Primary cilium: an elaborate structure that blocks cell division?

A primary cilium is a microtubule-based membranous protrusion found in almost all cell types. A primary cilium has a “9 + 0” axoneme that distinguishes this ancient organelle from the canonical motile “9 + 2” cilium. A primary cilium is the sensory center of the cell that regulates cell proliferation and embryonic development. The primary ciliary pocket is a specialized endocytic membrane domain in the basal region. The basal body of a primary cilium exists as a form of the centriole during interphase of the cell cycle. Although conventional thinking suggests that the cell cycle regulates centrosomal changes, recent studies suggest the opposite, that is, centrosomal changes regulate the cell cycle. In this regard, centrosomal kinase Aurora kinase A (AurA), Polo-like kinase 1 (Plk1), and NIMA related Kinase (Nek or Nrk) propel cell cycle progression by promoting primary cilia disassembly which indicates a non-mitotic function. However, the persistence of primary cilia during spermatocyte division challenges the dominate idea of the incompatibility of primary cilia and cell division. In this review, we demonstrate the detailed structure of primary cilia and discuss the relationship between primary cilia disassembly and cell cycle progression on the background of various mitotic kinases.     

Burrow ventilation and associated porewater irrigation by the polychaete Marenzelleria viridis

Burrow ventilation of benthic infauna generates water currents that irrigate the interstices of the sediments surrounding the burrow walls. Such activities have associated effects on biogeochemical processes affecting ultimately important ecosystem processes. In this study, the ventilation and irrigation behavior of Marenzelleria viridis, an invasive polychaete species in Europe, was analyzed using different approaches. M. viridis showed to perform two types of ventilation: (1) muscular pumping of water out of the burrow and (2) cilia pumping of water into the burrow. Flowmeter measurements presented muscular pumping in time averaged rates of 0.15 ml min⁻¹. Oxygen needle electrodes positioned above the burrow openings revealed that muscular undulation of the worm body pumps anoxic water out of the burrow. On the other hand, microscope observations of the animal showed that ventilation of oxygen-rich water in the burrow occurs by ciliary action. The volume of water irrigated by M. viridis appears to vary linearly within the first 24 h incubation, with rates ranging from 0.003 to 0.01 ml min⁻¹. From those rates we could estimate that the time averaged rate of cilia ventilation should be about 0.16 ml min⁻¹. Since the cilia pumping into the burrow occurs in periods of 24 ± 12 min and at 50-70% of the measured time, considerable amounts of water from deeper sediments may percolate upwards to the sediment surface. This water is rich in reduced compounds and nutrients and may have important associated ecological implications in the ecosystem (e.g. affecting redox conditions, organic matter degradation, benthic recruitment and primary production).     

Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica

EhCP-B9, a cysteine protease (CP) involved in Entamoeba histolytica virulence, is a potential target for disease diagnosis and drug design. After purification from inclusion bodies produced in Escherichia coli, the recombinant EhCP-B9 precursor (ppEhCP-B9) can be refolded using detergents as artificial chaperones. However, the conformational changes that occur during ppEhCP-B9 refolding remain unknown. Here, we comprehensively describe conformational changes of ppEhCP-B9 that are induced by various chemical detergents acting as chaperones, including non-ionic, zwitterionic, cationic and anionic surfactants. We monitored the effect of detergent concentration and incubation time on the secondary and tertiary structures of ppEhCP-B9 using fluorescence and circular dichroism (CD) spectroscopy. In the presence of non-ionic and zwitterionic detergents, ppEhCP-B9 adopted a β-enriched structure (ppEhCP-B9_β1) without proteolytic activity at all detergent concentrations and incubation times evaluated. ppEhCP-B9 also exhibits a β-rich structure in low concentrations of ionic detergents, but at concentrations above the critical micelle concentration (CMC), the protein acquires an α + β structure, similar to that of papain but without proteolytic activity (ppEhCP-B9_α + β1). Interestingly, only within a narrow range of experimental conditions in which SDS concentrations were below the CMC, ppEhCP-B9 refolded into a β-sheet rich structure (ppEhCP-B9_β2) that slowly transforms into a different type of α + β conformation that exhibited proteolytic activity (ppEhCP-B9_α + β2) suggesting that enzymatic activity is gained as slow transformation occurs.     

