Uptake of binary actin ADP-ribosylating toxins

The focus of this article is on the cellular uptake mechanism of the family of binary actin ADP-ribosylating toxins from clostridia. These toxins are special-type AB toxins, because they are composed of two nonlinked proteins, which have to assemble on the surface of eukaryotic cells to act cytotoxically. The enzymatically active component (A), ADP-ribosylates G-actin in the cytosol of target cells. This leads to a complete depolymerization of the actin filaments and, thereby, to rounding up of cultured cells. The second component of these toxins, the binding/translocation component (B), mediates the transport of the enzyme component into the cytosol.     

Functional comparison of the NAD binding cleft of ADP-ribosylating toxins

Although a common core structure forms the active site of ADP-ribosylating (ADPRT) toxins, the limited-sequence homology within this region suggests that different mechanisms are being used by toxins to perform their shared function. To explain differences in their mechanisms of NAD binding and hydrolysis, the functional interrelationship of residues predicted to perform similar functions in the beta3-strand of the NAD binding cleft of different ADPRT toxins was compared. Replacing Tyr54 in the A-subunit of diphtheria toxin (DTA) with a serine, its functional homologue in cholera toxin (CT), resulted in the loss of catalytic function but not NAD binding. The catalytic role of the aromatic portion of Tyr54 in the ADPRT reaction was confirmed by the ability of a Tyr54-to-phenylalanine DTA mutant to retain ADPRT activity. In reciprocal studies, positioning a tyrosine in the beta3-strand of the A1-subunit of CT (CTA1) caused both loss of function and altered structure. The restricted flexibility of the CTA1 active site relative to function became evident upon the loss of ADPRT activity when a conservative Val60-to-leucine mutation was performed. We conclude from our studies that DT and CT maintain a similar mechanism of NAD binding but differ in their mechanisms of NAD hydrolysis. The aromatic moiety at position 54 in DT is integral to NAD hydrolysis, while NAD hydrolysis in CT appears highly dependent on the precise positioning of specific residues within the beta3-strand of the active-site cleft.     

Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding

Five ADP-ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site-directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three-dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.     

Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex.

A member of the Bacillus-produced vegetative insecticidal proteins (VIPs) possesses high specificity against the major insect pest, corn rootworms, and belongs to a class of binary toxins and regulators of biological pathways distinct from classical A-B toxins. The 1.5 A resolution crystal structure of the enzymatic ADP-ribosyltransferase component, VIP2, from Bacillus cereus reveals structurally homologous N- and C-terminal alpha/beta domains likely representing the entire class of binary toxins and implying evolutionary relationships between families of ADP-ribosylating toxins. The crystal structure of the kinetically trapped VIP2-NAD complex identifies the NAD binding cleft within the C-terminal enzymatic domain and provides a structural basis for understanding the targeting and catalysis of the medically and environmentally important binary toxins. These structures furthermore provide specific experimental results to help resolve paradoxes regarding the specific mechanism of ADP-ribosylation of actin by implicating ground state destabilization and nicotinamide product sequestration as the major driving forces for catalysis.     

Serological study of yeast killer toxins by monoclonal antibodies

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.     

Retroviral insertional mutagenesis identifies a small protein required for synthesis of diphthamide, the target of bacterial ADP-ribosylating toxins

Retroviral insertional mutagenesis was used to produce a mutant Chinese hamster ovary cell line that is completely resistant to several different bacterial ADP-ribosylating toxins. The gene responsible for toxin resistance, termed diphtheria toxin (DT) and Pseudomonas exotoxin A (ETA) sensitivity required gene 1 (DESR1), encodes two small protein isoforms of 82 and 57 residues. DESR1 is evolutionally conserved and ubiquitously expressed. Only the longer isoform is functional because the mutant cell line can be complemented by transfection with the long but not the short isoform. We demonstrate that DESR1 is required for the first step in the posttranslational modification of elongation factor-2 at His(715) that yields diphthamide, the target site for ADP ribosylation by DT and ETA. KTI11, the analog of DESR1 in yeast, which was originally identified as a gene regulating the sensitivity of yeast to zymocin, is also required for diphthamide biosynthesis, implicating DESR1/KTI11 in multiple biological processes.     

Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2

Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme. Dph2 and Dph5 were previously identified. Here, we report the identification of the remaining three yeast proteins (Dph1, -3, and -4) and show that all five Dph proteins have either functional (Dph1, -2, -3, and -5) or sequence (Dph4) homologs in mammals. We propose a unified nomenclature for these proteins (e.g., HsDph1 to -5 for the human proteins) and their genes based on the yeast nomenclature. We show that Dph1 and Dph2 are homologous in sequence but functionally independent. The human tumor suppressor gene OVCA1, previously identified as homologous to yeast DPH2, is shown to actually be HsDPH1. We show that HsDPH3 is the previously described human diphtheria toxin and Pseudomonas exotoxin A sensitivity required gene 1 and that DPH4 encodes a CSL zinc finger-containing DnaJ-like protein. Other features of these genes are also discussed. The physiological function of diphthamide and the basis of its ubiquity remain a mystery, but evidence is presented that Dph1 to -3 function in vivo as a protein complex in multiple cellular processes.     

Mode of action of yeast toxins: energy requirement for Saccharomyces cerevisiae killer toxin.

The role of the energy status of the yeast cell in the sensitivity of cultures to two yeast toxins was examined by using 12K release from cells as a measure of toxin action. The Saccharomyces cerevisiae killer toxin bound to sensitive cells in the presence of drugs that interfered with the generation or use of energy, but it was unable to efflux 12K from the cells under these conditions. In direct contrast, the Torulopsis glabrata pool efflux-stimulating toxin induced efflux of the yeast 42K pool was insensitive to the presence of energy poisons in cultures. The results indicate that an energized state, maintained at the expense of adenosine 5'-triphosphate from either glycolytic or mitochondrial reactions, is required for the action of the killer toxin on the yeast cell.     

Use of killer toxins for computer-aided differentiation of Candida albicans strains

Killer toxins were isolated from eight selected killer yeasts. Their activity on 100 Candida albicans isolates of human and animal origin was studied. A computer aided system for differentiating C. albicans strains was developed. By using this system, it was possible to differentiate 14 biotypes of C. albicans isolates based on their susceptibility to the killer toxins.     

Inhibition of protein phosphatase-1 by clavosines A and B. Novel members of the calyculin family of toxins

Site-directed mutagenesis was used to investigate the mechanism of interaction between the catalytic subunit of human protein phosphatase-1 (PP-1cgamma) and members of the calyculin family of toxins. Clavosines A and B are related to calyculins but are glycosylated with a trimethoxy rhamnose group. We provide experimental evidence implicating Tyr-134 as an important residue in PP-1cgamma that mediates interactions with the calyculins. Mutation of Tyr-134 to Phe, to prevent hydrogen bond formation, resulted in a slight increase in sensitivity of PP-1cgamma to clavosines A and B and calyculin A. In contrast, a Y134A mutant was 10-fold less sensitive to inhibition by all three inhibitors. The greatest effect on inhibition was found by substituting an Asp for Tyr-134 in the phosphatase. Clavosine B inhibited PP-1cgamma Y134D with a 310-fold decrease in potency. Clavosine A and calyculin A were also markedly poorer inhibitors of this mutant. These results suggest that a hydrogen bond between Tyr-134 and the calyculins is unlikely to be essential for inhibitor binding to the phosphatase. The clavosines and calyculin A were tested for their ability to inhibit other mutants of PP-1cgamma (including Ile-133, Val-223, and Cys-291). Our mutagenesis studies provide an experimental basis for assessing models of calyculin binding found in the literature (Lindvall, M. K., Pihko, P. M., and Koskinen, A. M. (1997) J. Biol. Chem. 272, 23312-23316; Gupta, V., Ogawa, A. K., Du, X., Houk, K. N., and Armstrong, R. W. (1997) J. Med. Chem. 40, 3199-3206; Gauss, C. M., Sheppeck, I. J., Nairn, A. C., and Chamberlain, R. (1997) Bioorg. Med. Chem. 5, 1751-1773). A new model for clavosine and calyculin A binding to PP-1c is presented that is consistent with previous structure-function experiments and which accommodates key structural features of the clavosines, including the novel rhamnose moiety.     

