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beta-sheet pore

(76 References)

Heinz, C., H. Engelhardt, et al. (2003). "The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable beta -Sheet Domain." J Biol Chem 278(10): 8678-85.

            MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel beta-strands organized in a coherent domain. Heating to 92 and 112 degrees C was required to dissociate the MspA tetramer and to unfold the beta-sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming beta-barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6 m urea at 50 degrees C was reduced 13- and 30-fold, respectively, although the MspA tetramer and the beta-sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are beta-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications.


Alvarez, C., F. Casallanovo, et al. (2003). "Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces-Modulation of conformation and activity, and lipid-protein interaction." Chem Phys Lipids 122(1-2): 97-105.

            Sticholysins I and II (St I and St II) are water-soluble toxins produced by the sea anemone Stichodactyla helianthus. St I and St II bind to biological and model membranes containing sphingomyelin (SM), forming oligomeric pores that lead to leakage of internal contents. Here we describe functional and structural studies of the toxins aiming at the understanding at a molecular level of their mechanism of binding, as well as their effects on membrane permeabilization. St I and St II caused potassium leakage from red blood cells and temperature-dependent hemolysis, the activation energy of the process being lower for the latter toxin. Protein intrinsic fluorescence measurements provided evidence for toxin binding to model membranes composed of 1:1 (mol:mol) egg phosphatidyl choline (ePC):SM. The fluorescence intensity increased and the maximum emission wavelength decreased as a result of binding. The changes were quantitatively different for both toxins. Circular dichroism spectra showed that both St I and St II exhibit a high content of beta-sheet structure and that binding to model membranes did not alter the toxin's conformation to a large extent. Changing the lipid composition by adding 5 mol% of negatively charged phosphatidic acid (PA) or phosphatidyl glycerol (PG) had small, but detectable, effects on protein conformation. The influence of lipid composition on toxin-induced membrane permeabilization was assessed by means of fluorescence measurements of calcein leakage. The effect was larger for ePC:SM bilayers containing 5 mol% of negative curvature-inducing lipids. Electron paramagnetic resonance (EPR) spectra of intercalated fatty acid spin probes carrying the nitroxide moiety at different carbons (5, 7, 12, and 16) evidenced the occurrence of lipid-protein interaction. Upon addition of the toxins, two-component spectra were observed for the probe labeled at C-12. The broader component, corresponding to a population of strongly immobilized spin probes, was ascribed to boundary lipid. The contribution of this component to the total spectrum was larger for St II than for St I. Moreover, it was clearly detectable for the C-12-labeled probe, but it was absent when the label was at C-16, indicating a lack of lipid-protein interaction close to the lipid terminal methyl group. This effect could be either due to the fact that the toxins do not span the whole bilayer thickness or to the formation of a toroidal pore leading to the preferential interaction with acyl chain carbons closer to the phospholipids head groups.


Dennison, S. M., N. Greenfield, et al. (2002). "VSV transmembrane domain (TMD) peptide promotes PEG-mediated fusion of liposomes in a conformationally sensitive fashion." Biochemistry 41(50): 14925-34.

            Helical instability induced by gly residues in the transmembrane domain (TMD) of G protein, the fusion protein of vesicular stomatitis virus (VSV), was speculated to aid in the later steps of the fusion process, because G protein with ala's substituted for the two TMD gly's was inactive (Cleverley, D. Z., and Lenard, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3425-30). Here we examine the conformations of synthetic peptides corresponding to fusion-active (GGpep) and inactive (AApep; G's replaced by A's) TMDs by CD spectroscopy, and then their effects on the kinetics of poly (ethyleneglycol) (PEG)-mediated fusion of small unilamellar vesicles. GGpep and AApep both assumed history-dependent, non-interconvertible ordered structures. Both peptides were largely helical under all conditions if derived from trifluoroethanol solutions, and aggregated in a beta-sheet form if derived from acetonitrile solutions. In solvent, detergents or lipid bilayers, GGpep showed a greater range of secondary structural features than did AApep. The two peptides had large but different effects on PEG-mediated fusion. Both enhanced the rate but not the extent of lipid mixing. AApep significantly inhibited the extent of fusion pore formation while GGpep had no effect. The initial rate of fusion was enhanced 6-fold by GGpep and less than 2-fold by AApep. Addition of 5 mol % hexadecane overrode all peptide-induced effects. We suggest that both GGpep and hexadecane promote pore formation by stabilizing the nonlamellar structures in fusion intermediates or initial small pores. AApep, which had fewer nonhelical features in its CD spectrum than GGpep, actually inhibited fusion pore formation.


Das, G. and S. Matile (2002). "Transmembrane pores formed by synthetic p-octiphenyl beta-barrels with internal carboxylate clusters: regulation of ion transport by pH and Mg(2+)- complexed 8-aminonaphthalene-1,3,6-trisulfonate." Proc Natl Acad Sci U S A 99(8): 5183-8.

            Design, synthesis, and study of a synthetic barrel-stave supramolecule with p-octiphenyl "staves," beta-sheet "hoops," and hydrophobic exterior as well as internal carboxylate clusters are reported. Ion transport experiments indicate the formation of transmembrane pores at 5 < pH < 7 with nanomolar activity. Blockage of dye efflux from spherical bilayers by external Mg(OAc)(2) and internal 8-aminonaphthalene-1,3,6-trisulfonate is suggestive for weakly cooperative (n = 1.16) formation of aspartate-Mg(2+)-8-aminonaphthalene-1,3,6-trisulfonate complexes within the barrel-stave supramolecule (K(D) = 2.9 mM). Corroborative evidence from structural studies by circular dichroism spectroscopy is provided and discussed with emphasis of the importance of internal charge repulsion for pore formation and future applications toward binding and catalysis within supramolecular synthetic pores.


Chittchang, M., N. Salamat-Miller, et al. (2002). "Poly(L-lysine) as a model drug macromolecule with which to investigate secondary structure and microporous membrane transport, part 2: diffusion studies." J Pharm Pharmacol 54(11): 1497-505.

            Peptide drugs are hydrophilic in nature and so their preferred pathway of membrane transport is by the paracellular route, which primarily involves passive diffusion across intercellular pores. The objective of the present study was to investigate the effect of secondary structure on the aqueous diffusion of a model polypeptide, poly(L-lysine), through a microporous membrane. The primary aim was to systematically evaluate the variables (e.g. viscosity and/or hydrodynamic radius) that may contribute to the difference, if any, in the calculated values of the aqueous diffusion coefficient (D(aq)) for each conformer of poly(L-lysine). Variations in pH and temperature of the medium were used to induce secondary structural changes in poly(L-lysine). Transport studies were conducted for 3 h at 25 or 37 degrees C using side-by-side diffusion cells. Hydrophilic microporous polyester membranes with a 1-microm pore diameter were used to measure the free diffusion of each conformer. The values for the apparent permeability (P(app)) and D(aq) were calculated using standard equations. The viscosity of each conformer solution was determined and the hydrodynamic radius of each conformer was then estimated. At 25 degrees C, both P(app) and D(aq) of the alpha-helix conformer were approximately the same as those of the random coil conformer. In contrast, at 37 degrees C, the P(app) and the D(aq) of the beta-sheet conformer were significantly (P < 0.05) less than those of the random coil conformer. At 25 degrees C, the solutions containing primarily either the random coil or the-helix conformers had approximately the same viscosity. On the other hand, at 37 degrees C, the solutions containing the beta-sheet conformer had a significantly (P < 0.05) higher viscosity than when this conformer was absent. The random coil and the alpha-helix conformers appeared to have comparable sizes, whereas the hydrodynamic radius estimated for the beta-sheet conformer was significantly (P < 0.05) larger than those for the other two conformers. In summary, changing the secondary structure of poly(L-lysine) from the random coil to the alpha-helix did not affect its P(app) and intrinsic D(aq). On the other hand, appearance of the beta-sheet conformer significantly decreased the values of P(app) and D(aq). The differences appeared to result from the significantly higher solution viscosity as well as the extended structure associated with the beta-sheet conformer of poly(L-lysine). This strategy may represent a potential mechanism to sustain the delivery of therapeutic peptide drugs from a controlled drug delivery device.


Anguiano, M., R. J. Nowak, et al. (2002). "Protofibrillar islet amyloid polypeptide permeabilizes synthetic vesicles by a pore-like mechanism that may be relevant to type II diabetes." Biochemistry 41(38): 11338-43.

            Islet amyloid polypeptide (IAPP) and insulin are copackaged and cosecreted by pancreatic islet beta-cells. Non-insulin-dependent (type II) diabetes mellitus (NIDDM) is characterized by dysfunction and depletion of these beta-cells and also, in more than 90% of patients, amyloid plaques containing fibrillar IAPP. An aggregated but not necessarily fibrillar form of IAPP is toxic in cell culture, suggesting that prefibrillar oligomeric (protofibrillar) IAPP may be pathogenic. We report here that IAPP generates oligomeric species in vitro that are consumed as beta-sheet-rich fibrils grow. Protofibrillar IAPP, like protofibrillar alpha-synuclein, which is implicated in Parkinson's disease pathogenesis, permeabilizes synthetic vesicles by a pore-like mechanism. The formation of the IAPP amyloid pore is temporally correlated to the formation of early IAPP oligomers and its disappearance to the appearance of amyloid fibrils. Neither pores nor oligomers were formed by the nonfibrillogenic rat IAPP variant. The IAPP amyloid pore may be critical to the pathogenic mechanism of NIDDM, as other amyloid pores may be to Alzheimer's disease and Parkinson's disease.


Wong, K. K., F. S. Brinkman, et al. (2001). "Evaluation of a structural model of Pseudomonas aeruginosa outer membrane protein OprM, an efflux component involved in intrinsic antibiotic resistance." J Bacteriol 183(1): 367-74.

            The outer membrane protein OprM of Pseudomonas aeruginosa is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in Escherichia coli, was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914-919, 2000), demonstrating a distinctive architecture comprising outer membrane beta-barrel and periplasmic helical-barrel structures, which assemble differently from the common beta-barrel-only conformation of porins. Based on their sequence similarity, a similar content of alpha-helical and beta-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the "iris" mechanism of action for TolC and permit us now to form more focused hypotheses about the functional domains of OprM and its related family of efflux proteins.


Vie, V., N. Van Mau, et al. (2001). "Lipid-induced pore formation of the Bacillus thuringiensis Cry1Aa insecticidal toxin." J Membr Biol 180(3): 195-203.

            After activation, Bacillus thuringiensis (Bt) insecticidal toxin forms pores in larval midgut epithelial cell membranes, leading to host death. Although the crystal structure of the soluble form of Cry1Aa has been determined, the conformation of the pores and the mechanism of toxin interaction with and insertion into membranes are still not clear. Here we show that Cry1Aa spontaneously inserts into lipid mono- and bilayer membranes of appropriate compositions. Fourier Transform InfraRed spectroscopy (FTIR) indicates that insertion is accompanied by conformational changes characterized mainly by an unfolding of the beta-sheet domains. Moreover, Atomic Force Microscopy (AFM) imaging strongly suggests that the pores are composed of four subunits surrounding a 1.5 nm diameter central depression.


Nguyen, N. T., R. Maurus, et al. (2001). "Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes." Biochemistry 40(44): 13177-87.

            We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria. Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism. This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties. Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis. The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K. This latter loop is present only in type II citrate synthase sequences. Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network. Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer. The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases. This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation. Another surprising observation from the structure of type II E. coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site. Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur. Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure. Thus, acetylcoenzyme A binding to type II E. coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite. We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases. Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.


Methot, N., B. D. Ritchie, et al. (2001). "Structure of the pore-forming transmembrane domain of a ligand-gated ion channel." J Biol Chem 276(26): 23726-32.

            The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide (1)H/(2)H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm(-)1, respectively, in both (1)H(2)O and (2)H(2)O. The amide I band is very similar to the amide I bands observed in the spectra of alpha-helical proteins, such as myoglobin and bacteriorhodopsin, that lack beta structure and exhibit much less beta-sheet character than is observed in proteins with as little as 20% beta sheet. Curve-fitting estimates 75-80% alpha-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane alpha-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane alpha-helices.


McNay, J. L. and E. J. Fernandez (2001). "Protein unfolding during reversed-phase chromatography: I. Effect of surface properties and duration of adsorption." Biotechnol Bioeng 76(3): 224-32.

