Lipid-mediated inactivation of colicin E1 channels by calcium ions

Based on the model of a toroidal protein-lipid pore, the effect of calcium ions on colicin E1 channel was predicted. In electrophysiological experiments Ca2+ suppressed the activity of colicin E1 channels in membranes formed of diphytanoylphosphatidylglycerol, whereas no desorption of the protein occurred from the membrane surface. The effect of Ca2+ was not observed on membranes formed of diphytanoylphosphatidylcholine. Single-channel measurements revealed that Ca2+-induced reduction of the colicin-induced current across the negatively charged membrane was due to a decrease in the number of open colicin channels and not changes in their properties. In line with the toroidal model, the effect of Ca2+ on the colicin E1 channel-forming activity is explained by alteration of the membrane lipid curvature caused by electrostatic interaction of Ca2+ with negatively charged lipid head groups.     

On the role of lipid in colicin pore formation

Insights into the protein-membrane interactions by which the C-terminal pore-forming domain of colicins inserts into membranes and forms voltage-gated channels, and the nature of the colicin channel, are provided by data on: (i) the flexible helix-elongated state of the colicin pore-forming domain in the fluid anionic membrane interfacial layer, the optimum anionic surface charge for channel formation, and voltage-gated translocation of charged regions of the colicin domain across the membrane; (ii) structure-function data on the voltage-gated K(+) channel showing translocation of an arginine-rich helical segment through the membrane; (iii) toroidal channels formed by small peptides that involve local participation of anionic lipids in an inverted phase. It is proposed that translocation of the colicin across the membrane occurs through minimization of the Born charging energy for translocation of positively charged basic residues across the lipid bilayer by neutralization with anionic lipid head groups. The resulting pore structure may consist of somewhat short, ca. 16 residues, trans-membrane helices, in a locally thinned membrane, together with surface elements of inverted phase lipid micelles.     

Histidine 440 controls the opening of colicin E1 channels in a lipid-dependent manner

The in vitro activity of many pore-forming toxins, in particular, the rate of increase in the membrane conductance induced by the channel-forming domain (P178) of colicin E1 is maximum at an acidic pH. However, after P178 binding at acidic conditions, a subsequent pH shift from 4 to 6 on both sides of the planar bilayer lipid membrane caused a large increase in the trans-membrane current which was solely due to an increase in the number of open channels. This effect required the presence of anionic lipid. Replacing the His440 residue of P178 by alanine eliminated the pH-shift effect thereby showing that it is associated with deprotonation of this histidine residue. It was concluded that alkalinization-induced weakening of the electrostatic interactions between colicin and the membrane surface facilitates conformational changes required for the transition of membrane-bound colicin molecules to an active channel state.     

Secondary structure of the pore-forming colicin A and its C-terminal fragment. Experimental fact and structure prediction

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.     

毒素蛋白Colicin E1与磷脂膜的相互作用

多肽及蛋白质的插膜机制是目前分子生物学、细胞生物学研究中十分活跃的领域之一。本文通过荧光、圆二色等波谱学技术,深入地探讨了处于不同构象状态的毒素蛋白分子与磷脂膜作用后的构象变化。结果表明：带负电荷的磷脂膜对处于不同构象状态的ＣｏｌｉｃｉｎＥ１分子的二级结构有较强的诱导作用;这种作用是电荷依赖性的。处于不同构象状态的毒素蛋白分子在磷脂膜的诱导下均可不同程度恢复其天然状态下插膜时的构象。不同磷脂对ＣｏｌｉｃｉｎＥ１分子诱导的强弱依次为ＤＭＰＧ＞ＤＭＰＥ＞ＤＭＰＣ。ＣｏｌｉｃｉｎＥ１分子与磷脂膜的结合是紧密的,结合后的蛋白质有较强的抗变性能力。     

Uptake across the cell envelope and insertion into the inner membrane of ion channel-forming colicins in E coli.

Pore-forming colicins exert their lethal effect on E coli through formation of a voltage-dependent channel in the inner (cytoplasmic-membrane) thus destroying the energy potential of sensitive cells. Their mode of action appears to involve 3 steps: i) binding to a specific receptor located in the outer membrane; ii) translocation across this membrane; iii) insertion into the inner membrane. Colicin A has been used as a prototype of pore-forming colicins. In this review, the 3 functional domains of colicin A respectively involved in receptor binding, translocation and pore formation, are defined. The components of sensitive cells implicated in colicin uptake and their interactions with the various colicin A domains are described. The 3-dimensional structure of the pore-forming domain of colicin A has been determined recently. This structure suggests a model of insertion into the cytoplasmic membrane which is supported by model membrane studies. The role of the membrane potential in channel functioning is also discussed.     

Lipid dependence of the channel properties of a colicin E1-lipid toroidal pore

Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed "small" conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (C20) or thinner membranes, whereas it formed a novel "large" conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 A, were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid "flip-flop" and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.     

Acidic interaction of the colicin A pore-forming domain with model membranes of Escherichia coli lipids results in a large perturbation of acyl chain order and stabilization of the bilayer.

2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 136-amino-acid-residue COOH-terminal fragment of colicin A is endowed with ionophoric activity.