Mouse NKR-P1. A family of genes selectively coexpressed in adherent lymphokine-activated killer cells

NK cells are a subpopulation of large granular lymphocytes. They are able to recognize and lyse a wide variety of virally infected or neoplastic target cells without previous sensitization or MHC restriction. The molecules involved in target recognition and subsequent triggering of the killing process are still undefined. Recently, a 30-kDa protein highly expressed on rat NK cells and capable of mediating transmembrane signaling was identified and the gene coding for it cloned and sequenced. To better understand the role of this protein in NK cell-mediated cytotoxicity, we cloned its mouse homologue by cross-hybridization of the rat gene to a cDNA library generated from highly purified mouse lymphokine-activated NK cells. Three messages, differing in size and sequence and encoded by different genes, are specifically cotranscribed in mouse NK cells. The protein products of this gene family express the lectin-like motif characteristic of type II transmembrane molecules. Both the rat and mouse proteins have conserved tyrosine and serine residues in their cytoplasmatic portion that are potential phosphorylation sites. They also share a sequence that could be the binding site of the P56lck tyrosine kinase. These observations are consistent with the signaling function hypothesized for these proteins.     

A hypothesis for the mechanism of sodium channel opening by batrachotoxin and related toxins

The mechanism of action of one class of sodium channel opening agents (batrachotoxinin, veratridine, aconitine and grayanotoxin) is proposed to involve complexation of a triad of agent oxygen atoms with the ε-ammonium ion of a channel lysing side chain, holding open the mouth or exit of the ion channel. This idea complements the oxygen triad model derived by structural considerations (Masutani, T., Seyama, I., Narahashi, T. and Iwasa, J. (1981) J. Pharm. Exp. Therap. 217,812) and extended by crystal structure comparisons (Codding, P.W. (1983) J. Am. Chem. Soc. 105, 3172). The mechanism is based on results for acetylcholine receptor ion channel gating, structure and function, using single group rotation (SGR) theory (cf. Kosower, E.M. (1983) Biochem. Biophys. Res. Commun. 111, 1022 and in press (1983);FEBS Lett. (1983) 155, 245; ibid. 157, 144; Biophys. J. (1983) 45, in press).     

Evolution of the human killer cell inhibitory receptor family

Phylogenetic analysis of different domains of human natural killer cell inhibitory receptors (KIR) implicated both intragenic duplication and deletion of exons and interlocus recombination in the evolution of these receptors. In phylogenies of the extracellular immunoglobulin (Ig) superfamily C2-set domains and of the pre-membrane (PM) domain, KIR receptors having two C2-set domains and those having three such domains tended to form separate clusters. However, the phylogenies of the transmembrane (TM) and cytoplasmic (CYT) domains showed quite different topologies, suggesting that major sites of interlocus recombination have been between exon 6 (encoding PM) and exon 7 (encoding TM) and between exon 7 and exons 8-9 (encoding CYT). Examination of the pattern of nucleotide substitution in the exons encoding Ig C2-set domains supported the hypothesis that positive Darwinian selection has acted to diversify the residues within these domains that are involved in contact with class I MHC molecules.     

Exploring the common dynamics of homologous proteins. Application to the globin family

We present a procedure to explore the global dynamics shared between members of the same protein family. The method allows the comparison of patterns of vibrational motion obtained by Gaussian network model analysis. After the identification of collective coordinates that were conserved during evolution, we quantify the common dynamics within a family. Representative vectors that describe these dynamics are defined using a singular value decomposition approach. As a test case, the globin heme-binding family is considered. The two lowest normal modes are shown to be conserved within this family. Our results encourage the development of models for protein evolution that take into account the conservation of dynamical features.     

A common folding mechanism in the cytochrome c family

Of the globular proteins, cytochrome c (cyt c) has been used extensively as a model system for folding studies. Here we analyse the folding pathway of different cyt c proteins from prokaryotes and eukaryotes, and attempt to single out general correlations between structural determinants and folding mechanisms. Recent studies provide evidence that the folding pathway of several cyt c proteins involves the formation of a partially structured intermediate. Using state-of-the-art kinetic analysis on published data, we show that such a folding intermediate is an obligatory on-pathway species that might represent either a defined local minimum in the reaction coordinate or an unstable high-energy state. Available data also indicate that some essential structural features of the folding intermediate and transition states are highly conserved across this protein family. Thus, cyt c proteins share a consensus folding mechanism in spite of large differences in physico-chemical properties and thermodynamic stability. This novel outlook on the folding of cyt c can shed light on much published data and might offer a general scheme by which to plan new experiments.     