            Residue-level features of bovine pancreatic trypsin inhibitor (BPTI) unfolding on reversed-phase chromatography (RPC) surfaces were investigated using hydrogen-deuterium exchange labeling and NMR. A set of silica-based RPC surfaces was used to examine the influence of alkyl chain length and media pore size on adsorbed BPTI conformation. In all cases there was substantial unfolding in the adsorbed state; however, residual protection from exchange was consistently observed. Particle pore size did not influence conformation substantially for C4-alkyl modified silica; however, 120 A pore C18 media produced more hydrogen exchange than any other surface examined. In this case, the radius of curvature inside the pore approaches the size of the BPTI molecule. Generally, the pattern of hydrogen exchange protection was uniform; however, the beta-sheet region was selectively protected on the large-pore C18 media. The beta-sheet region forms a hydrophobic core that forms early when BPTI folds in solution. This suggests that partially unfolded states possessing a native-like structure play an important role in adsorption and elution in RPC. Finally, increased contact time with the surface before elution fostered unfolding and altered chromatographic behavior considerably.


Hotze, E. M., E. M. Wilson-Kubalek, et al. (2001). "Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane beta-sheet from a prepore intermediate." J Biol Chem 276(11): 8261-8.

            Perfringolysin O (PFO), a member of the cholesterol-dependent cytolysin family of pore-forming toxins, forms large oligomeric complexes comprising up to 50 monomers. In the present study, a disulfide bridge was introduced between cysteine-substituted serine 190 of transmembrane hairpin 1 (TMH1) and cysteine-substituted glycine 57 of domain 2 of PFO. The resulting disulfide-trapped mutant (PFO(C190-C57)) was devoid of hemolytic activity and could not insert either of its transmembrane beta-hairpins (TMHs) into the membrane unless the disulfide was reduced. Both the size of the oligomer formed on the membrane and its rate of formation were unaffected by the oxidation state of the Cys(190)-Cys(57) disulfide bond; thus, the disulfide-trapped PFO was assembled into a prepore complex on the membrane. The conversion of this prepore to the pore complex was achieved by reducing the C190-C57 disulfide bond. PFO(C190-C57) that was allowed to form the prepore prior to the reduction of the disulfide exhibited a dramatic increase in the rate of PFO-dependent hemolysis and the membrane insertion of its TMHs when compared with toxin that had the disulfide reduced prior mixing the toxin with membranes. Therefore, the rate-limiting step in pore formation is prepore assembly, not TMH insertion. These data demonstrate that the prepore is a legitimate intermediate during the insertion of the large transmembrane beta-sheet of the PFO oligomer. Finally, the PFO TMHs do not appear to insert independently, but instead their insertion is coupled.


Hirakura, Y. and B. L. Kagan (2001). "Pore formation by beta-2-microglobulin: a mechanism for the pathogenesis of dialysis associated amyloidosis." Amyloid 8(2): 94-100.

            Beta-2 microglobulin (beta 2M, molecular weight 10,000) is a 99 residue immune system protein which is part of the MHC Class I complex whose role is to present antigens to T cells. beta 2M serum levels rise dramatically in renal failure, and a syndrome called "dialysis associated amyloidosis" occurs with time in a majority of hemodialysis patients who exhibit beta 2M amyloid deposits in joints, bone and other organs. beta 2M can also induce Ca++ efflux from calvariae, collagenase production, and bone resorption. We report here that beta 2M formed relatively nonselective, long-lived, voltage independent ion channels in planar phospholipid bilayer membranes at physiologically relevant concentrations. The channels were inhibited by Congo red and blocked by zinc suggesting that they exist in an aggregated beta sheet state as is common with other amyloid fibril forming peptides. Multiple single channel conductances were seen suggesting that various oligomers of beta 2M may be capable of forming channel structures. We suggest that beta 2M channel formation may account for some of the pathophysiologic effects seen in dialysis associated amyloidosis. These findings lend further weight to the "channel hypothesis" of amyloid pathogenesis.


Cui, M., J. Shen, et al. (2001). "Brownian dynamics simulations of interaction between scorpion toxin Lq2 and potassium ion channel." Biophys J 80(4): 1659-69.

            The association of the scorpion toxin Lq2 and a potassium ion (K(+)) channel has been studied using the Brownian dynamics (BD) simulation method. All of the 22 available structures of Lq2 in the Brookhaven Protein Data Bank (PDB) determined by NMR were considered during the simulation, which indicated that the conformation of Lq2 affects the binding between the two proteins significantly. Among the 22 structures of Lq2, only 4 structures dock in the binding site of the K(+) channel with a high probability and favorable electrostatic interactions. From the 4 candidates of the Lq2-K(+) channel binding models, we identified a good three-dimensional model of Lq2-K(+) channel complex through triplet contact analysis, electrostatic interaction energy estimation by BD simulation and structural refinement by molecular mechanics. Lq2 locates around the extracellular mouth of the K(+) channel and contacts the K(+) channel using its beta-sheet rather than its alpha-helix. Lys27, a conserved amino acid in the scorpion toxins, plugs the pore of the K(+) channel and forms three hydrogen bonds with the conserved residues Tyr78(A-C) and two hydrophobic contacts with Gly79 of the K(+) channel. In addition, eight hydrogen-bonds are formed between residues Arg25, Cys28, Lys31, Arg34 and Tyr36 of Lq2 and residues Pro55, Tyr78, Gly79, Asp80, and Tyr82 of K(+) channel. Many of them are formed by side chains of residues of Lq2 and backbone atoms of the K(+) channel. Thirteen hydrophobic contacts exist between residues Met29, Asn30, Lys31 and Tyr36 of Lq2 and residues Pro55, Ala58, Gly79, Asp80 and Tyr82 of the K(+) channel. These favorable interactions stabilize the association between the two proteins. These observations are in good agreement with the experimental results and can explain the binding phenomena between scorpion toxins and K(+) channels at the level of molecular structure. The consistency between the BD simulation and the experimental data indicates that our three-dimensional model of Lq2-K(+) channel complex is reasonable and can be used in further biological studies such as rational design of blocking agents of K(+) channels and mutagenesis in both toxins and K(+) channels.


Athanasiadis, A., G. Anderluh, et al. (2001). "Crystal structure of the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina." Structure (Camb) 9(4): 341-6.

            BACKGROUND: Membrane pore-forming toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a membrane, undergoes a series of transformations, leading to an active, membrane-bound form. In contrast to bacterial toxins, no structure of a pore-forming toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinatoxin II (EqtII) may unravel a novel mechanism. RESULTS: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand beta sandwich fold with a hydrophobic core and a pair of alpha helices, each of which is associated with the face of a beta sheet. CONCLUSIONS: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the beta sandwich core. This segment includes a three-turn alpha helix that lies on the surface of a beta sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the membrane pore, whereas the beta sandwich structure remains unaltered and attaches to a membrane as do other structurally related extrinsic membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in membrane pore formation.


Ahting, U., M. Thieffry, et al. (2001). "Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria." J Cell Biol 153(6): 1151-60.

            Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of approximately 350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.


Shepard, L. A., O. Shatursky, et al. (2000). "The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins." Biochemistry 39(33): 10284-93.

            Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of membrane-penetrating toxins. The CDCs form large homooligomers (estimated to be comprised of up to 50 CDC monomers) that are responsible for generating a large pore in cholesterol-containing membranes of eukaryotic cells. The assembly of the PFO cytolytic complex was examined to determine whether it forms an oligomeric prepore complex on the membrane prior to the insertion of its membrane-spanning beta-sheet. A PFO oligomeric complex was formed on liposomes at both 4 degrees C and 37 degrees C and shown by SDS-agarose gel electrophoresis to be comprised of a large, comparatively homogeneous complex instead of a distribution of oligomer sizes. At low temperature, the processes of oligomerization and membrane insertion could be resolved, and PFO was found to form an oligomer without significant membrane insertion of its beta-hairpins. Furthermore, PFO was found to increase the ion conductivity through a planar bilayer by large and discrete stepwise changes in conductance that are consistent with the insertion of a preassembled pore complex into the bilayer. The combined results of these analyses strongly support the hypothesis that PFO forms a large oligomeric prepore complex on the membrane surface prior to the insertion of its transmembrane beta-sheet.


Possani, L. D., E. Merino, et al. (2000). "Peptides and genes coding for scorpion toxins that affect ion-channels." Biochimie 82(9-10): 861-8.

            Most scorpion toxins are ligand peptides that recognize and bind to integral membrane proteins known as ion-channels. To date there are at least 202 distinct sequences described, obtained from 30 different species of scorpions, 27 from the family Buthidae and three from the family Scorpionidae. Toxins that recognize potassium and chloride channels are usually from 29 to 41 amino acids long, stabilized by three or four disulfide bridges, whereas those that recognize sodium channels are longer, 60 to 76 amino acid residues, compacted by four disulfide bridges. Toxins specific for calcium channels are scarcely known and have variable amino acid lengths. The entire repertoire of toxins, independently of their specificity, was analyzed together by computational programs and a phylogenetic tree was built showing two separate branches. The K(+) and Cl(-) channel specific toxins are clustered into 14 subfamilies, whereas those of Na(+) and Ca(2+) specific toxins comprise at least 12 subfamilies. There are clear similarities among them, both in terms of primary sequence and the main three-dimensional folding pattern. A dense core formed by a short alpha helix segment and several antiparallel beta-sheet stretches, maintained by disulfide pairing, seems to be a common structural feature present in all toxins. The physiological function of these peptides is manifested by a blockage of ion passage through the channels or by a modification of the gating mechanism that controls opening and closing of the ion pore.


Koch, S. E., I. Bodi, et al. (2000). "Architecture of Ca(2+) channel pore-lining segments revealed by covalent modification of substituted cysteines." J Biol Chem 275(44): 34493-500.

            The cysteine accessibility method was used to explore calcium channel pore topology. Cysteine mutations were introduced into the SS1-SS2 segments of Motifs I-IV of the human cardiac L-type calcium channel, expressed in Xenopus oocytes and the current block by methanethiosulfonate compounds was measured. Our studies revealed that several consecutive mutants of motifs II and III are accessible to methanethiosulfonates, suggesting that these segments exist as random coils. Motif I cysteine mutants exhibited an intermittent sensitivity to these compounds, providing evidence for a beta-sheet secondary structure. Motif IV showed a periodic sensitivity, suggesting the presence of an alpha-helix. These studies reveal that the SS1-SS2 segment repeat in each motif have non-uniform secondary structures. Thus, the channel architecture evolves as a highly distorted 4-fold pore symmetry.


Kelly, S. J. and M. J. Jedrzejas (2000). "Structure and molecular mechanism of a functional form of pneumolysin: a cholesterol-dependent cytolysin from Streptococcus pneumoniae." J Struct Biol 132(1): 72-81.

            One of the key steps in understanding human disease arising from gram-positive bacteria lies in the mechanisms of the cholesterol-dependent cytolysins (CDCs). Pneumolysin (PLY), a CDC from Streptococcus pneumoniae, is of special importance due to the severe impacts of pneumococcal infections on mortality and morbidity worldwide. We have overexpressed, purified, and characterized PLY in its fully functional complex form with the enzyme bound to its receptor activator on target cells, cholesterol. The circular dichroism studies of PLY in solution with an excess of cholesterol show a change in the far UV spectrum consistent with a decrease in the beta-sheet and an increase in the random coil structures of the enzyme. Pore formation in membranes leading to cell lysis is the functional target for this cytolysin. The sedimentation velocity and equilibrium analyses of the cholesterol-bound enzyme show hydrodynamic properties different from those of the cholesterol-free form. The soluble form of the cholesterol-free enzyme exists in solution as a mixture of monomers and dimers, whereas the cholesterol-bound form exists only as a monomer. A mechanism of formation of PLY pores in the lipid bilayer of the target cells is discussed.


Huang, H. W. (2000). "Action of antimicrobial peptides: two-state model." Biochemistry 39(29): 8347-52.

            The argument and experimental evidence are presented for a two-state model that explains the action of both helical and beta-sheet antimicrobial peptides after they bind to the plasma membranes of cells. Each peptide has two distinct physical states of binding to lipid bilayers. At low peptide-to-lipid ratios (P/L), the peptide tends to adsorb in the lipid headgroup region in a functionally inactive state. At a P/L above a threshold value P/L, the peptide forms a multiple-pore state that is lethal to a cell. The susceptibility of a cell to an antimicrobial peptide depends on the value of P/L that is determined by the lipid composition of the cell membrane. This model provides plausible explanations for the experimental findings that the susceptibility of different bacteria to a peptide is not directly correlated to its binding affinity, different peptides preferentially kill different pathogens, and peptides exhibit varying levels of lytic activity against different eukaryotic cells.


