Structure-function relationships of a membrane pore forming toxin revealed by reversion mutagenesis

Cyt2Aa1 is a haemolytic membrane pore forming toxin produced by Bacillus thuringiensis subsp. kyushuensis. To investigate membrane pore formation by this toxin, second-site revertants of an inactive mutant toxin Cyt2Aa1-I150A were generated by random mutagenesis using error-prone PCR. The decrease in side chain length caused by the replacement of isoleucine by alanine at position 150 in the alphaD-beta4 loop results in the loss of important van der Waals contacts that exist in the native protein between I150 and K199 and L203 on alphaE. 28 independent revertants of I150A were obtained and their relative toxicity can be explained by the position of the residue in the structure and the effect of the mutation on side-chain interactions. Analysis of these revertants revealed that residues on alphaA, alphaB, alphaC, alphaD and the loops between alphaA and alphaB, alphaD and beta5, beta6 and beta7 are important in pore formation. These residues are on the surface of the molecule suggesting that they may participate in membrane binding and toxin oligomerization. Changing the properties of the amino acid side-chains of these residues could affect the conformational changes required to transform the water-soluble toxin into the membrane insertion competent state.     

Alanine-162 of Bacillus thuringiensis Cyt2Aa2 toxin is essential for membrane binding and oligomerisation

Cyt2Aa2 is a cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. It is specifically toxic to dipteran larvae in vivo and is also active against several cell types, such as erythrocytes. The active toxin is proposed to bind to the cell membrane, and membrane pore formation by toxin oligomerisation leads to cell lysis. This study aimed to characterise the role of residues (I139, S159, L160, S161, A162, D209 and V215) potentially involved in the membrane binding of Cyt2Aa2. All mutants, except I139A and V215A, showed similar characteristics to the wild-type toxin after proteinase K cleavage. Three mutants, S159A, L160A and S161A, showed high haemolytic activity but low toxicity against Aedes aegypti. Membrane interaction assays showed that these mutants could bind to rat red blood cells (rRBCs) and oligomerise. The mutant D209N had no haemolytic activity but was still mildly toxic to A. aegypti. The mutant A162V could not lyse rRBCs, even at high concentrations, and showed no toxicity against A. aegypti. Our data suggest that alanine 162 of the Cyt2Aa2 toxin is involved in membrane binding and oligomerisation. Substitution of this amino acid altered the conformation of the toxin and affected its biological activity.     

Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system

Abstract A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family. Region 1 comprises the central α-helix 5 of domain I and has been implicated in the pore formation activity of the toxin. Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity. Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions. Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.     

Analysis of the antigenic difference between Vero toxin 2 (VT2) and VT2 variant (VT2vh) of Verotoxin-producing Escherichia coli by a site-directed mutagenesis

Ouchterlony double gel diffusion analysis of the A and B subunits of purified Vero toxin 2 (VT2) and a variant of VT2 (VT2vh) demonstrated that the difference in antigenicity between VT2 and VT2vh is due to the difference in the B subunit of the two toxins. Analysis of mutants of VT2vh prepared by site-directed mutagenesis attributed the antigenic dissimilarity to the difference in the amino acid residue at position 24 of the B subunit.     

Quality control of Bacillus thuringiensis ssp. israelensis products based on toxin quantification with monoclonal antibodies

Quality control of Bacillus thuringiensis ssp. israelensis (Bti) products is currently based on international toxic units (ITUs). The potency of products is related to the activity of a standard (IPS-82, Institute Pasteur, Paris) assessed in bioassays using Aedes aegypti as a target host. The procedure is time consuming and costly, often producing variable results. The activity of Bti is based on four different insecticidal crystal proteins (ICPs): Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Monoclonal antibodies were produced using IPS-82 for immunisation and an enzyme-linked immunosorbent assay (ELISA) was developed. Antibodies were selected with specificity against Cry11A and Cyt1A. Cry4 specific antibodies could not distinguish between Cry4A and Cry4B. Within five replicate assessments of the three ICPs (in µg mg^-1 ICP protein), an error between 3 and 8% was recorded, whereas a 14% error was obtained comparing seven samples of the same production batch for ITUs mg^-1. The toxicity against A. aegypti expressed in ITUs correlated well with the results of the ELISA (correlation coefficient r Cyt 1=0.79; Cry 11=0.87; Cry 4=0.91) also when related to the sum of all ICPs (r=0.87). The ELISA can reduce efforts to determine Bti quality compared with the labour-intensive and variable ITU bioassay.     

Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of beta-barrel pore-forming toxins (beta-PFTs). By contrast, other beta-PFTs form homo-oligomeric pores; for example, the staphylococcal alpha-hemolysin (alpha HL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site-specific chemical modification during single-channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the alpha HL pore. It is also pertinent to the mechanism of assembly of beta-PFT pores and suggests new possibilities for engineering these proteins.     

Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules

Few methods exist for obtaining the internal dimensions of transmembrane pores for which 3-D structures are lacking or for showing that structures determined by crystallography reflect the internal dimensions of pores in lipid bilayers. Several approaches, involving polymer penetration and transport, have revealed limiting diameters for various pores. But, in general, these approaches do not indicate the locations of constrictions in the channel lumen. Here, we combine cysteine mutagenesis and chemical modification with sulfhydryl-reactive polymers to locate the constriction in the lumen of the staphylococcal alpha-hemolysin pore, a model protein of known structure. The rates of reaction of each of four polymeric reagents (MePEG-OPSS) of different masses towards individual single cysteine mutants, comprising a set with cysteines distributed over the length of the lumen of the pore, were determined by macroscopic current recording. The rates for the three larger polymers (1.8, 2.5, and 5.0 kD) were normalized with respect to the rates of reaction with a 1.0-kD polymer for each of the seven positions in the lumen. The rate of reaction of the 5.0-kD polymer dropped dramatically at the centrally located Cys-111 residue and positions distal to Cys-111, whether the reagent was applied from the trans or the cis side of the bilayer. This semi-quantitative analysis sufficed to demonstrate that a constriction is located at the midpoint of the pore lumen, as predicted by the crystal structure, and although the constriction allows a 2.5-kD polymer to pass, transport of a 5.0-kD molecule is greatly restricted. In addition, PEG chains gave greater reductions in pore conductance when covalently attached to the narrower regions of the lumen, permitting further definition of the interior of the pore. The procedures described here should be applicable to other pores and to related structures such as the vestibules of ion channels.     

Directional degradation of β-chitin by chitinase A1 revealed by a novel reducing end labelling technique

A novel procedure for labelling the molecular ends of β-chitin crystals has been established. By introducing a hydrazide derivative of biotin at the reducing end of a chitin chain, followed by a specific interaction between biotin and streptavidin coupled with a colloidal gold particle, the chain directionality of β-chitin microcrystals could be directly visualized by transmission electron microscopy. This method allowed to certify the parallelism of the chitin chains in the β-chitin microcrystals, and also to label the reducing tips of β-chitin microcrystals degraded by Bacillus circulans chitinase A1. With these substrates, the labelling occurred only at their tapered tip, which indicates that the digestion of these crystals proceeded from their reducing end. The generalization of this new labelling method to other polysaccharide crystals is discussed.     

Dual mechanisms involved in development of diverse biological activities of islet-activating protein,pertussis toxin,as revealed by chemical modification of lysine residues in the toxin molecule

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein composed of an A-protomer and a B-oligomer. There seem to be at least two molecular mechanisms by which IAP exerts its various effects in vivo and in vitro. On the one hand, some of the effects were not significantly affected by acetamidination of the ε-amino groups of the lysine residues in the molecule. These include the activities in vitro (1) catalyzing ADP-ribosylation of one of the membrane proteins directly, (2) enhancing membrane adenylate cyclase activity in C6 cells, (3) reversing receptor-mediated inhibition of insulin or glycerol release from pancreatic islets of adipocytes, respectively, and the activities in vivo (4) inhibiting epinephrine-induced hyperglycemia, (5) potentiating glucose-induced hyperinsulinemia, (6) reducing hypertension and increasing the heart rate in genetically hypertensive rats. These activities are concluded to develop as a result of ADP-ribosylation catalyzed by the A-protomer which is rendered accessible to its intramembrane substrate thanks to the associated B-oligomer moiety. Thus, neither the enzymic activity of the A-protomer nor the transporting activity of the B-oligomer needs free amino groups of the lysine residues in the IAP molecule. On the other hand, additional effects of IAP, such as (1) mitogenic, (2) lymphocytosis-promoting, (3) histamine-sensitizing, (4) adjuvant and (5) vascular permeability increasing, were markedly suppressed by acetamidination of the intrapeptide lysine residues. The free ε-amino group of lysine would play an indispensable role in the firm (or divalent) attachment of the B-oligomer of IAP to the cell surface that is responsible for development of these activities.     

