved in all SV2 isoforms across vertebrates—is essential for BoNT/A1 binding to neurons and for its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an antibotulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications, thereby achieving highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors.

Toxin A (TcdA) and toxin B (TcdB) are two key virulence factors secreted by Clostridioides difficile , which is listed as an urgent threat by the CDC. These two large homologous exotoxins are mainly responsible for diseases associated with C. difficile infection (CDI) with symptoms ranging from diarrhea to life threatening pseudomembranous colitis. Single-domain camelid antibodies (VHHs) AH3 and AA6 are two potent antitoxins against TcdA, which when combined with two TcdB-targeting VHHs showed effective protection against both primary and recurrent CDI in animal models. Here, we report the co-crystal structures of AH3 and AA6 when they form complexes with the glucosyltransferase domain (GTD) and a fragment of the delivery and receptor-binding domain (DRBD) of TcdA, respectively. Based on these structures, we find that AH3 binding enhances the overall stability of the GTD and interferes with its unfolding at acidic pH, and AA6 may inhibit the pH-dependent conformational changes in the DRBD that is necessary for pore formation of TcdA. These studies reveal two functionally critical epitopes on TcdA and shed new insights into neutralizing mechanisms and potential development of epitope-focused vaccines against TcdA.