Guennoun, S. and J. D. Horisberger (2000). "Structure of the 5th transmembrane segment of the Na,K-ATPase alpha subunit: a cysteine-scanning mutagenesis study." FEBS Lett 482(1-2): 144-8.

            To study the structure of the pathway of cations across the Na, K-ATPase, we applied the substituted cysteine accessibility method to the putative 5th transmembrane segment of the alpha subunit of the Na,K-ATPase of the toad Bufo marinus. Only the most extracellular amino acid position (A(796)) was accessible from the extracellular side in the native Na,K-pump. After treatment with palytoxin, six other positions (Y(778), L(780), S(782), P(785), E(786) and L(791)), distributed along the whole length of the segment, became readily accessible to a small-size methanethiosulfonate compound (2-aminoethyl methanethiosulfonate). The accessible residues are not located on the same side of an alpha-helical model but the pattern of reactivity would rather suggest a beta-sheet structure for the inner half of the putative transmembrane segment. These results demonstrate the contribution of the 5th transmembrane segment to the palytoxin-induced channel and indicate which amino acid positions are exposed to the pore of this channel.


Sliwinski-Korell, A., H. Engelhardt, et al. (1999). "Oligomerization and structural changes of the pore-forming Pseudomonas aeruginosa cytotoxin." Eur J Biochem 265(1): 221-30.

            Pseudomonas aeruginosa produces a pathogenic factor, the 29-kDa pore-forming protein cytotoxin. Nonspecific oligomers of cytotoxin up to the hexamer, induced by oxidative crosslinking or detergent micellae, were based on intermolecular disulfide bridges. SDS induced tetramer, hexamer and mainly pentamers that were resistant to reducing conditions, indicating an additional oligomerization mechanism. Functional oligomerization after incubation with different membranes resulted in an oligomer of approximately 145 kDa that was identified as the pentamer by comparison with the SDS-induced oligomers. Covalent modification with diethylpyrocarbonate showed that histidine residues are indispensable for functional pentamerization. Pentamer formation was not influenced by the lipid composition of the liposomes tested, indicating that rising membrane fluidity did not increase oligomerization. The secondary structure of cytotoxin determined by spectroscopy is characterized by approximately 50% beta-sheet, 20% beta-turn, 10% alpha-helix and 20% remaining structure. Contact with detergent micellae or liposomes induced a reorganization of beta-structure associations, as observed by attenuated total reflection-Fourier transform infrared spectroscopy. Electron microscopy and principle component analysis of the cytotoxin monomer demonstrated a tapered molecule of 11 nm in length and a maximum width of 3.5 nm. These results classify the cytotoxin as a pore-forming toxin, rich in antiparallel beta-structure, that needs to oligomerize and inserts into membranes; it is very similar to the Staphylococcus aureus alpha-toxin.


Shimada, Y., M. Nakamura, et al. (1999). "C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin." J Biol Chem 274(26): 18536-42.

            Perfringolysin O (theta-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the beta-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in theta-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native theta-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in beta-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of theta-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.


Shai, Y. (1999). "Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides." Biochim Biophys Acta 1462(1-2): 55-70.

            Permeation of the cell membrane leading to cell death is a mechanism used by a large number of membrane-lytic peptides. Some are linear, mostly helical, and others contain one or more disulfide bonds forming beta-sheet or both beta-sheet and alpha-helix structures. They are all soluble in solution but when they reach the target membrane, conformational changes occur which let them associate with and lyse the membrane. Some lytic peptides are not cell-selective and lyse different microorganisms and normal mammalian cells, while others are specific to either type of cells. Despite extensive studies, the mode of action of membrane-lytic peptides is not fully understood and the basis for their selectivity towards specific target cells is not known. Many studies have shown that peptide-lipid interactions leading to membrane permeation play a major role in their activity. Membrane permeation by amphipathic alpha-helical peptides has been proposed to occur via one of two general mechanisms: (i) transmembrane pore formation via a 'barrel-stave' mechanism; and (ii) membrane destruction/solubilization via a 'carpet' mechanism. This review, which is focused on the different stages of membrane permeation induced by representatives of amphipathic alpha-helical antimicrobial and cell non-selective lytic peptides distinguishes between the 'carpet' mechanism, which holds for antimicrobial peptides versus the 'barrel-stave' mechanism, which holds for cell non-selective lytic peptides.


Renisio, J. G., Z. Lu, et al. (1999). "Solution structure of potassium channel-inhibiting scorpion toxin Lq2." Proteins 34(4): 417-26.

            Lq2 is a unique scorpion toxin. Acting from the extracellular side, Lq2 blocks the ion conduction pore in not only the voltage- and Ca2+ -activated channels, but also the inward-rectifier K+ channels. This finding argues that the three-dimensional structures of the pores in these K+ channels are similar. However, the amino acid sequences that form the external part of the pore are minimally conserved among the various classes of K+ channels. Because Lq2 can bind to all the three classes of K+ channels, we can use Lq2 as a structural probe to examine how the non-conserved pore-forming sequences are arranged in space to form similar pore structures. In the present study, we determined the three-dimensional structure of Lq2 using nuclear magnetic resonance (NMR) techniques. Lq2 consists of an alpha-helix (residues S10 to L20) and a beta-sheet, connected by an alphabeta3 loop (residues N22 to N24). The beta-sheet has two well-defined anti-parallel strands (residues G26 to M29 and residues K32 to C35), which are connected by a type I' beta-turn centered between residues N30 and K31. The N-terminal segment (residues Z1 to T8) appears to form a quasi-third strand of the beta-sheet.


Pajatsch, M., C. Andersen, et al. (1999). "Properties of a cyclodextrin-specific, unusual porin from Klebsiella oxytoca." J Biol Chem 274(35): 25159-66.

            The function of CymA, 1 of the 10 gene products involved in cyclodextrin uptake and metabolism by Klebsiella oxytoca, was characterized. CymA is essential for growth on cyclodextrins, but it can also complement the deficiency of a lamB (maltoporin) mutant of Escherichia coli for growth on linear maltodextrins, indicating that both cyclic and linear oligosaccharides are accepted as substrates. CymA was overproduced in E. coli and purified to apparent homogeneity. CymA is a component of the outer membrane, is processed from a signal peptide-containing precursor, and possesses a high content of antiparallel beta-sheet. Incorporation of CymA into lipid bilayers and conductance measurements revealed that it forms ion-permeable channels, which exhibit a substantial current noise. CymA-induced membrane conductance decreased considerably upon addition of alpha-cyclodextrin. Titration experiments allowed the calculation of a half-saturation constant, K(S), of 28 microM for its binding to CymA. CymA assembled in vitro to two-dimensionally crystalline tubular membranes, which, on electron microscopy, are characterized by a p1-related two-sided plane group. The crystallographic unit cell contains four monomeric CymA molecules showing a central pore. The lattice parameters are a = 16.1 nm, b = 3.8 nm, gamma = 93 degrees. CymA does not form trimeric complexes in lipid membranes and shows no tendency to trimerize in solution. CymA thus is an atypical porin with novel properties specialized to transfer cyclodextrins across the outer membrane.


Oren, Z., J. Hong, et al. (1999). "A comparative study on the structure and function of a cytolytic alpha-helical peptide and its antimicrobial beta-sheet diastereomer." Eur J Biochem 259(1-2): 360-9.

            Antimicrobial peptides which adopt mainly or only beta-sheet structures have two or more disulfide bonds stabilizing their structure. The disruption of the disulfide bonds results in most cases in a large decrease in their antimicrobial activity. In the present study we examined the effect of d-amino acids incorporation on the structure and function of a cytolytic alpha-helical peptide which acts on erythrocytes and bacteria. The influence of a single or double d-amino acid replacement in alpha-helical peptides on their structure was reported previously in 50% 2,2,2, trifluoroethanol/water [Krause et al. (1995) Anal. Chem. 67, 252-258]. Here we used Attenuated Total Reflectance Fourier-Transform Infrared (ATR-FTIR) spectroscopy and found that the predominant structure of the wild-type peptide is alpha-helix in phospholipid membranes, whereas the structure of the diastereomer is beta-sheet. However, the linear, beta-sheet diastereomer preserved its cytolytic activity on bacteria but not on erythrocytes. Previous studies have shown that the ability of antimicrobial peptides to lyse bacteria but not normal mammalian cells correlated with their ability to disintegrate preferentially negatively charged, but not zwitterionic phospholipid membranes. In contrast, the diastereomer described here disrupts zwitterionic and negatively charged vesicles with similar potencies to those of the hemolytic wild-type peptide. Interestingly, whereas addition of a positive charge to the N-terminus of the wild-type peptide (which caused a minor effect on its structure) increased activity only towards some of the bacteria tested, similar modification in the diastereomer increased activity towards all of them. Furthermore, the modified wild-type peptide preserved its potency to destabilize zwitterionic and negatively charged vesicles, whereas the modified diastereomer had a reduced potency on zwitterionic vesicles but increased potency on negatively charged vesicles. Overall our results suggest that this new class of antimicrobial diastereomeric peptides bind to the membrane in 'carpet-like' manner followed by membrane disruption and breakdown, rather than forming a transmembrane pore which interfere with the bacteria potential. These studies also open a way to design new broad-spectrum antibacterial peptides.


Miyazawa, A., Y. Fujiyoshi, et al. (1999). "Nicotinic acetylcholine receptor at 4.6 A resolution: transverse tunnels in the channel wall." J Mol Biol 288(4): 765-86.

            The nicotinic acetylcholine (ACh) receptor is the neurotransmitter-gated ion channel responsible for the rapid propagation of electrical signals between cells at the nerve/muscle synapse. We report here the 4.6 A structure of this channel in the closed conformation, determined by electron microscopy of tubular crystals of Torpedo postsynaptic membranes embedded in amorphous ice. The analysis was conducted on images recorded at 4 K with a 300 kV field emission source, by combining data from four helical families of tubes (-16,6; -18,6; -15,7; -17,5), and applying three-dimensional corrections for lattice distortions. The study extends earlier work on the same specimen at 9 A resolution.Several features having functional implications now appear with better definition. The gate of the channel forms a narrow bridge, consisting of no more than one or two rings of side-chains, across the middle portion of the membrane-spanning pore. Tunnels, framed by twisted beta-sheet strands, are resolved in the extracellular wall of the channel connecting the water-filled vestibule to the putative ACh-binding pockets. A set of narrow openings through which ions can flow are resolved between alpha-helical segments forming part of the cytoplasmic wall of the channel. It is suggested that the extracellular tunnels are access routes to the binding pockets for ACh, and that the cytoplasmic openings serve as filters to exclude anions and other impermeant species from the vicinity of the pore. Both transverse pathways are likely to be important in achieving a rapid postsynaptic response.


Menestrina, G., V. Cabiaux, et al. (1999). "Secondary structure of sea anemone cytolysins in soluble and membrane bound form by infrared spectroscopy." Biochem Biophys Res Commun 254(1): 174-80.

            Attenuated total reflection (ATR) Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of two pore-forming cytolysins from the sea anemone Stichodactyla helianthus and their interaction with lipid membranes. Frequency component analysis of the amide I' band indicated that these peptides are composed predominantly of beta structure, comprising 44-50% beta-sheet, 18-20% beta-turn, 12-15% alpha-helix, and 19-22% random coil. Upon interaction with lipid membranes a slight increase in the alpha-helical and beta-sheet structures was observed with a concomitant decrease of the unordered structure. Polarisation experiments indicated that both toxins had some disordering effect on the lipid layers. The dichroic ratio of the alpha-helical component of the membrane-bound toxin was 3.0-3.3, indicating that this element was oriented with an angle of 38 degrees-42 degrees with respect to the normal to the plane of the crystal surface, thus resulting almost parallel to the mean direction of the lipid chains.


Marvin, L., E. De, et al. (1999). "Isolation, amino acid sequence and functional assays of SGTx1. The first toxin purified from the venom of the spider scodra griseipes." Eur J Biochem 265(2): 572-9.

