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1.
Clostridium perfringens epsilon-toxin shows structural similarity to the pore-forming toxin aerolysin 总被引:1,自引:0,他引:1
Cole AR Gibert M Popoff M Moss DS Titball RW Basak AK 《Nature structural & molecular biology》2004,11(8):797-798
Epsilon-toxin from Clostridium perfringens is a lethal toxin. Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism. Here we report the crystal structure of epsilon-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila. Pore-forming toxins can change conformation between soluble and transmembrane states. By comparing the two toxins, we have identified regions important for this transformation. 相似文献
2.
Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae 总被引:2,自引:0,他引:2
Jiménez-Juárez N Muñoz-Garay C Gómez I Saab-Rincon G Damian-Almazo JY Gill SS Soberón M Bravo A 《The Journal of biological chemistry》2007,282(29):21222-21229
Pore-forming toxins are biological weapons produced by a variety of living organisms, particularly bacteria but also by insects, reptiles, and invertebrates. These proteins affect the cell membrane of their target, disrupting permeability and leading eventually to cell death. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. The Cry toxins produced by Bacillus thuringiensis are widely used as insecticides. These proteins have been recognized as pore-forming toxins, and their primary action is to lyse midgut epithelial cells in their target insect. To exert their toxic effect, a prepore oligomeric intermediate is formed leading finally to membrane-inserted oligomeric pores. To understand the role of Cry oligomeric pre-pore formation in the insecticidal activity we isolated point mutations that affected toxin oligomerization but not their binding with the cadherin-like, Bt-R(1) receptor. We show the helix alpha-3 in domain I contains sequences that could form coiled-coil structures important for oligomerization. Some single point mutants in this helix bound Bt-R(1) receptors with similar affinity as the wild-type toxin, but were affected in oligomerization and were severally impaired in pore formation and toxicity against Manduca sexta larvae. These data indicate the pre-pore oligomer and the toxin pore formation play a major role in the intoxication process of Cry1Ab toxin in insect larvae. 相似文献
3.
《Molecular membrane biology》2013,30(2):45-64
Protein toxins are soluble molecules secreted by pathogenic bacteria which act at the plasma membrane or in the cytoplasm of target cells. They must therefore interact with a membrane at some point, either to modify its permeability properties or to reach the cytoplasm. As a consequence, toxins have the built-in capacity to adopt two generally incompatible states: water-soluble and transmembrane. Irrespective of their origin or function, the membrane interacting domain of most protein toxins seems to have adopted one out of two structural strategies to be able to undergo this metamorphosis. In the first group of toxins the membrane interacting domain has the structural characteristics of most known membrane proteins, I.e. it contains hydrophobic and amphipathic α-hellces long enough to span a membrane. To render this ‘membrane protein’ water-soluble during the initial part of its life the hydrophobic hellces are sheltered from the solvent by a barrel of amphipathic helices. In the second group of toxins the opposite strategy is adopted. The toxin is an intrinsically soluble protein and is composed mainly of β-structure. These toxins manage to become membrane proteins by oligomerizing in order to combine amphipathic β-sheet to generate sufflclent hydropho-bicity for membrane insertion to occur. Toxins from this latter group are thought to perforate the lipid bilayer as a β-barrel such as has been described for bacterial porins, and has recently been shown for staphylococcal α-toxin. The two groups of toxins will be described in detail through the presentation of examples. Particular attention will be given to the β-structure toxins, since four new structures have been solved over the past year: the staphyloccocal α-toxin channel, the anthrax protective antigen protoxin, the anthrax protective antigen-soluble heptamer and the CytB protoxin. Structural similarities with mammalian proteins implicated in the immune response and apoptosis will be discussed. Peptide toxins will not be covered in this review. 相似文献
4.
