Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins

Interactions among the three structural domains of Bacillus 1huringiensis Cn.l toxins %~ere investigated by functional analysis of chinieric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain 1 or domain III among CrylAb, Cn,lAc, CrylC, and CrylE. The activity of the purified trypsinactivated chimeric toxins was evaluiated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only CrylC was active against these cells and only chimeras possessing domain II from CrylC were functional. Combination of domain 1 from CrylE Niith domains Il and III from CrylC, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was frorn Cr31M or CrylAc were slightly smaller than those formed by toxins in which domain I was from CrylC. The properties of the pores formed by the chimeras are therefore likely to result froin an interaction between domain I and domain II or 111. Domain III appears to modulate the activity of the chimeric toxins: combination of domain 111 from CrylAb with domains 1 and II of CrylC gave a protein which was more strongly active than CrylC. (Résumé d'auteur

Clostridium perfringensepsilon toxin H149A mutant as a platform for receptor binding studies

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity

Louse (Insecta : Phthiraptera) mitochondrial 12S rRNA secondary structure is highly variable

Lice are ectoparasitic insects hosted by birds and mammals. Mitochondrial 12S rRNA sequences obtained from lice show considerable length variation and are very difficult to align. We show that the louse 12S rRNA domain III secondary structure displays considerable variation compared to other insects, in both the shape and number of stems and loops. Phylogenetic trees constructed from tree edit distances between louse 12S rRNA structures do not closely resemble trees constructed from sequence data, suggesting that at least some of this structural variation has arisen independently in different louse lineages. Taken together with previous work on mitochondrial gene order and elevated rates of substitution in louse mitochondrial sequences, the structural variation in louse 12S rRNA confirms the highly distinctive nature of molecular evolution in these insects

Eukaryotic translation elongation factor 1A (eEF1A) domain I from S. cerevisiae is required but not sufficient for inter-species complementation

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisia

Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex.

Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products

A Combination of Two Human Monoclonal Antibodies Limits Fetal Damage by Zika Virus in Macaques

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement in vitro (ADE) and extend their half-lives. Here we report on prophylactic co-administration of the Fc-modified antibodies to pregnant rhesus macaques challenged 3 times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV

NMR measurements on intercalated 3R-TaS2.Ix (I=NH3 and N2H4)

Proton nuclear magnetic resonance was used to probe the environment of the intercalant molecule in low dimensional (aeolotropic) solids 3RI-TaS2(NH3)2/3 and 3RII-TaS2(N2H4)4/3, and, specifically to look for phase transitions associated with changes in superlattice geometry. The proton resonance frequency for the intercalated molecules in the temperature interval 200 to 300K indicates that there are different magnetic environments in three temperature domains. (i) There is only one resonance field for molecules in isotropic magnetic environments. (ii) The high- and low-temperature domains (I and III) have molecules in only two different environments, whereas in the middle temperature domain (II) there are at least four different magnetic environments. (iii) The different magnetic environments measured by the ratio of anisotropic to isotropic resonance absorption intensity (rai) indicate that the ratios rai are constants of the sample in domains I and II. (iv) At low temperatures (domain III), rai depends on magnetic field polarisation effects and the temperature treatment of the sample. The authors conclude that the different magnetic environments observed for ammonia and hydrazine appear to be determined by the host, octahedrally coordinate TaS2