            A new toxin (SGTx1) was purified from the venom of the spider Scodra griseipes by a combination of gel filtration and reverse-phase chromatography. The complete amino acid sequence of SGTx1, TCRYLFGGCKTTADCCKHLACRSDGKYCAWDGTF, was established by direct automated Edman degradation, and is in perfect agreement with the molecular mass of 3775 Da found by mass spectrometry. The primary structure of SGTx1 exhibited sequence identity with other spider toxins such as hanatoxin (76%), TxP5 toxin (32%) and huwentoxin (26%). The six cysteines in the sequence suggested three disulfide bridges, the presence of which was demonstrated by mass spectrometry after dithiothreitol reduction. Analysis of secondary structure using circular dichroism spectrometry yielded more than 50% beta-sheet and about 15-20% beta-turn. The extent of the beta-content and the presence of disulfide bridges suggest a structure of interconnected beta-strands. In addition, a study of membrane/toxin interactions was carried out by reconstitution in planar lipid bilayers and by antibacterial assays. SGTx1 displays moderate pore-forming ability (conductance of about 100 pS in 1 M NaCl), but antibacterial activity was not observed against Gram-positive or Gram-negative strains. As a preliminary assay, the activity of SGTx1 was investigating using electrophysiological measurements. At 0.15 microM, SGTx1 reversibly inhibits more than 40% of outward potassium currents in rat cerebellum granular cells. This result is reminiscent with the effect described for hanatoxin extracted from the venom of Grammostola spatulata.


Li, N., M. Erman, et al. (1999). "Structure of Ustilago maydis killer toxin KP6 alpha-subunit. A multimeric assembly with a central pore." J Biol Chem 274(29): 20425-31.

            Ustilago maydis is a fungal pathogen of maize, some strains of which secrete killer toxins. The toxins are encoded by double-stranded RNA viruses in the cell cytoplasm. The U. maydis killer toxin KP6 contains two polypeptide chains, alpha and beta, having 79 and 81 amino acids, respectively, both of which are necessary for its killer activity. The crystal structure of the alpha-subunit of KP6 (KP6alpha) has been determined at 1.80-A resolution. KP6alpha forms a single domain structure that has an overall shape of an ellipsoid with dimensions 40 A x 26 A x 21 A and belongs to the alpha/beta-sandwich family. The tertiary structure consists of a four-stranded antiparallel beta-sheet, a pair of antiparallel alpha-helices, a short strand along one edge of the sheet, and a short N-terminal helix. Although the fold is reminiscent of toxins of similar size, the topology of KP6alpha is distinctly different in that the alpha/beta-sandwich motif has two right-handed betaalphabeta split crossovers. Monomers of KP6alpha assemble through crystallographic symmetries, forming a hexamer with a central pore lined by hydrophobic N-terminal helices. The central pore could play an important role in the mechanism of the killing action of the toxin.


Le, Y., S. Gagneten, et al. (1999). "Nuclear targeting determinants of the phage P1 cre DNA recombinase." Nucleic Acids Res 27(24): 4703-9.

            The Cre DNA recombinase of bacteriophage P1 has become a useful tool for genomic manipulation in mice and other eukaryotes. Because Cre is of prokaryotic origin, the 38 kDa protein has been presumed to gain access to the eukaryotic nucleus simply because it is sufficiently small to pass through the nuclear pore by passive diffusion. Instead, we show here that Cre carries nuclear targeting determinants that efficiently direct Cre entry into the nucleus of mammalian cells. Fusions of Cre with green fluorescent protein (GFP) identified two regions that are necessary for nuclear localization. Region I contains a cluster of basic amino acids that is essential for nuclear localization and which resembles a bipartite-like nuclear localization signal. Region II exhibits a beta-sheet structure with which the bipartite motif may interact. However, neither region is by itself sufficient for nuclear localization. Nuclear transport in vitro with a 98 kDa GFP-Cre fusion protein shows that Cre does not gain access to the nucleus by passive diffusion, but instead enters the nucleus by means of an energy-dependent process. Thus, Cre is one of the few prokaryotic proteins that have been shown to carry determinants that allow it to target the eukaryotic nucleus.


Kasianowicz, J. J., D. L. Burden, et al. (1999). "Genetically engineered metal ion binding sites on the outside of a Channel's transmembrane beta-barrel." Biophys J 76(2): 837-45.

            We are exploring the ability of genetically engineered versions of the Staphylococcus aureus alpha-hemolysin (alphaHL) ion channel to serve as rationally designed sensor components for analytes including divalent cations. We show here that neither the hemolytic activity nor the single channel current of wild-type alphaHL was affected by [Zn(II)] </= 1 mM. Binding sites for the divalent cations were formed by altering the number and location of coordinating side chains, e.g., histidines and aspartic acids, between positions 126 and 134, inclusive. Several mutant alphaHLs exhibited Zn(II)-induced current noise that varied with Zn(II) concentration. At a fixed divalent cation concentration, the current fluctuation kinetics depended on the analyte type, e.g., Zn(II), Cu(II), Ni(II), and Co(II). We also show that the ability of Zn(II) to change the mutant channel current suggests that the pore's topology is beta-sheet and that position 130 is near the turn at the trans mouth. Both conclusions are consistent with the crystal structure of WT-alphaHL oligomerized in detergent. Our results, in the context of the channel's crystal structure, suggest that conductance blockades were caused by Zn(II) binding to the outside surface of the pore. Thus, analyte-induced current blockades alone might not establish whether an analyte binding site is inside a pore.


Wigley, W. C., S. Vijayakumar, et al. (1998). "Transmembrane domain of cystic fibrosis transmembrane conductance regulator: design, characterization, and secondary structure of synthetic peptides m1-m6." Biochemistry 37(3): 844-53.

            Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) give rise to cystic fibrosis (CF), the most common genetic disease in the Caucasian population. CFTR is organized into five putative domains, including two that are predicted to be transmembrane and consist of six membrane-spanning segments each. CFTR mediates regulated anion transport across the apical membrane of epithelial cells. The pore through which CFTR transports its solutes is thought to be formed by some combination of the amino-terminal membrane-spanning segments. Although these sequences are predicted to be alpha-helical in secondary structure, to date, no direct structural evidence has been presented testing this hypothesis. Here, we present the biophysical characterization of six peptides (m1-m6) representing the predicted amino-terminal membrane-spanning domain of CFTR. The peptides can be incorporated into liposomes and are soluble in SDS micelles and trifluoroethanol (TFE). FTIR and CD spectroscopy indicate all six peptides adopt a stable, predominantly alpha-helical secondary structure in these environments. In contrast, peptide m6 undergoes a shift from alpha-helix to beta-sheet when dissolved in 20% methanol. Additionally, the peptides show an increase in beta-sheet in TFE, a known inducer of alpha-helices, relative to that seen in the nativelike environments. These results have implications for the folding of this complex membrane protein and suggest that the possible functional role of m6 is manifested through a shift in secondary structure.


Vetter, I. R., M. W. Parker, et al. (1998). "Crystal structure of a colicin N fragment suggests a model for toxicity." Structure 6(7): 863-74.

            BACKGROUND: Pore-forming colicins are water-soluble bacteriocins capable of binding to and translocating through the Escherichia coli cell envelope. They then undergo a transition to a transmembrane ion channel in the cytoplasmic membrane leading to bacterial death. Colicin N is the smallest pore-forming colicin known to date (40 kDa instead of the more usual 60 kDa) and the crystal structure of its membrane receptor, the porin OmpF, is already known. Structural knowledge of colicin N is therefore important for a molecular understanding of colicin toxicity and is relevant to toxic mechanisms in general. RESULTS: The crystal structure of colicin N reveals a novel receptor-binding domain containing a six-stranded antiparallel beta sheet wrapped around the 63 A long N-terminal alpha helix of the pore-forming domain. The pore-forming domain adopts a ten alpha-helix bundle that has been observed previously in the pore-forming domains of colicin A, la and E1. The translocation domain, however, does not appear to adopt any regular structure. Models for receptor binding and translocation through the outer membrane are proposed based on the structure and biochemical data. CONCLUSIONS: The colicin N-ompF system is now the structurally best-defined translocation pathway. Knowledge of the colicin N structure, coupled with the structure of its receptor, OmpF, and previously published biochemical data, limits the numerous possibilities of translocation and leads to a model in which the translocation domain inserts itself through the porin pore, the receptor-binding domain stays outside and the pore-forming domain translocates along the outer wall of the trimeric porin channel.


Tsigelny, I., S. K. Mahata, et al. (1998). "Mechanism of action of chromogranin A on catecholamine release: molecular modeling of the catestatin region reveals a beta-strand/loop/beta-strand structure secured by hydrophobic interactions and predictive of activity." Regul Pept 77(1-3): 43-53.

            A novel fragment of chromogranin A, known as 'catestatin' (bovine chromogranin A344-364), inhibits catecholamine release from chromaffin cells and noradrenergic neurons by acting as a non-competitive nicotinic cholinergic antagonist, and may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. To characterize how this activity depends on the peptide's structure, we searched for common 3-dimensional motifs for this primary structure or its homologs. Catestatin's primary structure bore significant (29-35.5% identity, general alignment score 44-57) sequence homology to fragment sequences within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, IPKM, and 2IG2. Each of these sequences exists in nature as a beta-strand/loop/beta-strand structure, stabilized by hydrophobic interactions between the beta-strands. The catestatin structure was stable during molecular dynamics simulations. The catestatin loop contains three Arg residues, whose electropositive side chains form the terminus of the structure, and give rise to substantial uncompensated charge asymmetry in the molecule. A hydrophobic moment plot revealed that catestatin is the only segment of chromogranin A predicted to contain amphiphilic beta-strand. Circular dichroism in the far ultraviolet showed substantial (63%) beta-sheet structure, especially in a hydrophobic environment. Alanine-substitution mutants of catestatin established a crucial role for the three central arginine residues in the loop (Arg351, Arg353, and Arg358), though not for two arginine residues in the strand region toward the amino-terminus. [125I]Catestatin bound to Torpedo membranes at a site other than the nicotinic agonist binding site. When the catestatin structure was 'docked' with the extracellular domain of the Torpedo nicotinic cholinergic receptor, it interacted principally with the beta and delta subunits, in a relatively hydrophobic region of the cation pore extracellular orifice, and the complex of ligand and receptor largely occluded the cation pore, providing a structural basis for the non-competitive nicotinic cholinergic antagonist properties of the peptide. We conclude that a homology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular spatial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous nicotinic cholinergic antagonist.


Shepard, L. A., A. P. Heuck, et al. (1998). "Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy." Biochemistry 37(41): 14563-74.

            Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the fluorescent dye N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water. PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound to membranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans the membrane in an antiparallel beta-sheet conformation. The alternating exposure of these residues to the hydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded, amphipathic beta-sheet in the membrane-bound PFO that creates a stable interface between the pore and the membrane. This same region packs as three short alpha-helices in the soluble, monomeric form of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition in which three alpha-helices unfold to form a membrane-spanning amphipathic beta-sheet.


Rossjohn, J., S. C. Feil, et al. (1998). "Aerolysin--a paradigm for membrane insertion of beta-sheet protein toxins?" J Struct Biol 121(2): 92-100.

            The determination of the crystal structure of the bacterial protein proaerolysin provided the first view of a pore-forming toxin constructed mainly from beta-sheet. The structure that was obtained and subsequent crystallographic and biochemical studies have together allowed us to explain how the toxin is transformed from a water-soluble dimer to a heptameric transmembrane pore. Recent discoveries of structural similarities between aerolysin and other toxins suggest that the structure/function studies we have made may prove useful in understanding the actions of a number of pore-forming proteins.


Prevost, G., D. A. Colin, et al. (1998). "[Pore-forming leukotoxins from Staphylococcus aureus: variability of the target cells and 2 pharmacological processes]." Pathol Biol (Paris) 46(6): 435-41.

            The staphylococcal bi-component leukotoxins constitute a family included in the super-family of the beta-sheet-structured pore-forming toxins. They may be produced by Staphylococcus aureus and by Staphylococcus intermedius and their target cells vary according to the molecules. The mode of action proceeds by the sequential binding of the class S proteins, then by that of the class F proteins at the surface of the membranes. Then, the activation of cellular calcium-channels precedes the pore formation which seems to be sensitive to several monovalent cations. The cell response is inflammatory and includes the neosynthesis as well as the secretion of leukotriene B4, interleukin -8, histamine. The injection of leukotoxins to rabbits generates cell chemotaxis , vasodilatation, and tissue necrosis. The association of the production of leukotoxins with clinical syndromes concerns several aspects of the pathology of S. aureus, and confers to these leukotoxins an important role of virulence factors.


Ohnishi, M., T. Hayashi, et al. (1998). "Purification and characterization of procytotoxin of Pseudomonas aeruginosa. Dimer to monomer conversion of protoxin by proteolytic activation." J Biol Chem 273(1): 453-8.