The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor. 总被引:8,自引:2,他引:6
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The inositol-1,4,5-triphosphate (InsP3) receptor consists of a homotetramer of highly conserved 313 kd subunits that contain multiple transmembrane regions in the C-terminal part of the protein. The receptor was expressed in COS cells and its domain structure was studied by mutagenesis. Deletion of the transmembrane regions from the receptor results in the synthesis of a soluble receptor protein that efficiently binds InsP3 but which instead of associating into homotetramers remains monomeric. This result suggests a role for the transmembrane regions in the association of the receptor subunits into tetramers but not in ligand binding. To localize the ligand binding site, further cDNAs encoding truncated receptor proteins were constructed. Assays of InsP3 binding to these truncated InsP3 receptors revealed that sequences in the N-terminal fourth of the InsP3 receptor are sufficient for ligand binding. Accordingly, each subunit of the InsP3 receptor homotetramer contains an independent ligand binding site that is located on the N-terminal ends of each subunit and is separated from the putative channel-forming transmembrane regions by greater than 1400 amino acids. Gel filtration experiments demonstrate a large conformational change of the receptor as a function of ligand binding, suggesting a mechanism by which ligand binding might cause channel opening. 相似文献
5.
膜蛋白跨膜区预测方法的评价 总被引:6,自引:0,他引:6
基因组计划所产生的大量蛋白质序列迫切需要从理论上预测跨膜区。对现有预测跨膜区的方法进行评价 ,不仅可以帮助生物学家选择合适的方法 ,而且可以为生物信息学家发展新算法提供指导。采用了最新的膜蛋白数据库作为基本测试集合并选择了水溶性蛋白序列作为对照组 ,对目前已经公开发表且提供网上服务的跨膜区预测方法进行了评价和分析。经过分析比较 ,HMMTOP在所有的方法中综合预测效果最佳 相似文献
6.
SNARE proteins and fusogenic viral membrane proteins represent the major classes of integral membrane proteins that mediate fusion of eukaryotic lipid bilayers. Although both classes have different primary structures, they share a number of basic architectural features. There is ample evidence that the fusogenic function of representative fusion proteins is influenced by the primary structure of the single transmembrane domain (TMD) and the region linking it to the soluble assembly domains. Here, we used comprehensive non-redundant datasets to examine potential over- and underrepresentation of amino acid types in the TMDs and flanking regions relative to control proteins that share similar biosynthetic origins. Our results reveal conserved overall and/or site-specific enrichment of β-branched residues and Gly within the TMDs, underrepresentation of Gly and Pro in regions flanking the TMD N-terminus, and overrepresentation of the same residue types in C-terminal flanks of SNAREs and viral fusion proteins. Furthermore, the basic Lys and Arg are enriched within SNARE N-terminal flanking regions. These results suggest evolutionary conservation of key structural features of fusion proteins and are discussed in light of experimental findings that link these features to the fusogenic function of these proteins. 相似文献
7.
J D Pédelacq L Maveyraud G Prévost L Baba-Moussa A González E Courcelle W Shepard H Monteil J P Samama L Mourey 《Structure (London, England : 1993)》1999,7(3):277-287
BACKGROUND: Leucocidins and gamma-hemolysins are bi-component toxins secreted by Staphylococcus aureus. These toxins activate responses of specific cells and form lethal transmembrane pores. Their leucotoxic and hemolytic activities involve the sequential binding and the synergistic association of a class S and a class F component, which form hetero-oligomeric complexes. The components of each protein class are produced as non-associated, water-soluble proteins that undergo conformational changes and oligomerization after recognition of their cell targets. RESULTS: The crystal structure of the monomeric water-soluble form of the F component of Panton-Valentine leucocidin (LukF-PV) has been solved by the multiwavelength anomalous dispersion (MAD) method and refined at 2.0 A resolution. The core of this three-domain protein is similar to that of alpha-hemolysin, but significant differences occur in regions that may be involved in the mechanism of pore formation. The glycine-rich stem, which undergoes a major rearrangement in this process, forms an additional domain in LukF-PV. The fold of this domain is similar to that of the neurotoxins and cardiotoxins from snake venom. CONCLUSIONS: The structure analysis and a multiple sequence alignment of all toxic components, suggest that LukF-PV represents the fold of any water-soluble secreted protein in this family of transmembrane pore-forming toxins. The comparison of the structures of LukF-PV and alpha-hemolysin provides some insights into the mechanism of transmembrane pore formation for the bi-component toxins, which may diverge from that of the alpha-hemolysin heptamer. 相似文献
8.