Putative roles of cilia in polycystic kidney disease

The last 10 years has witnessed an explosion in research into roles of cilia in cystic renal disease. Cilia are membrane-enclosed finger-like projections from the cell, usually on the apical surface or facing into a lumen, duct or airway. Ten years ago, the major recognised functions related to classical “9 + 2” cilia in the respiratory and reproductive tracts, where co-ordinated beating clears secretions and assists fertilisation respectively. Primary cilia, which have a “9 + 0” arrangement lacking the central microtubules, were anatomical curiosities but several lines of evidence have implicated them in both true polycystic kidney disease and other cystic renal conditions: ranging from the homology between Caenorhabditis elegans proteins expressed on sensory cilia to mammalian polycystic kidney disease (PKD) 1 and 2 proteins, through the discovery that orpk cystic mice have structurally abnormal cilia to numerous recent studies wherein expression of nearly all cyst-associated proteins has been reported in the cilia or its basal body. Functional studies implicate primary cilia in mechanosensation, photoreception and chemosensation but it is the first of these which appears most important in polycystic kidney disease: in the simplest model, fluid flow across the apical surface of renal cells bends the cilia and induces calcium influx, and this is perturbed in polycystic kidney disease. Downstream effects include changes in cell differentiation and polarity. Pathways such as hedgehog and Wnt signalling may also be regulated by cilia. These data support important roles for cilia in the pathogenesis of cystic kidney diseases but one must not forget that the classic polycystic kidney disease proteins are expressed in several other locations where they may have equally important roles, such as in cell-cell and cell-matrix interactions, whilst it is not just aberrant cilia signalling that can lead to de-differentiation, loss of polarity and other characteristic features of polycystic kidney disease. Understanding how cilia fit into the other aspects of polycystic kidney disease biology is the challenge for the next decade. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

A structural investigation of complex I and I + III2 supercomplex from Zea mays at 11-13 Å resolution: Assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I

The projection structures of complex I and the I + III₂ supercomplex from the C₄ plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 Å. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric γ-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the γ-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant “type II” particle has a 15 Å shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant “type I” particle. The I + III₂ supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of “type I”. This would mean that the “type II” particles are not involved in the supercomplex formation and, hence, could have a different physiological role.     

The complete mitogenome of Apocheima cinerarius (Lepidoptera: Geometridae: Ennominae) and comparison with that of other lepidopteran insects

The complete mitochondrial genome (mitogenome) of a female flightless geometrid moth Apocheima cinerarius was found to be 15,722 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. The A + T content of the complete mitogenome is 80.83%. The AT skew value ([A − T] / [A + T]) is 0.027. The 13 PCGs of the mitogenome start with typical ATN codons, except for cox1 with the start codon CGA. All the tRNA genes have typical cloverleaf secondary structures, except for trnSer(AGN). The secondary structures of rrnL and rrnS were predicted. Six structural domains including conserved regions (IV, V) and variable regions (I, II, III, VI) were identified in the secondary structure of rrnL. The secondary structure of rrnS consists of 3 structural domains. The control region of A. cinerarius begins with conserved motifs of “ATAGA” + 19-bp poly T. It also contains a microsatellite-like (TA)₂₆, a stem-and-loop structure, and a poly-A stretch. Phylogenetic analysis showed that Geometroidea is more closely related to Bombycoidea than to Noctuoidea. A. cinerarius is more closely related to Biston panterinaria than to Phthonandria atrilineata, which is in accordance with the conventional morphology-based classification.     