Mating-type locus control of killer toxins from Kluyveromyces lactis and Pichia acaciae

Killer-toxin complexes produced by Kluyveromyces lactis and Pichia acaciae inhibit cell proliferation of Saccharomyces cerevisiae. Analysis of their actions in haploid MATalpha cells revealed that introduction of the opposite mating-type locus (MATa) significantly suppressed antizymosis. Together with resistance expressed by MATa/MATalpha diploids, the reciprocal action of MATa or MATalpha in haploids of opposite mating types suggests that these killer toxins may be subject to MAT locus control. Congruently, derepressing the silent mating-type loci, HMR and HML, by removing individual components of the histone deacetylase complex Sir1-4, either by transposon-tagging or by chemically inactivating the histone deacetylase catalytic subunit Sir2, yields toxin resistance. Consistent with MAT control of toxin action, killer-toxin-insensitive S. cerevisiae mutants (kti) become mating-compromised despite resisting the toxins' cell-cycle effects. Mating inhibition largely depends on the time point of toxin application to the mating mixtures and is less pronounced in Elongator mutants, whose resistance to the toxins' cell-cycle effects is the result of toxin-target process deficiencies. In striking contrast, non-Elongator mutants defective in early-response events such as toxin import/activation hardly recover from toxin-induced mating inhibition. This study reveals a novel effect of yeast killer toxins on mating and sexual reproduction that is independent of their impact on cellular proliferation and cell-cycle progression.     

A common catalytic mechanism for proteins of the HutI family

Imidazolonepropionase (HutI) (imidazolone-5-propanote hydrolase, EC 3.5.2.7) is a member of the amidohydrolase superfamily and catalyzes the conversion of imidazolone-5-propanoate to N-formimino-L-glutamate in the histidine degradation pathway. We have determined the three-dimensional crystal structures of HutI from Agrobacterium tumefaciens (At-HutI) and an environmental sample from the Sargasso Sea Ocean Going Survey (Es-HutI) bound to the product [ N-formimino-L-glutamate (NIG)] and an inhibitor [3-(2,5-dioxoimidazolidin-4-yl)propionic acid (DIP)], respectively. In both structures, the active site is contained within each monomer, and its organization displays the landmark feature of the amidohydrolase superfamily, showing a metal ligand (iron), four histidines, and one aspartic acid. A catalytic mechanism involving His265 is proposed on the basis of the inhibitor-bound structure. This mechanism is applicable to all HutI forms.     

CDC/MACPF家族成孔毒素研究进展

成孔毒素(Pore-forming toxins,PFTs)通常与细菌等原核生物的致病机理相关,在真核生物中,这类蛋白在免疫、胚胎发育、神经细胞迁移等方面发挥着重要作用。其中球状蛋白成孔毒素中的两大家族,胆固醇结合细胞溶素(Cholesterol-dependent cytolysins,CDCs)和攻膜复合体/穿孔素超家族(Membrane attack complex/perforin superfamily,MACPF)以其独特的分子构型和功能受到研究者的关注。CDCs由革兰氏阳性细菌产生,在细菌的侵染过程中发挥裂解靶细胞膜的作用;在真核生物中,MACPF蛋白同时具有裂解和非裂解两种形式。目前,对于以产气荚膜梭菌溶血素(PFO)为代表的CDCs蛋白的结构及分子机制了解得较为透彻,近年来晶体学及生物化学研究揭示,尽管CDCs和MACPF这两大家族成孔毒素在氨基酸序列水平上存在较大差异,但它们共同的折叠模式预示:MACPF蛋白以一种类似于CDCs的成孔机制来行使其裂解靶膜的功能。文章对这两大家族成孔毒素的结构、成孔机制以及目前的研究热点问题予以综述,为该领域今后的研究提供有益参考。     

Yeast killer toxins,molecular mechanisms of their action and their applications

Killer toxins secreted by some yeast strains are the proteins that kill sensitive cells of the same or related yeast genera. In recent years, many new yeast species have been found to be able to produce killer toxins against the pathogenic yeasts, especially Candida albicans. Some of the killer toxins have been purified and characterized, and the genes encoding the killer toxins have been cloned and characterized. Many new targets including different components of cell wall, plasma membrane, tRNA, DNA and others in the sensitive cells for the killer toxin action have been identified so that the new molecular mechanisms of action have been elucidated. However, it is still unknown how some of the newly discovered killer toxins kill the sensitive cells. Studies on the killer phenomenon in yeasts have provided valuable insights into a number of fundamental aspects of eukaryotic cell biology and interactions of different eukaryotic cells. Elucidation of the molecular mechanisms of their action will be helpful to develop the strategies to fight more and more harmful yeasts.