Stehling EG da Silveira WD Campos TA Brocchi M Pertinhez TA Spisni A 《Protein expression and purification》2008,57(1):88-94
Scorpion venoms contain toxic peptides that recognize K(+) channels of excitable and non-excitable cells. These toxins comprise three structurally distinct groups designated alpha-KTx, beta-KTx, and gamma-KTx. It is highly desirable to develop systems for the expression of these toxins for further physiological and structural studies. In this work, an expression vector (pTEV3) was constructed by inserting protein D (major capsid of phage lambda) and TEV protease recognition site into plasmid pET21d DNA sequences. Three alpha-KTx toxins (OsK2, PbTx1, and BmKK3) were cloned into vector pTEV3 and expressed as soluble fusion proteins. The fractions containing the purified fusion proteins (protein D-toxin) were treated with TEV protease to remove protein D. The resulting toxins were analyzed by MALDI-TOF Mass Spectrometry. The results showed that the vector is appropriate for the expression of the target toxins in soluble form and that ion exchange purification of these toxins by flow-through recovery is possible. Analysis by MALDI-TOF Mass Spectrometry of Osk2 demonstrated that this toxin was expressed in its native form, as suggested by the values expected for the presence of two disulfide bridges. 相似文献
9.
Rosado CJ Kondos S Bull TE Kuiper MJ Law RH Buckle AM Voskoboinik I Bird PI Trapani JA Whisstock JC Dunstone MA 《Cellular microbiology》2008,10(9):1765-1774
Pore-forming toxins (PFTs) are commonly associated with bacterial pathogenesis. In eukaryotes, however, PFTs operate in the immune system or are deployed for attacking prey (e.g. venoms). This review focuses upon two families of globular protein PFTs: the cholesterol-dependent cytolysins (CDCs) and the membrane attack complex/perforin superfamily (MACPF). CDCs are produced by Gram-positive bacteria and lyse or permeabilize host cells or intracellular organelles during infection. In eukaryotes, MACPF proteins have both lytic and non-lytic roles and function in immunity, invasion and development. The structure and molecular mechanism of several CDCs are relatively well characterized. Pore formation involves oligomerization and assembly of soluble monomers into a ring-shaped pre-pore which undergoes conformational change to insert into membranes, forming a large amphipathic transmembrane β-barrel. In contrast, the structure and mechanism of MACPF proteins has remained obscure. Recent crystallographic studies now reveal that although MACPF and CDCs are extremely divergent at the sequence level, they share a common fold. Together with biochemical studies, these structural data suggest that lytic MACPF proteins use a CDC-like mechanism of membrane disruption, and will help understand the roles these proteins play in immunity and development. 相似文献
10.
O Shatursky A P Heuck L A Shepard J Rossjohn M W Parker A E Johnson R K Tweten 《Cell》1999,99(3):293-299
Perfringolysin O (PFO), a water-soluble monomeric cytolysin secreted by pathogenic Clostridium perfringens, oligomerizes and forms large pores upon encountering cholesterol-containing membranes. Whereas all pore-forming bacterial toxins examined previously have been shown to penetrate the membrane using a single amphipathic beta hairpin per polypeptide, cysteine-scanning mutagenesis and multiple independent fluorescence techniques here reveal that each PFO monomer contains a second domain involved in pore formation, and that each of the two amphipathic beta hairpins completely spans the membrane. In the soluble monomer, these transmembrane segments are folded into six alpha helices. The insertion of two transmembrane hairpins per toxin monomer and the major change in secondary structure are striking and define a novel paradigm for the mechanism of membrane insertion by a cytolytic toxin. 相似文献
11.