Complete mitochondrial genome of the stonefly Cryptoperla stilifera Sivec (Plecoptera: Peltoperlidae) and the phylogeny of Polyneopteran insects

We present the complete mitogenome of a stonefly, Cryptoperla stilifera Sivec (Plecoptera; Peltoperlidae). The mitogenome was a circular molecule consisting of 15,633 nucleotides, 37 genes and a A + T-rich region. C. stilifera mitogenome was similar to Pteronarcys princeps mitogenome (Plecoptera; Pteronarcyidae). All transfer RNA genes (tRNAs) had typical cloverleaf secondary structures except for trnSer (AGN), where the stem-loop structure of the dihydrouridine (DHU) arm was missing. The A + T-rich region of C. stilifera had two stem-loops and each had two interlink. Three conserved sequence blocks (CSBs) were present in the A + T-rich regions of C. stilifera, Peltoperla tarteri and Peltoperla arcuata. Moreover, many polynucleotide stretches (Poly N, N = A, T and C) in the A + T-rich region of C. stilifera Phylogenetic relationships of Polyneopteran species were constructed based on the nucleotide sequences of 13 protein coding genes (PCGs). Both maximum likelihood (ML) and Bayesian inference (BI) analyses supported Grylloblattodea as the sister group to Plecoptera + Dermaptera and Embiidina and Phasmatodea as sister groups.     

Ca 2+-induced self-assembly in designed peptides with optimally spaced gamma-carboxyglutamic acid residues

We have previously elucidated a new paradigm for the metal ion-induced helix-helix assembly in the natural γ-carboxyglutamic acid (Gla)-containing class of conantokin (con) peptides, typified by con-G and a variant of con-T, con-T[K7Gla], independent of the hydrophobic effect. In these “metallo-zipper” structures, Gla residues spaced at i, i + 4, i + 7, i + 11 intervals, which is similar to the arrangement of a and d residues in typical heptads of coiled-coils, coordinate with Ca²⁺ and form specific antiparallel helical dimers. In order to evaluate the common role of Gla residues in peptide self-assembly, we extend herein the same Gla arrangement to designed peptides: NH₂-(γLSγEAK)₃-CONH₂ (peptide 1) and NH₂-γLSγEAKγLSγQANγLSγKAE-CONH₂ (peptide 2). Peptide 1 and peptide 2 exhibit no helicity alone, but undergo structural transitions to helical conformations in the presence of a variety of divalent cations. Sedimentation equilibrium ultracentrifugation analyses showed that peptide 1 and peptide 2 form helical dimers in the presence of Ca²⁺, but not Mg²⁺. Folding and thiol-disulfide rearrangement assays with Cys-containing peptide variants indicated that the helical dimers are mixtures of antiparallel and parallel dimers, which is different from the strict antiparallel strand orientations of con-G and con-T[K7γGla] dimers. These findings suggest that the Gla arrangement, i, i + 4, i + 7, i + 11, i + 14, plays a key role in helix formation, without a strict adherence to strand orientation of the helical dimer.     

The complete mitochondrial genome of Gomphocerus tibetanus Uvarov, 1935 (Orthoptera: Acrididae: Gomphocerinae)

The complete nucleotide sequence of the mitochondrial genome (mitogenome) of Gomphocerus tibetanus Uvarov, 1935 (Orthoptera: Acrididae: Gomphocerinae) was determined. It is 15,571 bp in length and contains 74.8% A + T. All Gomphocerus tibetanus protein-coding sequences start with a typical ATN codon. The usual termination codons (TAA and TAG) were found from 13 PCGs except COI and COII which took incomplete codon T as termination codons. All tRNA genes could be folded into the typical cloverleaf secondary structure, except tRNA^Ser(AGN) lacking of dihydrouridine (D) arm. The sizes of the large and small ribosomal RNA genes are 1313 and 822 bp, respectively. The A + T content of the A + T-rich region is 82.3%. A preliminary analysis on characteristics of Gomphocerinae mitogenome was made by comparision among three Gomphocerinae mitogenomes and Locusta migratoria.     