            Cytotoxin of Pseudomonas aeruginosa is a cytolytic toxin that forms a pore on the target membrane by oligomerizing into a pentamer. This toxin is produced as an inactive precursor (proCTX) and is converted to an active form by proteolytic cleavage at the C terminus. We purified proCTX to apparent homogeneity and characterized it in a comparison with the active toxin. ProCTX bound to the erythrocyte membrane but did not form an oligomer on the membrane, hence the lack of hemolytic activity in proCTX. Circular dichroic experiments showed that active and proCTX have similar beta-sheet dominant structures. Intrinsic fluorescence analysis indicated that a molecule-buried tryptophan residue(s) of proCTX was exposed to the surface of the molecule as a result of conversion to the active form. In analytical gel filtration, chemical cross-linking, and analytical ultracentrifugation experiments, dimer to monomer conversion occurred with proteolytic activation.


Hill, K., K. Model, et al. (1998). "Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins [see comment]." Nature 395(6701): 516-21.

            The mitochondrial outer membrane contains machinery for the import of preproteins encoded by nuclear genes. Eight different Tom (translocase of outer membrane) proteins have been identified that function as receptors and/or are related to a hypothetical general import pore. Many mitochondrial membrane channel activities have been described, including one related to Tim23 of the inner-membrane protein-import system; however, the pore-forming subunit(s) of the Tom machinery have not been identified until now. Here we describe the expression and functional reconstitution of Tom40, an integral membrane protein with mainly beta-sheet structure. Tom40 forms a cation-selective high-conductance channel that specifically binds to and transports mitochondrial-targeting sequences added to the cis side of the membrane. We conclude that Tom40 is the pore-forming subunit of the mitochondrial general import pore and that it constitutes a hydrophilic, approximately 22 A wide channel for the import of preproteins.


Ferreras, M., F. Hoper, et al. (1998). "The interaction of Staphylococcus aureus bi-component gamma-hemolysins and leucocidins with cells and lipid membranes." Biochim Biophys Acta 1414(1-2): 108-26.

            Staphylococcus aureus gamma-hemolysins (HlgA, HlgB and HlgC) and Panton-Valentine leucocidins (LukS-PV and LukF-PV) are bi-component toxins forming a protein family with some relationship to alpha-toxin. Active toxins are couples formed by taking one protein from each of the two subfamilies of the S-components (LukS-PV, HlgA and HlgC) and the F-components (LukF-PV and HlgB). We compared the mode of action of the six possible couples on leukocytes, red blood cells and model lipid membranes. All couples were leucotoxic on human monocytes, whereas only four couples (HlgA+HlgB, HlgC+HlgB, LukS-PV+HlgB and HlgA+LukF-PV) were hemolytic. Toxins HlgA+HlgB and HlgC+HlgB were also able to induce permeabilisation of model membranes by forming pores via oligomerisation. The presence of membrane-bound aggregates, the smallest and most abundant of which had molecular weight and properties similar to that formed by alpha-toxin, was detected by SDS-PAGE. By infrared spectroscopy in the attenuated total reflection configuration (FTIR-ATR), the secondary structure of both components and of the aggregate were determined to be predominantly beta-sheet and turn with small variations among different toxins. Polarisation experiments indicated that the structure of the membrane complex was compatible with the formation of a beta-barrel oriented perpendicularly to the plane of the membrane, similar to that of porins. The couple LukS-PV+LukF-PV was leucotoxic, but not hemolytic. When challenged against model membranes it was able to bind to the lipid vesicles and to form the aggregate with the beta-barrel structure, but not to increase calcein permeability. Thus, the pore-forming effect correlated with the hemolytic, but not with the complete leucotoxic activity of these toxins, suggesting that other mechanisms, like the interaction with endogenous cell proteins, might also play a role in their pathogenic action.


Brazier, S. P., B. Ramesh, et al. (1998). "Secondary structure analysis of the putative membrane-associated domains of the inward rectifier K+ channel ROMK1." Biochem J 335 ( Pt 2): 375-80.

            The inward rectifier K+ channels contain two putative membrane-spanning domains per subunit (M1, M2) and a 'pore' (P) region, which is similar to the H5 domain of voltage-gated K+ channels. Here we have used Fourier transform infrared (FTIR) and CD spectroscopy to analyse the secondary structures of synthetic peptides corresponding to the M1, M2 and P regions of ROMK1 in aqueous solution, in organic solvents and in phospholipid membranes. A previous CD study was unable to provide any structural data on a similar P peptide [Ben-Efraim and Shai (1997) Biophys. J. 72, 85-96]. However, our FTIR and CD spectroscopic analyses indicate that this peptide adopts an alpha-helical structure when reconstituted into dimyristoyl phosphatidylcholine vesicles and lysophosphatidyl choline (LPC) micelles as well as in trifluoroethanol (TFE) solvent. This result is in good agreement with a previous study on a peptide corresponding to the pore domain of a voltage-gated K+ channel [Haris, Ramesh, Sansom, Kerr, Srai and Chapman (1994) Protein Eng. 7, 255-262]. FTIR spectra of the M1 peptide in LPC micelles displayed a strong absorbance characteristic of an intermolecular beta-sheet structure, suggesting aggregation of the M1 peptide. Sucrose gradient centrifugation was used to separate aggregated peptide from peptide incorporated into micelles in an unaggregated manner; subsequent analysis by FTIR suggested that the M1 peptide adopted an alpha-helical structure when incorporated into phospholipid membranes. FTIR and CD spectra of the M2 peptide in phospholipids and high concentrations of TFE suggest that this peptide adopts an alpha-helical structure. The structural data obtained in these experiments have been used to propose a model for the structure of the membrane-associated core (M1-P-M2) of the inward rectifier K+ channel protein.


Scanlon, M. J., D. Naranjo, et al. (1997). "Solution structure and proposed binding mechanism of a novel potassium channel toxin kappa-conotoxin PVIIA." Structure 5(12): 1585-97.

            BACKGROUND: kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins. PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. RESULTS: The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore. Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA. CONCLUSIONS: Although the structure of PVIIA is considerably different to that of the alphaK scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.


Rossjohn, J., S. C. Feil, et al. (1997). "Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form." Cell 89(5): 685-92.

            The mechanisms by which proteins gain entry into membranes is a fundamental problem in biology. Here, we present the first crystal structure of a thiol-activated cytolysin, perfringolysin O, a member of a large family of toxins that kill eukaryotic cells by punching holes in their membranes. The molecule adopts an unusually elongated shape rich in beta sheet. We have used electron microscopy data to construct a detailed model of the membrane channel form of the toxin. The structures reveal a novel mechanism for membrane insertion. Surprisingly, the toxin receptor, cholesterol, appears to play multiple roles: targeting, promotion of oligomerization, triggering a membrane insertion competent form, and stabilizing the membrane pore.


Meunier, O., M. Ferreras, et al. (1997). "A predicted beta-sheet from class S components of staphylococcal gamma-hemolysin is essential for the secondary interaction of the class F component." Biochim Biophys Acta 1326(2): 275-86.

            Site-directed mutagenesis was performed on genes encoding HlgA and HlgC, two of the three proteins expressed from the staphylococcal y-hemolysin locus, which originate two pore-forming toxins (HlgA + HlgB, HlgC + HlgB). As related proteins, HlgA and HlgC were found to bind first to cell membranes. Amino acid substitutions concerned residues that would predictably disrupt a 13 amino acid conserved beta-sheet of the Chou and Fasman secondary structure prediction. The mutation of a threonin into an aspartic acid residue from HlgA (T28D) and from HlgC (T30D) that would break this predicted N-terminal structure lowered dramatically the biological activities on purely lipidic vesicles, erythrocytes and polymorphonuclear cells. The change in secondary structure was confirmed by Fourier Transformed Infrared spectroscopy. The binding of mutated and native proteins at the same kind of sites onto polymorphonuclear cells was evidenced with flow cytometry and fluorescein-labelled anti-class S antibodies or wild type HlgA or HlgC. However, the subsequent binding of fluorescein-labelled HlgB to membrane-bound mutated HlgA or HlgC complexes was inhibited. In conclusion, the first binding of class S components is essential for the subsequent binding of class F components, and a predicted beta-sheet seems to be at least one of the functional domains involved.


Matsuzaki, K., S. Yoneyama, et al. (1997). "Membrane permeabilization mechanisms of a cyclic antimicrobial peptide, tachyplesin I, and its linear analog." Biochemistry 36(32): 9799-806.

            Tachyplesin I (T-SS), an antimicrobial peptide from Tachypleus tridentatus, has a cyclic antiparallel beta-sheet structure maintained by two disulfide bridges. The peptide effectively permeabilizes both bacterial and artificial lipid membranes. T-Acm, a linear analog peptide with the four SH groups protected by acetamidomethyl groups, exhibits a much weaker membrane-permeabilizing activity in spite of a greater disruption of the lipid organization [Matsuzaki, K., Nakayama, M., Fukui, M., Otaka, A., Funakoshi, S., Fujii, N., Bessho, K., & Miyajima, K. (1993) Biochemistry 32, 11704-11710]. To clarify the efficient permeabilization mechanism of T-SS, we studied the interactions of both peptides with liposomes and planar lipid bilayers. The cyclic peptide capable of spanning the bilayer (ca. 3 nm length) was found to form an anion-selective pore and translocate across the bilayer coupled with the pore formation. A cis-negative transmembrane potential facilitated the pore formation compared with the cis-positive potential. In contrast, the linear peptide failed to translocate. Instead, it impaired the membrane barrier by disrupting the lipid organization with morphological changes in the vesicles.


Liemann, S., I. Bringemeier, et al. (1997). "Crystal structure of the C-terminal tetrad repeat from synexin (annexin VII) of Dictyostelium discoideum." J Mol Biol 270(1): 79-88.

            Synexin (annexin VII) is a cytosolic Ca(2+)-binding protein that promotes membrane fusion and forms voltage-regulated ion channels in artificial and natural membranes. The crystal structure of the C-terminal tetrad repeat from recombinant synexin (annexin VII) of Dictyostelium discoideum was solved to 2.45 A resolution. The protein crystallized in a dimeric form with two molecules joined face-to-face by their convex sides. Mainly hydrogen bonds and van der Waals contacts are involved in dimer formation, while not Ca2+ is bound to the conserved Ca(2+)-binding sites. The truncated N terminus is folded into a short antiparallel beta-sheet, from which the side-chain of Tyr111 penetrates sideways into the central, hydrophilic pore and may directly affect the ion channel activity. In order to investigate the structure of the missing N-terminal domain, we synthesized a 37-membered peptide of the N-terminal tail, (GYPPQQ)6G. CD and NMR studies showed a random coil conformation of the peptide in solution, suggesting for the synexin N terminus the lack of a well-ordered, three-dimensional fold.


Clancy, J. P., Z. Bebok, et al. (1997). "Purification, characterization, and expression of CFTR nucleotide-binding domains." J Bioenerg Biomembr 29(5): 475-82.

            The nucleotide binding domains (NBDs) within CFTR were initially predicted to lie in the cell cytoplasm, and to gate anion permeability through a pore that was present in membrane spanning alpha helices of the overall polypeptide. Our studies designed to characterize CFTR suggest several important features of the isolated nucleotide binding domain. NBD-1 appears to bind nucleotides with similar affinity to the full-length CFTR protein. In solution, the domain contains a high beta sheet content and self-associates into ordered polymers with molecular mass greater than 300,000 Daltons. The domain is very lipophilic, disrupts liposomes, and readily enters the planar lipid bilayer. Clinically important mutations in the domain may disrupt the nucleotide binding capabilities of the protein, either through a direct effect on the nucleotide binding site, or through effects that influence the overall folding of the domain in vitro. Finally, after expression in human epithelial cells (including epithelial cells from a CF patient), the first nucleotide binding domain targets the plasma membrane even in the absence of other constituents of full-length CFTR and mediates anion permeability in these cells.


Blanc, E., J. M. Sabatier, et al. (1997). "Solution structure of maurotoxin, a scorpion toxin from Scorpio maurus, with high affinity for voltage-gated potassium channels." Proteins 29(3): 321-33.