Structural characterization of the pore forming protein TatAd of the twin-arginine translocase in membranes by solid-state N-NMR

The transmembrane protein TatA is the pore forming unit of the twin-arginine translocase (Tat), which has the unique ability of transporting folded proteins across the cell membrane. This ATP-independent protein export pathway is a recently discovered alternative to the general secretory (Sec) system of bacteria. To obtain insight in the translocation mechanism, the structure and alignment in the membrane of the well-folded segments 2-45 of TatA_d from Bacillus subtilis was studied here. Using solid-state NMR in bicelles containing anionic lipids, the topology and orientation of TatA_d was determined in an environment mimicking the bacterial membrane. A wheel-like pattern, characteristic for a tilted transmembrane helix, was observed in ¹⁵N chemical shift /¹⁵N-¹H dipolar coupling correlation NMR spectra. Analysis of this PISA wheel revealed a 14-16 residue long N-terminal membrane-spanning helix which is tilted by 17° with respect to the membrane normal. In addition, comparison of uniformly and selectively ¹⁵N-labeled TatA_2-45 samples allowed determination of the helix polarity angle.     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. II. Effect of membrane lipid composition in a liposome system

In the first paper of this series, it was shown that a toxin from the sea anemone Stoichactis helianthus increased the permeability of black lipid membranes due to transmembrane channel formation. In the present study, we have used liposomes to examine the reactivity of the toxin with different phospholipids. Membrane damage was assessed by measuring the release of ⁸⁶Rb⁺ and ¹⁴C-labeled membrane lipid. For the different lipids, the rank order of marker release was: sphingomyelin > C18: 2 phosphatidylcholine > C18: 1 phosphatidylcholine > C18: 0 phosphatidylcholine > C16: 0 phosphatidylcholine = C14: 0 phosphatidylcholine. In C14: 0 and C16: 0 phosphatidylcholine liposomes there was no ¹⁴C-labeled lipid release and only 13 to 16% ⁸⁶Rb⁺ release which corresponds to the ⁸⁶Rb⁺ content in the outermost aqueous shell of multilamellar liposomes. This indicates that membrane damage was limited to the outermost bilayer. In liposomes prepared with the other lipids, the extent of release of both markers increased proportionately with the length and the degree of unsaturation of the lipids' acyl side chains. Sphingomyelin liposomes were the most susceptible with 47% of the ¹⁴C-labeled lipid marker and 90% of the ⁸⁶Rb⁺ marker being released. The large extent of ¹⁴C-labeled lipid release is attributed to a detergent-like activity of the toxin which presumably is due to the amphipathic nature of the protein. Thus, the toxin can inflict membrane damage in two ways: (1) channel formation, and (2) detergent action. The importance of one mechanism or the other apparently varies depending on membrane structure and lipid composition.     

Spatial distribution of cytoplasmic domains of the Mg-transporter MgtE,in a solution lacking Mg,revealed by paramagnetic relaxation enhancement

MgtE is a prokaryotic Mg²⁺ transporter that controls cellular Mg²⁺ concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg²⁺-free and Mg²⁺-bound forms. The Mg²⁺-binding sites lay at the interface of the 2 domains, making the Mg²⁺-bound form compact and globular. In the Mg²⁺-free structure, however, the domains are far apart, and the Mg²⁺-binding sites are destroyed. Therefore, it is unclear how Mg²⁺-free MgtE changes its conformation to accommodate Mg²⁺ ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg²⁺ in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg²⁺-bound crystal structure, facilitating efficient capture of Mg²⁺ with increased intracellular Mg²⁺ concentration, which is necessary to close the gate.