Analysis of three leafminers' complete mitochondrial genomes

Liriomyza trifolii (Burgess), Liriomyza huidobrensis (Blanchard), and Liriomyza bryoniae (Kaltenbach), are three closely related and economically important leafminer pests in the world. This study examined the complete mitochondrial genomes of L. trifolii, L. huidobrensis and L. bryoniae, which were 16141 bp, 16236 bp and 16183 bp in length, respectively. All of them displayed 37 typical animal mitochondrial genes and an A + T-rich region. The genomes were highly compact with only 60–68 bp of non-coding intergenic spacer. However, considerable differences in the A + T-rich region were detected among the three species. Results of this study also showed the two ribosomal RNA genes of the three species had very limited variable sites and thus should not provide much information in the study of population genetics of these species. Data generated from three leafminers' complete mitochondrial genomes should provide valuable information in studying phylogeny of Diptera, and developing genetic markers for species identification in leafminers.     

Ciliary sieving and active ciliary response in capture of particles by suspension-feeding brachiopod larvae

Larvae of a brachiopod, Glottidia pyramidata, used at least two ciliary mechanisms to capture algal cells upstream from the lateral band of cilia that produces a feeding/swimming current. (1) Filtration: the larvae retained algal cells on the upstream (frontal) side of a sieve composed of a row of stationary laterofrontal cilia. Movement of the laterofrontal cilia could not be observed during capture or rejection of particles, but the laterofrontal cilia can bend toward the beating lateral cilia, a possible mechanism for releasing rejected particles from the ciliary sieve. (2) Localized changes of ciliary beat: the larvae may also concentrate particles by a local change in beat of lateral cilia in response to particles. The evidence is that the beat of lateral cilia changed coincident with captures of algal cells and that captured particles moved on paths consistent with a current redirected toward the frontal side of the tentacle by an induced local reversal of the lateral cilia. The change of beat of lateral cilia could have been an arrest rather than a reversal of ciliary beat, however. The similar ciliary bands in adult and larval lophophorates (brachiopods, phoronids, and bryozoans) suggest that these animals share a range of ciliary behaviours. The divergent accounts of ciliary feeding of lophophorates could be mostly the result of different authors observing different aspects of ciliary feeding.     

The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke

Mucus propelling cilia are excitable by many stimulants, and have been shown to increase their beating frequency up to threefold, by physiological extracellular stimulants, such as adenosine-triphosphate, acetylcholine, and others. This is thought to represent the evolutionary adaptation of mucociliary systems to the need of rapid and efficient cleansing the airways of foreign particles. However, the mucus transport velocity depends not only on the beat frequency of the cilia, but on their beat pattern as well, especially in the case of mucus bearing cilia that beat in a complex, three-dimensional fashion. In this study, we directly measured the force applied by live ciliary tissues with an atomic force microscope, and found that it increases linearly with the beating frequency. This implies that the arc swept by the cilia during their effective stroke remains unchanged during frequency increase, thus leading to a linear dependence of transport velocity on the beat frequency. Combining the atomic force microscope measurements with optical measurements, we have indications that the recovery stroke is performed on a less inclined plane, leading to an effective shortening of the overall path traveled by the cilia tip during this nontransporting phase of their beat pattern. This effect is observed to be independent of the type of stimulant (temperature or chemical), chemical (adenosine-triphosphate or acetylcholine), or concentration (1 μM-100 μM), indicating that this behavior may result from internal details of the cilium mechanical structure.     