            Maurotoxin (MTX), purified from the scorpionid Scorpio maurus is a potent ligand for potassium channels. It shows a broad specificity as being active on Kv1.1 (Kd = 37 nM), Kv1.2 (Kd = 0.8 nM), Kv1.3 (Kd = 150 nM) voltage-gated potassium channels, as well as on small-conductance calcium-activated potassium channels. It has a unique disulfide pairing among the scorpion toxins family. The solution structure of MTX has been determined by 2D-NMR techniques, which led to the full description of its 3D conformation: a bended helix from residues 6 to 16 connected by a loop to a two-stranded antiparallel beta sheet (residues 23 to 26 and 28 to 31). The interaction of MTX with the pore region of the Kv1.2 potassium channel has been modeled according to their charge anisotropy. The structure of MTX is similar to other short scorpion toxins despite its peculiar disulfide pairing. Its interaction with the Kv1.2 channel involves a dipole moment, which guides and orients the toxin onto the pore, toward the binding site, and which thus is responsible for the specificity.


Wright, C. S. and G. Hester (1996). "The 2.0 A structure of a cross-linked complex between snowdrop lectin and a branched mannopentaose: evidence for two unique binding modes." Structure 4(11): 1339-52.

            BACKGROUND: Galanthus nivalis agglutinin (GNA), a mannose-specific lectin from snowdrop bulbs, is a tetrameric member of the family of Amaryllidaceae lectins that exhibit antiviral activity towards HIV. Its subunits are composed of three pseudo-symmetrically related beta sheet domains, each with a conserved mannose-binding site. Crystal structures of monosaccharide and disaccharide complexes of GNA have revealed that all 12 binding sites of the tetramer are functional, and that the degree of occupancy is dependent on the availability of subsidiary interactions from neighboring subunits. The complex of GNA with a branched mannopentaose ((Manalpha1,6-(alpha1, 3-Man)Man-alpha1,6-(alpha1,3-Man)Man) described here simulates a more biologically relevant complex. RESULTS: Two unique mannopentaose binding modes co-exist in the tetragonal structure (1 subunit/asymmetric unit) of the complex. In one, the conserved monosaccharide-binding pocket in domain 1 (CRD 1) is utilized for cross-linkage of twofold related GNA dimers by the outer 3,6 tri-Man arm, which alternates between two orientations consistent with crystal symmetry. Inter-linked dimers assemble helically along the 41 crystal axis forming a pore-like structure. In the second binding mode, the complete 3,6 tri-Man arm binds to an extended binding region in domain 3 (CRD 3) with subsites for each terminal Man and the internal Man positioned in the conserved monosaccharide pocket. The two remaining mannose residues are not visible in either binding mode. CONCLUSIONS: This structure provides insights into possible mechanisms of the cross-linkage that is known to occur when lectins interact with specific multivalent cell surface receptors during events such as agglutination and mitogenic stimulation. By virtue of the large number of sites available for mannose binding, GNA has multiple possibilities of forming unique lattice structures. The two distinctly different binding modes observed in this study confirm that high affinity mannose binding occurs only at the two domain sites located near dimer interfaces.


Shao, L., K. W. Kinnally, et al. (1996). "Circular dichroism studies of the mitochondrial channel, VDAC, from Neurospora crassa." Biophys J 71(2): 778-86.

            The protein that forms the voltage-gated channel VDAC (or mitochondrial porin) has been purified from Neurospora crassa. At room temperature and pH 7, the circular dichoism (CD) spectrum of VDAC suspended in octyl beta-glucoside is similar to those of bacterial porins, consistent with a high beta-sheet content. When VDAC is reconstituted into phospholipid liposomes at pH 7, a similar CD spectrum is obtained and the liposomes are rendered permeable to sucrose. Heating VDAC in octyl beta-glucoside or in liposomes results in thermal denaturation. The CD spectrum irreversibly changes to one consistent with total loss of beta-sheet content, and VDAC-containing liposomes irreversibly lose sucrose permeability. When VDAC is suspended at room temperature in octyl beta-glucoside at pH < 5 or in sodium dodecyl sulfate at pH 7, its CD spectrum is consistent with partial loss of beta-sheet content. The sucrose permeability of VDAC-containing liposomes is decreased at low pH and restored at pH 7. Similarly, the pH-dependent changes in the CD spectrum of VDAC suspended in octyl beta-glucoside also are reversible. These results suggest that VDAC undergoes a reversible conformational change at low pH involving reduced beta-sheet content and loss of pore-forming activity.


Ortells, M. O. and G. G. Lunt (1996). "A mixed helix-beta-sheet model of the transmembrane region of the nicotinic acetylcholine receptor." Protein Eng 9(1): 51-9.

            We have modelled the transmembrane region of the alpha 7 nicotinic acetylcholine receptor as a mixed alpha-helical/beta-sheet structure. The model was mainly based on the crystal structure of a pore-forming toxin, heat-labile enterotoxin. This is a pentameric protein having a central pore or channel composed of five alpha-helices, one from each of the 5 B subunits that form this pentamer. The remainder of this structure is beta-sheet, loops and a short alpha-helix, not included in the model. The model uses this channel as a template to build the transmembrane region, from M1 to the middle of M3. The remainder of M3 and M4 were built de novo as alpha-helices. Great consideration was given to labelling data available for the transmembrane region. In general terms, the shape of the model agrees very well with that obtained independently by electron microscopic analysis and the secondary structure predicted by the model is in accord with that estimated independently by Fourier transform infrared spectroscopy. The M2 helical region of the model is only slightly kinked, contrary to what is inferred from electron microscopic analysis, but has the same overall shape and form. On the membrane face of the model, the presence of deep pockets may provide the structural basis for the distinction between annular and non-annular lipid binding sites. Also, the transmembrane region is clearly asymmetric in the direction perpendicular to the membrane, and this may have strong influence on the surrounding lipid composition of each leaflet of the cytoplasmic membrane.


Moniatte, M., F. G. van der Goot, et al. (1996). "Characterisation of the heptameric pore-forming complex of the Aeromonas toxin aerolysin using MALDI-TOF mass spectrometry." FEBS Lett 384(3): 269-72.

            Aerolysin, a virulence factor secreted by Aeromonas hydrophila, is representative of a group of beta-sheet toxins that must form stable homooligomers in order to be able to insert into biological membranes and generate channels. Electron microscopy and image analysis of two-dimensional membrane crystals had previously revealed a structure with 7-fold symmetry suggesting that aerolysin forms heptameric oligomers [Wilmsen et al. (1992) EMBO J. 11, 2457-2463]. However, this unusual molecularity of the channel remained to be confirmed by an independent method since low-resolution electron crystallography had led to artefactual data for other pore-forming toxins. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to measure the mass of the aerolysin oligomer preparation. A mass of 333 850 Da was measured, fitting very well with a heptameric complex (expected mass: 332 300 Da). These results confirm the earlier evidence that the aerolysin oligomer is a heptamer and also show that MALDI-TOF mass spectrometry could be a valuable tool to study non-covalent association of proteins.


Li, J., P. A. Koni, et al. (1996). "Structure of the mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation." J Mol Biol 257(1): 129-52.

            The delta-endotoxin CytB, found in parasporal inclusions of Bacillus thuringiensis subspecies kyushuensis, is a membrane pore-forming protein which is lethal to the larvae of Dipteran insects and broadly cytolytic in vitro. The crystal structure of CytB in the protoxin form has been determined by isomorphous replacement using heavy-atom derivatives of both the wild-type protein and an engineered cysteine mutant. The atomic model comprising residues 19 to 245 and 28 bound water molecules has been refined at 2.6 angstrom resolution to a crystallographic R-factor of 19.7% and a free R-factor of 26.1%. CytB has a single domain of alpha/beta architecture but a novel connectivity comprising two outer layers of alpha-helix hairpins wrapped around a mixed beta-sheet. In the protoxin form, CytB is a dimer linked by the intertwined N-terminal strands in a continuous, 12-stranded beta-sheet. Proteolytic processing cleaves the intertwined beta-strands to release the active CytB as a monomer, as well as removing the C-terminal tail to uncover the three-layered core. The homologous toxin CytA should show the same fold. Mutations in CytA that inhibit expression map to the dimer contacts and to the tip of helix pair A-B in contact with the sheet, apparently preventing correct folding. Mutations that inhibit toxicity map to the edge of the beta-sheet adjoining the helix pair C-D and to the sheet face, while mutations on the helix surfaces have no effect. Therefore segments forming the sheet, rather than the amphiphilic but short helices, are responsible for membrane binding and pore formation. A conformational change is postulated by which the helix pair C-D peels away from the sheet to lie on the membrane surface, while the sheet region rearranges to form an oligomeric trans-membrane pore.


Chakrabarti, S. R., K. Chaudhuri, et al. (1996). "Porins of Vibrio cholerae: purification and characterization of OmpU." J Bacteriol 178(2): 524-30.

            Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity. The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded. The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units. Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit. Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein. The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium. The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU. Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated E. coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes. OmpU is also not antigenically related to OmpF. Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V. cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium. OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol. 170:2575-2583, 1988).


Wei, J. and G. D. Fasman (1995). "A poly(ethylene glycol) water-soluble conjugate of porin: refolding to the native state." Biochemistry 34(19): 6408-15.

            Porin, from Rhodabacter capsulatus, was chemically modified with methoxypoly(ethylene glycol) (m-PEG; molecular mass = 5000 Da) succinimidyl carbonate to yield methoxypoly(ethylene glycol)-porin (m-PEG-SC-Porin), as previously reported for bacteriorhodopsin [Sirokman, G., & Fasman, G. D. (1993) Protein Sci. 3, 1101-1170]. The m-poly(ethylene glycol)-porin (m-PEG-SC-Porin 50) conjugate, containing one poly(ethylene glycol) chain, was water soluble. The secondary structure of the conjugate in water was mainly random coil. Circular dichroism spectroscopy showed it was predominantly in the beta-pleated sheet structure in 0.6% octyltetraoxyethylene and 0.3 M LiCl, as was porin. A proteoliposome, containing the isolated porin conjugate, was prepared to measure permeability of the sugar stachyose. The m-PEG-SC-Porin 50 proteoliposome of porin maintained the permeability for the sugar, as did the proteoliposome of porin. The swelling rate of the conjugate versus the sugar was lower than it was for porin. This indicated that a pore in the conjugate exists but perhaps with a slightly different pore size. The refolding of the conjugate was studied by stepwise addition of trifluoroethanol (TFE) to lower the dielectric constant, simulating the insertion of porin into the membrane. An alpha-helical structure that did not exist in the native porin was formed with the m-PEG-SC-Porin 50, upon the addition of TFE, and the helicity increased with increasing concentrations of TFE. The m-PEG-SC-Porin 50 could be stepwise refolded to the native conformation, predominantly in the beta-sheet conformation, by the addition of hexafluoro-2-propanol in the 5-10% concentration range.(ABSTRACT TRUNCATED AT 250 WORDS)


Lala, A. K. and S. M. Raja (1995). "Photolabeling of a pore-forming toxin with the hydrophobic probe 2-[3H]diazofluorene. Identification of membrane-inserted segments of Staphylococcus aureus alpha-toxin." J Biol Chem 270(19): 11348-57.

            The identification of membrane-inserted segments of pore-forming soluble proteins is crucial to understanding the action of these proteins at the molecular level. A distinct member of this class of proteins is alpha-toxin, a 293-amino acid-long 33-kDa hemolytic toxin secreted by Staphylococcus aureus that can form pores in both artificial and natural membranes. We have studied the interaction of alpha-toxin with single bilayer vesicles prepared from asolectin using a hydrophobic photoactivable reagent, 2-[3H]diazofluorene ([3H]DAF) (Pradhan, D., and Lala, A. K. (1987) J. Biol. Chem. 262, 8242-8251). This reagent readily partitions into the membrane hydrophobic core and on photolysis labels the lipid and protein segments that penetrate the membrane. Current models on the mode of action of alpha-toxin indicate that, on interaction with membranes, alpha-toxin forms an oligomer, which represents the active pore. In keeping with these models, we observe that [3H]DAF photolabels the membrane-bound alpha-toxin oligomer. Cyanogen bromide fragmentation of [3H]DAF-labeled alpha-toxin gave several fragments, which were subjected to Edman degradation. We could thus sequence residues 1-19, 35-60, 114-139, 198-231, and 235-258. Radioactive analysis and phenylthiohydantoin-derivative analysis during sequencing permitted analysis of DAF insertion sites. The results obtained indicated that the N and C termini (residues 235-258) have been extensively labeled. The putative pore-forming glycine-rich central hinge region was poorly labeled, indicating that the apposing side of the lumen of the pore does not form the lipid-protein interface. The DAF labeling pattern indicated that the major structural motif in membrane-bound alpha-toxin was largely beta-sheet.