Alongside the well-studied membrane spanning helices, alpha-helical transmembrane (TM) proteins contain several functionally and structurally important types of substructures. Here, existing 3D structures of transmembrane proteins have been used to define and study the concept of reentrant regions, i.e. membrane penetrating regions that enter and exit the membrane on the same side. We find that these regions can be divided into three distinct categories based on secondary structure motifs, namely long regions with a helix-coil-helix motif, regions of medium length with the structure helix-coil or coil-helix and regions of short to medium length consisting entirely of irregular secondary structure. The residues situated in reentrant regions are significantly smaller on average compared to other regions and reentrant regions can be detected in the inter-transmembrane loops with an accuracy of approximately 70% based on their amino acid composition. Using TOP-MOD, a novel method for predicting reentrant regions, we have scanned the genomes of Escherichia coli, Saccharomyces cerevisiae and Homo sapiens. The results suggest that more than 10% of transmembrane proteins contain reentrant regions and that the occurrence of reentrant regions increases linearly with the number of transmembrane regions. Reentrant regions seem to be most commonly found in channel proteins and least commonly in signal receptors. 相似文献
12.
Oh KJ Barbuto S Meyer N Kim RS Collier RJ Korsmeyer SJ 《The Journal of biological chemistry》2005,280(1):753-767
The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of delta-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an alpha-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes. 相似文献
13.
Elizabeth M. Fozo Kira S. Makarova Svetlana A. Shabalina Natalya Yutin Eugene V. Koonin Gisela Storz 《Nucleic acids research》2010,38(11):3743-3759
Small, hydrophobic proteins whose synthesis is repressed by small RNAs (sRNAs), denoted type I toxin–antitoxin modules, were first discovered on plasmids where they regulate plasmid stability, but were subsequently found on a few bacterial chromosomes. We used exhaustive PSI-BLAST and TBLASTN searches across 774 bacterial genomes to identify homologs of known type I toxins. These searches substantially expanded the collection of predicted type I toxins, revealed homology of the Ldr and Fst toxins, and suggested that type I toxin–antitoxin loci are not spread by horizontal gene transfer. To discover novel type I toxin–antitoxin systems, we developed a set of search parameters based on characteristics of known loci including the presence of tandem repeats and clusters of charged and bulky amino acids at the C-termini of short proteins containing predicted transmembrane regions. We detected sRNAs for three predicted toxins from enterohemorrhagic Escherichia coli and Bacillus subtilis, and showed that two of the respective proteins indeed are toxic when overexpressed. We also demonstrated that the local free-energy minima of RNA folding can be used to detect the positions of the sRNA genes. Our results suggest that type I toxin–antitoxin modules are much more widely distributed among bacteria than previously appreciated. 相似文献
14.
Coupled proteolytic and mass spectrometry studies indicate a novel topology for the glycine receptor 总被引:2,自引:0,他引:2
Members of the heteropentameric ligand-gated ion channel superfamily rapidly mediate signaling across the synaptic cleft. Sequence analysis and limited experimental studies have yielded a topological model containing four transmembrane alpha-helices, labeled M1 to M4, and a large soluble, extracellular N-terminal domain. This model persists to date despite some recent structural studies that suggest it may be inappropriate. In this study, the topology of the glycine receptor was probed by limited proteolysis coupled to mass spectrometry. Of particular note, accessible cleavage sites within the putative M1 and M3 transmembrane helices were identified. Membrane-associated fragments within the postulated globular extracellular N-terminal domain were also observed. This report presents several key details incorporated in a new topological model and is the first direct experimental evidence that a subset of the transmembrane regions are too short to be membrane-spanning alpha-helices; rather, these regions are proposed to be a mix of alpha-helices and beta-sheets. This report is also the first to exploit the capability of mass spectrometry to probe critically the topology of a class of membrane proteins of unknown structure. 相似文献
15.
Hong Wing Lee Hong Ching Lee Lawrence K. Lee Erdahl T. Teber 《Journal of biomolecular structure & dynamics》2013,31(2):308-318
Major advances have been made in the prediction of soluble protein structures, led by the knowledge-based modeling methods that extract useful structural trends from known protein structures and incorporate them into scoring functions. The same cannot be reported for the class of transmembrane proteins, primarily due to the lack of high-resolution structural data for transmembrane proteins, which render many of the knowledge-based method unreliable or invalid. We have developed a method that harnesses the vast structural knowledge available in soluble protein data for use in the modeling of transmembrane proteins. At the core of the method, a set of transmembrane protein decoy sets that allow us to filter and train features recognized from soluble proteins for transmembrane protein modeling into a set of scoring functions. We have demonstrated that structures of soluble proteins can provide significant insight into transmembrane protein structures. A complementary novel two-stage modeling/selection process that mimics the two-stage helical membrane protein folding was developed. Combined with the scoring function, the method was successfully applied to model 5 transmembrane proteins. The root mean square deviations of the predicted models ranged from 5.0 to 8.8?Å to the native structures. 相似文献
16.