Na(+)-ATPase and protein kinase C are targets to 1-O-hexadecylphosphocoline (miltefosine) in Trypanosoma cruzi

Miltefosine has been shown to be a very active compound against Trypanosoma cruzi. Here, we evaluated the effects of miltefosine on the activity of the Na⁺-ATPase and protein kinase C (PKC) present in the plasma membrane of T. cruzi. Furosemide (2 mM), a specific inhibitor of Na⁺-ATPase, abolished the growth of T. cruzi showing a crucial role of this enzyme to parasite growth. Miltefosine inhibited the Na⁺-ATPase activity with IC₅₀ = 18 ± 5 μg mL⁻¹. This effect was shown to be reversible, dependent on the pH and Ca²⁺. The inhibition was not observed when the membranes were solubilized with 0.1% deoxycholate, suggesting that the interaction between the enzyme and membrane phospholipids might be important for the drug effect. Miltefosine also inhibited the parasite PKC activity, but through a Na⁺-ATPase-independent way. Altogether the results indicate that miltefosine inhibits T. cruzi growth through, at least in part, the inhibition of both Na⁺-ATPase and PKC activities.     

Toxoplasma gondii: Recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay

In this study, for the first time, the evaluation of Toxoplasma gondii full-length recombinant GRA5 antigen for the serodiagnosis of human toxoplasmosis is shown. The recombinant GRA5 antigen as a fusion protein containing His-tag at both terminals was obtained using an Escherichia coli expression system. The usefulness of rGRA5 for the diagnosis of toxoplasmosis in an ELISA was tested on a total of 189 sera from patients with different stages of the infection and 31 sera from sero-negative individuals, obtained during routine diagnostic tests. Anti-GRA5 IgG antibodies were detected in 70.9% of all seropositive serum samples. This result was comparable to ELISA using a Toxoplasma lysate antigen (TLA) and six combinations of recombinant antigens. The sensitivity of IgG ELISA calculated from all positive serum samples was similar for TLA (94.2%), rMAG1 + rSAG1 + rGRA5 (92.6%), rGRA2 + rSAG1 + rGRA5 (93.1%) and rROP1 + rSAG1 + rGRA5 (94.2%) cocktails, whereas the sensitivity of cocktails without rGRA5 antigens was lower giving 82.0%, 86.2% and 87.8%, respectively. Thus, the present study showed that the full-length rGRA5 is suitable for use as a component of an antigen cocktail for the detection of anti-T. gondii IgG antibodies.     

Mutations in Radial Spoke Head Protein Genes RSPH9 and RSPH4A Cause Primary Ciliary Dyskinesia with Central-Microtubular-Pair Abnormalities

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, “9+2”-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish.     

Biological activities of ACL-I and physicochemical properties of ACL-II, lectins isolated from the marine sponge Axinella corrugata

Lectin II from the marine sponge Axinella corrugata (ACL-II) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel, followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-II is a lectin with an Mr of 80 kDa and 78 kDa, estimated by SDS-PAGE and by FPLC on Superose 12 HR column, respectively. ACL-II mainly agglutinates native rabbit erythrocytes and this hemagglutinating activity is independent of Ca^2 +, Mg^2 + and Mn^2 +, but is inhibited by d-galactose, chitin and N-acetyl derivatives, with the exception of GalNAc. ACL-II is stable for up to 65 °C for 30 min, with a better stability at a pH range of 2 to 6. In contrast, ACL-I displays a strong mitogenic and cytotoxic effect.     

The association of IL1α and IL1β polymorphisms with susceptibility to systemic lupus erythematosus: A meta-analysis