Haris, P. I. and D. Chapman (1995). "The conformational analysis of peptides using Fourier transform IR spectroscopy." Biopolymers 37(4): 251-63.

            Fourier transform infrared spectroscopy (FTIR) can be used for conformational analysis of peptides in a wide range of environments. Measurements can be performed in aqueous solution, organic solvents, detergent micelles as well as in phospholipid membranes. Information on the secondary structure of peptides can be derived from the analysis of the strong amide I band. Orientation of secondary structural elements within a lipid bilayer matrix can be determined by means of polarized attenuated total reflectance-FTIR spectroscopy. Hydrogen-deuterium exchange can be monitored by the analysis of the amide II band. This review gives some example of peptide systems studied by FTIR spectroscopy. Studies on alamethicin and alpha-aminoisobutyric acid containing peptides have shown that FTIR spectroscopy is a sensitive tool for identifying 3(10)-helical structures. Changes in the structure of the magainins upon interaction with charged lipids were detected using FTIR spectroscopy. Tachyplesin is an example of a beta-sheet containing membrane active peptide. Polarized ir spectroscopy reveals that the antiparallel beta-sheet structures of tachyplesin are oriented parallel to the membrane surface. Synthesis of peptides corresponding to functionally/structurally important regions of large proteins is becoming increasingly popular. FTIR spectroscopy has been used to analyze the structure of synthetic peptides corresponding to the ion-selective pore of the voltage-gated potassium channel. In biomembrane systems these peptides adopt a highly helical structure. Under conditions, where these peptides are aggregated the presence of some intermolecular beta-sheet structure can also be detected.


Haris, P. I., D. Chapman, et al. (1995). "A Fourier-transform infrared spectroscopic investigation of the hydrogen-deuterium exchange and secondary structure of the 28-kDa channel-forming integral membrane protein (CHIP28)." Eur J Biochem 233(2): 659-64.

            Fourier-transform infrared spectroscopy (FTIR) has been employed to investigate the structural properties of the 28-kDa channel-forming integral membrane protein (CHIP28) present in phospholipid vesicles suspended in aqueous media. This study reports the FTIR spectra of this membrane protein present in H2O and 2H2O. The secondary structure of the protein was determined and found to consist of 36% alpha-helical and 42% beta-sheet structures. These results are in close agreement with the results of a previous CD study [Van Hoek, A. N., Wiener, M., Bicknese, S., Miercke, L., Biwersi, J. & Verkman, A. S. (1993) Biochemistry 32, 11,847-11,856]. However, the results differ from those given in an FTIR analysis by the same workers who recorded FTIR spectra of the CHIP28 protein in a dehydrated state. An unusually high extent of hydrogen-deuterium exchange of the peptide groups of this protein occurs. The magnitude of the spectral changes observed upon exposure of the protein to 2H2O is greater than has been observed with any other membrane protein previously studied. Thus, over 80% of the peptide groups exchange within 5 min and the amide I band maximum shifts to low frequency by approximately 20 cm-1. This high hydrogen-deuterium exchange observed with the CHIP28 protein is consistent with the presence of an aqueous pore within the protein structure.


Forst, S., J. Waukau, et al. (1995). "Functional and regulatory analysis of the OmpF-like porin, OpnP, of the symbiotic bacterium Xenorhabdus nematophilus." Mol Microbiol 18(4): 779-89.

            The function and novel regulation of OpnP of the symbiotic/pathogenic bacterium, Xenorhabdus nematophilus was studied. In vitro pore-function analysis of purified OpnP indicated that the single-channel-conductance values were similar to that measured for the porin protein, OmpF, of Esherichia coli. Nucleotide sequence analysis revealed that the mature OpnP protein contained 348 amino acid residues and shared 55% amino acid sequence identity with OmpF. Similar to ompF, opnP mapped between asnS and aspC. The 16 transmembrane beta-sheet structures and the internal loop 3 were highly conserved, while the remaining external loop domains were more divergent. Primer extension analysis identified the start site of transcription of opnP. A sigma 70-type promoter, a perfect 20 bp OmpR-binding site, and a binding site for the antisense molecule, micF RNA, were found in the upstream region of opnP. While the overall sequence identity of the asn-opnP-aspC region was high, the intergenic region between asnS and opnP had diverged markedly. The asnS-opnP region was 313 bp shorter than the intergenic region between asnS and ompF and lacked the OmpR-binding site that is required for ompF repression by high osmolarity in E. coli. Results from osmolarity-shift experiments indicated that OpnP was not repressed by high osmolarity. It was also found that OpnP was thermally regulated.


Deb, A., D. Bhattacharyya, et al. (1995). "A 25-kDa beta-lactam-induced outer membrane protein of Vibrio cholerae. Purification and characterization." J Biol Chem 270(7): 2914-20.

            A 25-kDa outer membrane protein, induced following treatment of Vibrio cholerae cells with beta-lactam antibiotics and constituting about 8-10% of the total outer membrane proteins of beta-lactam-resistant mutants, has been purified to homogeneity. It is a basic (pI 8.5) protein rich in beta-sheet structure and is a homodimer, the monomers being held together by hydrophobic interactions. The effective hydrophobicity of the protein is low, and a large part of the protein is exposed on the surface of the outer membrane. The protein does not have beta-lactamase or autolytic activity and is not a penicillin-binding protein. The Stoke's radius of the 25-kDa protein (26 A) is comparable to the pore size of the V. cholerae OmpF-like porin. Proteoliposome swelling assay showed that the 25-kDa protein might block the pores of OmpF through which beta-lactam antibiotics normally enter the cells. Twenty-two amino acid residues from the N-terminal end of the 25-kDa protein have been sequenced, and a 32-mer oligonucleotide probe was synthesized using the amino acid residues 2-12. This probe was used to identify the gene encoding the 25-kDa protein. The beta-lactam-resistant cells are insensitive to changes in the osmolarity of the growth medium in contrast to the wild type cells which exhibit osmoregulation of OmpF and OmpC synthesis. All beta-lactam-resistant mutants examined are resistant to novobiocin.


Andersson, M., T. Curstedt, et al. (1995). "An amphipathic helical motif common to tumourolytic polypeptide NK-lysin and pulmonary surfactant polypeptide SP-B." FEBS Lett 362(3): 328-32.

            The tumour-lysing and antimicrobial polypeptide NK-lysin and the pulmonary surfactant-associated polypeptide SP-B exhibit 24% residue identities (49% similarities), including six half-cystine residues in the same disulphide bonding pattern, and similar far-UV circular dichroism spectra corresponding to 45-55% alpha-helix and 20-25% beta-sheet structures. From this, we conclude that the conformations of NK-lysin and SP-B are similar. In contrast, the functional properties of the two proteins are dissimilar: SP-B does not exhibit antibacterial activity and NK-lysin does not significantly effect phospholipid spreading at an air/water interface. Saposins, which solubilize lipids and activate lysosomal hydrolases, the pore-forming amoebapores, and parts of acid sphingomyelinase and acyloxyacylhydrolase, also share 18-27% sequence identities with NK-lysin (and SP-B), including the six conserved half-cystine residues. The inclusion of NK-lysin extends the family of saposin-like polypeptides, all members of which appear to interact with lipids. Strictly conserved structural features with implications for helix topology and lipid interactions are observed.


Shinozaki, K., K. Anzai, et al. (1994). "Ion channel activity of a synthetic peptide with a primary structure corresponding to the presumed pore-forming region of the voltage dependent potassium channel." Biochem Biophys Res Commun 198(2): 445-50.

            A 26-mer peptide of which the sequence contains the presumed pore forming region of the Shaker K+ channel (H5 region) was chemically synthesized. The peptide was found to interact and penetrate lipid membranes based on the fluorescence of Trp residues of the peptide in the presence and absence of liposomes. The secondary structure and the ion channel forming ability of the peptide were measured by CD spectroscopy and by a planar bilayer technique, respectively. The secondary structure of the peptide was composed of a mixture of an alpha-helix, beta-sheet, beta-turn, and a random coil. The content of beta-sheet structure was increased by the presence of liposomes. In planar bilayers, the peptide formed anion-selective ion channels with a larger conductance than that of the native Shaker K+ channel. These results suggest that the H5 region of the Shaker K+ channel can penetrate into lipid bilayers and form ion channel structures by itself, but it requires other structural components to reproduce the native characteristics of the K+ channel.


Nar, H., R. Huber, et al. (1994). "Three-dimensional structure of 6-pyruvoyl tetrahydropterin synthase, an enzyme involved in tetrahydrobiopterin biosynthesis." Embo J 13(6): 1255-62.

            The crystal structure of rat liver 6-pyruvoyl tetrahydropterin synthase has been solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.4% at 2.3 A resolution. 6-Pyruvoyl tetrahydrobiopterin synthase catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. The functional enzyme is a hexamer of identical subunits. The 6-pyruvoyl tetrahydropterin synthase monomer folds into a sequential, four-stranded, antiparallel beta-sheet with a 25 residue, helix-containing insertion between strands 1 and 2 at the bottom of the molecule, and a segment between strands 2 and 3 forming a pair of antiparallel helices, layered on one side of the beta-sheet. Three 6-pyruvoyl tetrahydropterin synthase monomers form an unusual 12-stranded antiparallel beta-barrel by tight association between the N- and C-terminal beta-strands of two adjacent subunits. The barrel encloses a highly basic pore of 6-12 A diameter. Two trimers associate in a head-to-head fashion to form the active enzyme complex. The substrate-binding site is located close to the trimer-trimer interface and comprises residues from three monomers: A, A' and B. A metal-binding site in the substrate-binding pocket is formed by the three histidine residues 23, 48 and 50 from one 6-pyruvoyl tetrahydropterin synthase subunit. Close to the metal, but apparently not liganding it, are residues Cys42, Glu133 (both from A) and His89 (from B), which might serve as proton donors and acceptors during catalysis.


Van Hoek, A. N., M. Wiener, et al. (1993). "Secondary structure analysis of purified functional CHIP28 water channels by CD and FTIR spectroscopy." Biochemistry 32(44): 11847-56.

            The integral membrane protein CHIP28 is an important water channel in erythrocytes and kidney tubule epithelia and is a member of a family of channel/pore proteins including the lens protein MIP26. The purposes of this study were to purify functional, delipidated CHIP28 to homogeneity and to determine secondary structure by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). CHIP28 was initially purified and delipidated by anion-exchange chromatography following solubilization of N-lauroylsarcosine-stripped erythrocyte membranes with beta-octylglucoside (OG); MIP26 was initially purified and delipidated by anion-exchange chromatography following solubilization of urea-stripped bovine lens membranes by monomyristoylphosphatidylcholine. CHIP28 (glycosylated and nonglycosylated) and MIP26 were purified further by high-performance size-exclusion chromatography, eluting in OG as apparent dimers and tetramers, respectively. Proteoliposomes reconstituted with purified CHIP28 were highly water-permeable, with an osmotic water permeability Pf of 0.04 cm/s at 10 degrees C that was inhibited by 0.1 mM HgCl2. Proteoliposomes reconstituted with MIP26 had a low Pf of 0.005 cm/s. CD spectra of CHIP28 in OG or in reconstituted proteoliposomes gave a maximum at 193 nm and minima at 208 and 222 nm. Spectral decomposition using protein basis spectra gave 40 +/- 5% alpha-helix and 43 +/- 3% beta-sheet and -turn. HgCl2 did not affect the CD spectrum of CHIP28. Attenuated total reflectance FTIR of air-dried, membrane-associated CHIP28 gave 38 +/- 5% alpha-helix and 40 +/- 4% beta-sheet and -turn by spectral decomposition of the amide I resonance. For comparison, CD of MIP26 in OG gave 49 +/- 7% alpha-helix and 32 +/- 12% beta-sheet and -turn; FTIR gave 32 +/- 8% alpha-helix and 45 +/- 6% beta-sheet and -turn. Analysis of CHIP28 and MIP26 sequence data by the generalized hydropathy method of Jahnig [Jahnig, F. (1990) Trends Biochem. Sci. 15, 93-95] predicted 39-47% alpha-helix and 15-20% beta-structures. These results establish procedures to obtain large quantities of pure CHIP28 and MIP26 in functional forms and provide evidence for multiple membrane-spanning alpha-helices or mixed alpha/beta-domains.


Unwin, N. (1993). "Nicotinic acetylcholine receptor at 9 A resolution." J Mol Biol 229(4): 1101-24.