Möller S Mix E Blüggel M Serrano-Fernández P Koczan D Kotsikoris V Kunz M Watson M Pahnke J Illges H Kreutzer M Mikkat S Thiesen HJ Glocker MO Zettl UK Ibrahim SM 《In silico biology》2005,5(3):295-311
The existence of a soluble splice variant for a gene encoding a transmembrane protein suggests that this gene plays a role in intercellular signalling, particularly in immunological processes. Also, the absence of a splice variant of a reported soluble variant suggests exclusive control of the solubilisation by proteolytic cleavage. Soluble splice variants of membrane proteins may also be interesting targets for crystallisation as their structure may be expected to preserve, at least partially, their function as integral membrane proteins, whose structures are most difficult to determine. This paper presents a dataset derived from the literature in an attempt to collect all reported soluble variants of membrane proteins, be they splice variants or shedded. A list of soluble variants is derived in silico from Ensembl. These are checked on their presence in multiple organisms and their number of membranespanning regions is inspected. The findings then are confirmed by a comparison with identified proteins of a recent global proteomics study of human blood plasma. Finally, a tool to determine novel soluble variants by proteomics is provided. 相似文献
17.
Alternatively spliced soluble coxsackie-adenovirus receptors inhibit coxsackievirus infection 总被引:4,自引:0,他引:4
Dörner A Xiong D Couch K Yajima T Knowlton KU 《The Journal of biological chemistry》2004,279(18):18497-18503
18.
Iacovache I Degiacomi MT Pernot L Ho S Schiltz M Dal Peraro M van der Goot FG 《PLoS pathogens》2011,7(7):e1002135
Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function. 相似文献
19.
Specific interactions between alpha-helical transmembrane segments are important for folding and/or oligomerization of membrane proteins. Previously, we have shown that most transmembrane helix-helix interfaces of a set of crystallized membrane proteins are structurally equivalent to soluble leucine zipper interaction domains. To establish a simplified model of these membrane-spanning leucine zippers, we studied the homophilic interactions of artificial transmembrane segments using different experimental approaches. Importantly, an oligoleucine, but not an oligoalanine, se- quence efficiently self-assembled in membranes as well as in detergent solution. Self-assembly was maintained when a leucine zipper type of heptad motif consisting of leucine residues was grafted onto an alanine host sequence. Analysis of point mutants or of a random sequence confirmed that the heptad motif of leucines mediates self-recognition of our artificial transmembrane segments. Further, a data base search identified degenerate versions of this leucine motif within transmembrane segments of a variety of functionally different proteins. For several of these natural transmembrane segments, self-interaction was experimentally verified. These results support various lines of previously reported evidence where these transmembrane segments were implicated in the oligomeric assembly of the corresponding proteins. 相似文献
20.
Véronique Cabiaux Christian Wolff Jean-Marie Ruysschaert 《International journal of biological macromolecules》1997,21(4):285-298
Bacterial toxins are secreted as soluble proteins. However, they have to interact with a cell lipid membrane either to permeabilize the cells (pore forming toxins) or to enter into the cytosol to express their enzymatic activity (translocation toxins). The aim of this review is to suggest that the strategies developed by toxins to insert in a lipid membrane is mediated by their structure. Two categories, which contains both pore forming and translocation toxins, are emerging: alpha helical proteins containing hydrophobic domains and beta sheets proteins in which no hydrophobicity can be clearly detected. The first category would rather interact with the membrane through multi-spanning helical domains whereas the second category would form a beta barrel in the membrane. 相似文献