Many epidemiological studies have investigated IL1α and IL1β polymorphisms with SLE risk, but no conclusions are available because of conflicting results. This meta-analysis was performed to more precisely estimate the relationships. The databases of PubMed updated to September 1st, 2012 were retrieved. Odds ratio (OR) and corresponding 95% confidence interval (95% CI) as effect size were calculated by a fixed- or random-effect model. In total, six case–control studies for IL1β − 511C/T, four studies for IL1β + 3953C/T, three studies for IL1α − 889C/T and three studies for IL1α + 4845G/T were involved in this analysis. The results indicated that for IL1α − 889C/T polymorphism T allele was associated with decreased risk of SLE (OR (95% CI)) (T vs. C: 0.802 (0.679–0.949); TT + CT vs. CC: 0.615 (0.380–0.995); TT vs. CC: 0.679 (0.466–0.989)). However, when analysis for TT vs. CT + CC was conducted, the result indicated that IL1α − 889C/T polymorphism was not associated with SLE (OR (95% CI): 0.847 (0.595–1.205)). Combined analysis indicated that IL1β − 511C/T polymorphism was not overall associated with risk of SLE (OR (95% CI)) (T vs. C: 1.113 (0.954–1.298); TT vs. CT + CC: 1.146 (0.889–1.447); TT + CT vs. CC: 1.145 (0.903–1.452); TT vs. CC: 1.255 (0.928–1.698)). When subgroup analysis for Asian ethnicity was conducted, the results indicated that IL1β − 511C/T polymorphism was associated with SLE only for TT vs. CT + CC (OR (95% CI): 1.468 (1.001–2.152)), but was not associated for T vs. C (OR (95% CI): 1.214 (0.955–1.544)), TT + CT vs. CC (OR (95% CI): 1.112 (0.765–1.615)) and TT vs.CC (OR (95% CI): 1.411 (0.896–2.222)). In addition, overall analyses indicated that IL1β + 3953C/T and IL1α + 4845G/C polymorphisms were also not associated with risk of SLE (OR (95% CI)) (for IL1β + 3953C/T T vs. C: 0.996 (0.610–1.626), TT vs. CT + CC: 0.658 (0.318–1.358), TT + CT vs. CC: 1.021 (0.618–1.687), TT vs. CC: 0.640 (0.309–1.325); for IL1α + 4845G/T T vs. G: 1.067 (0.791–1.440), TT + GT vs. GG: 0.934 (0.646–1.351)).This study inferred that IL1α − 889C/T polymorphism might be moderately associated with SLE, but no sufficient evidence was available to support any associations between IL1β + 3953C/T or IL1α + 4845G/C polymorphisms and SLE. We could not draw a definite conclusion between IL1β − 511C/T polymorphism and risk of SLE owing to the limited data. Further large sample-sized studies should be required.     

Conformational changes and reaction of clostridial glycosylating toxins

The crystal structures of the catalytic fragments of ‘lethal toxin’ from Clostridium sordellii and of ‘α-toxin’ from Clostridium novyi have been established. Almost half of the residues follow the chain fold of the glycosyl-transferase type A family of enzymes; the other half forms large α-helical protrusions that are likely to confer specificity for the respective targeted subgroup of Rho proteins in the cell. In the crystal, the active center of α-toxin contained no substrates and was disassembled, whereas that of lethal toxin, which was ligated with the donor substrate UDP-glucose and cofactor Mn^2 +, was catalytically competent. Surprisingly, the structure of lethal toxin with Ca^2 + (instead of Mn^2 +) at the cofactor position showed a bound donor substrate with a disassembled active center, indicating that the strictly octahedral coordination sphere of Mn^2 + is indispensable to the integrity of the enzyme. The homologous structures of α-toxin without substrate, distorted lethal toxin with Ca^2 + plus donor, active lethal toxin with Mn^2 + plus donor and the homologous Clostridium difficile toxin B with a hydrolyzed donor have been lined up to show the geometry of several reaction steps. Interestingly, the structural refinement of one of the three crystallographically independent molecules of Ca^2 +-ligated lethal toxin resulted in the glucosyl half-chair conformation expected for glycosyl-transferases that retain the anomeric configuration at the C1″ atom. A superposition of six acceptor substrates bound to homologous enzymes yielded the position of the nucleophilic acceptor atom with a deviation of < 1 Å. The resulting donor-acceptor geometry suggests that the reaction runs as a circular electron transfer in a six-membered ring, which involves the deprotonation of the nucleophile by the β-phosphoryl group of the donor substrate UDP-glucose.