            The nicotinic acetylcholine receptor is a cation-selective, ligand-gated ion channel, involved in signal transmission at the chemical synapse. This paper reports the three-dimensional appearance of the channel in the closed conformation, at 9 A resolution. The structure was determined by electron microscopy of tubular crystals of Torpedo postsynaptic membranes embedded in amorphous ice. The analysis was carried out by averaging data from separate images, using helical diffraction methods. The images were recorded over a wide range of defocus (7000 to 18,800 A) so that all spacings in the object were well sampled. Tubes of only one kind ((-16.6) helical family) were processed, so that the Fourier terms could be averaged directly in reciprocal space. The three-dimensional map, obtained from 26 images, resolves some elements of secondary structure within the five protein subunits. In the synaptic part of each subunit, about 30 A from the bilayer surface, there is a group of three rods that are oriented predominantly perpendicular to the plane of the bilayer and twist around each other as in a left-handed coil. These rods presumably are alpha-helices. Two of them line the entrance to the channel, and the third is on the outside. The distinctive appearance of the alpha subunits in this region suggests that the rods may be involved in forming the binding pocket for acetylcholine. In the bilayer-spanning part of each subunit there is only one rod clearly visible, which forms the wall lining the pore, and so is assumed to be the transmembrane helix, M2. This rod does not form a straight path through the lipid bilayer, but bends, or kinks, near its mid-point, where it is closest to the axis of the pore, and tilts radially outwards on either side. It is flanked on the lipid-facing sides by a continuous rim of density, which seems likely to be composed of beta-sheet. A tentative alignment is made between the three-dimensional densities and the sequence of M2, based on correlation of the appearance of the rods with a special pattern of amino acid residues in the sequence. This alignment places the charged groups at the ends of M2 symmetrically on either side of the bilayer, and a highly conserved leucine residue (Leu251 of the alpha subunit) at the level of the kink.(ABSTRACT TRUNCATED AT 400 WORDS)


Chevalier, G., H. Duclohier, et al. (1993). "Purification and characterization of protein H, the major porin of Pasteurella multocida." J Bacteriol 175(1): 266-76.

            Protein H (B. Lugtenberg, R. van Boxtel, D. Evenberg, M. de Jong, P. Storm, and J. Frik, Infect. Immun. 52:175-182, 1986) is the major polypeptide of the outer membrane of Pasteurella multocida, a bacterium pathogenic for humans and animals. We have purified this protein to homogeneity by size exclusion chromatography after selective extraction with surfactants and demonstrated its pore-forming ability after reincorporation into planar lipid bilayers. In these experiments, the current through the pores was a linear function of the applied voltage in the range of -50 to +50 mV. Voltages beyond +/- 50 mV tended to partially close the channels, giving rise to apparent negative resistances. These observations suggest that protein H channels are probably not voltage regulated in vivo. With the patch clamp technique, single-channel conductance fluctuations of 0.33 nS were recorded in 1 M KCl. Electrophoretic and circular dichroism analyses showed that protein H forms homotrimers stable in sodium dodecyl sulfate at room temperature, with a high content of beta-sheet secondary structure. Upon boiling, the trimers were fully dissociated into monomers with an increase of alpha helix and irregular structure, at the expense of beta sheets. The apparent molecular mass of fully denatured monomers ranged between 37 and 41.8 kDa, depending on the electrophoretic system used for analysis. The trimeric arrangement of protein H was confirmed by image analysis of negatively stained, two-dimensional crystal arrays. This morphological study revealed, in agreement with electrophoretical data, a trimeric structure with an overall diameter of 7.7 nm. Each monomer appeared to contain a pore with an average diameter of 1 nm. Quantitative comparisons revealed that the amino acid composition (hydropathy index of -0.40) and the N-terminal sequence (determined over 36 residues) of protein H are similar to those of bacterial general porins, notably porin P2 of Haemophilus influenzae. We conclude from this set of structural and functional data that protein H of P. multocida is a pore-forming protein related to the superfamily of the nonspecific bacterial porins.


Chen, X. J., M. K. Lee, et al. (1993). "Site-directed mutations in a highly conserved region of Bacillus thuringiensis delta-endotoxin affect inhibition of short circuit current across Bombyx mori midguts." Proc Natl Acad Sci U S A 90(19): 9041-5.

            Bacillus thuringiensis delta-endotoxins (Cry toxins) are insecticidal proteins of approximately 65 kDa in the proteolytically processed and active form. The structure of one of these toxins, CryIIIA, has been determined by Li et al. [Li, J., Carroll, J. & Ellar, D. J. (1991) Nature (London) 353, 815-821] and contains three domains. It is believed that other delta-endotoxins adopt similar three-dimensional structure. Li et al. proposed that the first domain is the membrane pore-forming domain. Previous work from our laboratory has shown that the second domain is the receptor binding domain, but the function of the third domain is unclear. Site-directed mutagenesis was used to convert the "arginine face" of one of five highly conserved regions, QRYRVRIRYAS of CryIAa (residues 525-535), to selected other residues. This sequence corresponds to the beta-sheet 17 of CryIIIA in the third domain. Mutations in the second and third arginine positions resulted in structural alterations in the protein and were poorly expressed in Escherichia coli. Toxins from genes mutated to replace lysine for the first and fourth arginines were unaltered in expression and structure, as measured by trypsin activation, CD spectra, and receptor binding, but were substantially reduced in their insecticidal properties and inhibition of short circuit current across Bombyx mori midguts. It is proposed that this region plays a role in toxin function as an ion channel.


Persechini, P. M., D. M. Ojcius, et al. (1992). "Channel-forming activity of the perforin N-terminus and a putative alpha-helical region homologous with complement C9." Biochemistry 31(21): 5017-21.

            Cytolytic lymphocytes are endowed with a pore-forming protein called perforin. Recently, a cytolytic domain was located in the first 34 residues of the perforin N-terminus. It has been proposed that the first 19 residues are composed of a 3-domain structure including a putative amphipathic beta-sheet and that the 19 residues are sufficient for cytolytic activity. This model has now been tested by synthesizing peptides covering different portions of the N-terminus, and testing their ability to lyse lipid vesicles or increase the conductance of lipid bilayers or plasma membranes. It was found that the putative beta-sheet is indispensable for lytic activity and that the first 19 residues of the N-terminus are required for optimal lytic activity but that shorter peptides, containing only 16 residues, can form pores in lipid bilayers and cell membranes. A putative amphipathic alpha-helix from the central portion of perforin, homologous to complement C9, is nonlytic to lipid vesicles, but it can form pores in lipid bilayers. Taken together, these results support the model that the perforin N-terminus is important in initial pore formation and that the putative alpha-helical domain may be involved in subsequent perforin polymerization into large pores.


Monoi, H. (1991). "Effective pore radius of the gramicidin channel. Electrostatic energies of ions calculated by a three-dielectric model." Biophys J 59(4): 786-94.

            Electrostatic calculation of the gramicidin channel is performed on the basis of a three-dielectric model in which the peptide backbone of the channel is added as a third dielectric region to the conventional two-dielectric channel model (whose pore radius is often referred to as the effective pore radius reff). A basic principle for calculating electrostatic fields in three-dielectric models is introduced. It is shown that the gramicidin channel has no unique value of reff. The reff with respect to the "self-image energy" (i.e., the image energy in the presence of a single ion) is 2.6-2.7 A, slightly depending upon the position of the ion (the least-square value over the whole length of the pore is 2.6 A). In contrast, the reff with respect to the electric potential due to an ion (and hence the reff with respect to the interaction energy between two ions) is dependent upon the distance s of separation; it ranges from 2.6 to greater than 5 A, increasing with an increase in s. However, for the purpose of rough estimation, the reff with respect to the self-image energy can also be used in calculating the electric potential and the interaction energy, because the error introduced by this approximation is an overestimation of the order of 30% at most. It is also shown that the apparent dielectric constant for the interaction between two charges depends markedly upon the positions of the charges. In the course of this study, the dielectric constant and polarizability of the peptide backbone in the beta-sheet structure is estimated to be 10 and 8.22 A3.


Goormaghtigh, E., L. Vigneron, et al. (1991). "Secondary structure of the membrane-bound form of the pore-forming domain of colicin A. An attenuated total-reflection polarized Fourier-transform infrared spectroscopy study." Eur J Biochem 202(3): 1299-305.

            The structure of the pore-forming domain of the bacterial toxin colicin A was studied by attenuated total-reflection polarized Fourier-transform infrared spectroscopy. This channel-forming fragment interacts with dimyristoylglycerophosphoglycerol (Myr2GroPGro) vesicles and forms disk-like complexes. Analysis of the shape of the amide I' band indicates that its secondary structure is not affected by the pH 5.0-7.2. However, 5-10% of the peptide amino acids adopt an alpha-helical structure upon complex formation with Myr2GroPGro, while the random-coil and beta-sheet structure contents decrease. Interestingly, the increase in alpha-helical content is essentially due to an increase in the high-frequency component of the alpha-helical domain of amide I'. The fact that only this component was 90 degrees polarized (i.e. the helix is parallel to the acyl chain) suggests that only this particular type of helix is associated with the Myr2GroPGro bilayer.


Brunen, M., H. Engelhardt, et al. (1991). "The major outer membrane protein of Acidovorax delafieldii is an anion-selective porin." J Bacteriol 173(13): 4182-7.

            The major outer membrane protein (Omp34) of Acidovorax delafieldii (formerly Pseudomonas delafieldii) was purified to homogeneity and was characterized biochemically and functionally. The polypeptide has an apparent molecular weight (Mr) of 34,000, and it forms stable oligomers at pH 9.0 in the presence of 10% octylpolyoxyethylene or 2% lithium dodecyl sulfate below 70 degrees C. The intact protein has a characteristic secondary structure composition, as revealed by Fourier transforming infrared spectroscopy (about 60% beta sheet). These features and the amino acid composition are typical for porins. The purified Omp34 is associated with 1 to 2 mol of lipopolysaccharide per mol of the monomer. Pore-forming activity was demonstrated with lipid bilayer experiments. Single-channel and selectivity measurements showed that the protein forms highly anion-selective channels. The unusual dependence of the single-channel conductance on salt concentration suggests that the porin complexes bear positive surface charges, accumulating negatively charged counterions at the pore mouth.


Batenburg, A. M., R. Brasseur, et al. (1988). "Characterization of the interfacial behavior and structure of the signal sequence of Escherichia coli outer membrane pore protein PhoE." J Biol Chem 263(9): 4202-7.

            The behavior of the chemically synthesized PhoE signal peptide and signal peptide fragments on hydrophilic-hydrophobic interfaces was studied with circular dichroism and monolayer techniques. The experimental results were compared with computer-calculated predictions of peptide structure, orientation, and molecular area. The complete signal sequence was found to aggregate in a beta-sheet structure when introduced in an aqueous environment; on the other hand, in sodium dodecyl sulfate micelles approximately 75% alpha-helical structure was observed. Assuming this to reflect the actual structure in a peptide monolayer and taking into account the orientations predicted for the fragments, the measured molecular areas suggest a looped orientation of the signal sequence with both N and C terminus in the water phase.


Pattus, F., F. Heitz, et al. (1985). "Secondary structure of the pore-forming colicin A and its C-terminal fragment. Experimental fact and structure prediction." Eur J Biochem 152(3): 681-9.

            Conformational investigations, using circular dichroism, on the pore-forming protein, colicin A (Mr 60 000), and a C-terminal bromelain fragment (Mr 20 000) were undertaken to estimate their secondary structure and to search for pH-dependent conformational changes. Colicin A and the bromelain peptide are mainly alpha-helical with an enrichment of the alpha-helical content in the C-terminal domain carrying the ionophoric activity. The non-negligible beta-sheet structure in the C-terminal domain is unstable and is easily transformed into alpha-helix upon decreasing the polarity of the solvent. No evidence of pH-dependent conformational modification, correlated with modification of colicin A activity, could be obtained. The secondary structure estimated on the basis of experimental data favoured a model in which the pore is built of a minimal number of six transmembrane alpha-helical segments. Search for such segments in the amino acid sequence of the C-terminal domain of colicin A was carried out by combining secondary structure prediction methods with hydrophobicity and hydrophobic movement calculations. Similar calculations on the C-terminal domains of colicin E1 and IB indicate a common structure of the pores formed by colicin A, E1 and IB. Only two or three putative transmembrane segments could be selected in the sequences of colicin A, IB or E1. As a result, it is concluded that the channel is probably not built by a single colicin molecule but more likely by an oligomer.


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