DNA regions encoding the various domains of a protein can be expressed as separate entities by inserting at appropriate sites a 'STOP-Shine-Dalgarno-sequence-ATG' cassette encoding a termination codon, a Shine-Dalgano sequence and an initiation codon within the structural gene. This technique has been used to obtain a 137-amino-acid-residue pore-forming protein designated DA70C comprising the final 136-amino-acid-residue COOH-terminal of colicin A preceded by an NH2-terminal methionine. Da70C was correctly expressed but poorly released to the extracellular medium. Its purification involved, as a final step, a partition in Triton X-114 thus demonstrating that hydrophobic regions are exposed in this protein. The ability of DA70C to form ion channels in planar lipid bilayers was investigated and pore properties were analyzed. The results indicate that helices 1-3 of the 204-amino-acid-residue colicin pore-forming domain (containing 10 alpha-helices) are not involved in ion conduction through the channel. However, they are important in maintaining the stability of the soluble state of the COOH-terminal domain.     

Tryptophan-dependent sensitized photoinactivation of colicin E1 channels in bilayer lipid membranes

The bacterial toxin colicin E1 is known to induce voltage-gated currents across a planar bilayer lipid membrane. In the present study, it is shown that the colicin-induced current decreased substantially upon illumination of the membrane in the presence of the photosensitizer, aluminum phthalocyanine. This effect was almost completely abolished by the singlet oxygen quencher, sodium azide. Using single tryptophan mutants of colicin E1, Trp495 was identified as the amino acid residue responsible for the sensitized photodamage of the colicin channel activity. Thus, the distinct participation of a specific amino acid residue in the sensitized photoinactivation of a defined protein function was demonstrated. It is suggested that Trp495 is critical for the translocation and/or anchoring of the colicin channel domain in the membrane.     

Induction of lipid flip-flop by colicin E1 — a hallmark of proteolipidic pore formation in liposome membranes

The addition of the channel-forming domain of colicin E1 to liposomes elicited the transmembrane diffusion (flip-flop) of lipids concomitant to the release of the fluorescent dye from liposomes. Good correlation was found between kinetic and concentration dependences of the two processes. Both the liposome leakage and the lipid flip-flop were stimulated upon alkalinization of the buffer solution after colicin binding at acidic pH. These results in combination with the analysis of the data on colicin binding to liposomes provide evidence in favor of the validity of the toroidal (proteolipidic) pore model as the mechanism of colicin channel formation.     

On the nature of the unfolded intermediate in the in vitro transition of the colicin E1 channel domain from the aqueous to the membrane phase.

The transition of the colicin E1 channel polypeptide from a water-soluble to membrane-bound state occurs in vitro at acid pH values that are associated with an unfolded channel structure whose properties qualitatively resemble those of a "molten globule," or "compact unfolded," intermediate state. The role of such a state for activity was tested by comparing the pH dependence of channel-induced solute efflux and the amplitude of the near-UV CD spectrum. The requirement of a partly unfolded state for activity was shown by the coincidence of the onset of channel activity measured for 4 different lipid compositions with the decrease in near-UV CD amplitude as a function of pH. Tertiary constraints on the 3 tryptophans of the colicin channel, assayed by the amplitude of the near-UV CD spectrum, are retained over the pH range 3-4 where channel activity could be measured and, as well, at pH 2. In addition, the tryptophan fluorescence emission spectrum is virtually unchanged over the pH range 2-6. The temperature independence of the near-UV spectrum at pH 3-6 up to 70 degrees C implies that the colicin E1 channel polypeptide is more stable than that of colicin A. A transition between 53 and 58 degrees C in the amplitude of the near-UV CD is consistent with preservation of part of the hydrophobic core in a destabilized state at pH 2. Thus, the unfolded state associated with colicin activity at acidic pH has the properties of a "compact unfolded" state, having some, but not all of the properties of a "molten globule."(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional domains of colicin A

A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed. The receptor-binding properties of each protein were also analysed. From these results, we suggest that the NH₂-terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane. The central region of colicin A (residues 173 to 336) contains the receptor-binding domain. The COOH-terminal domain (residues 389 to 592) carries the pore-forming activity.     

A very short peptide makes a voltage-dependent ion channel: the critical length of the channel domain of colicin E1

Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH-terminal fragments which have channel-forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel-forming fragment obtained by CNBr cleavage of the wild-type molecule consists of the C-terminal 152 amino acids. By the use of oligonucleotide-directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site. The smallest of the CNBr-cleaved C-terminal fragments with channel-forming activity, in planar bilayer membranes, was generated by cleavage at new Met position 428 and has 94 amino acids, whereas a 75 amino acid peptide produced by cleavage of a new Met at position 447 did not have channel activity. The NH2-terminus of the channel-forming domain of colicin E1 appears therefore to lie between residues 428 and 447. Since, however, the last six C-terminal residues of the colicin can be removed without changing activity, the number of amino acids necessary to form the channel is 88 or less. In addition, the unique Cys residue in colicin E1 was replaced by Gly, and nine mutants were then made with Cys placed at sequential locations along the peptide for eventual use as sulfhydryl attachment sites to determine the local environment of the replaced amino acid. In the course of making 21 mutants, eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule. It has been proposed previously that the conformation of the colicin E1 channel is a barrel formed from five or six alpha-helices, each having 20 amino acids spanning the membrane and two to four residues making the turn at the boundary of the membrane. Our finding that 88 amino acids can make an active channel, combined with recently reported stoichiometric evidence that the channel is a monomer excludes this model and adds significant constraints which can be used in building a molecular model of the channel.     

Ion selectivity of colicin E1: III. Anion permeability

The antibiotic protein colicin E1 forms ion channels in planar lipid bilayers that are capable of conducting monovalent organic cations having mean diameters of at least 9 Å. Polyvalent organic cations appear to be completely impermeant, regardless of size. All permeant ions, whether large or small, positively or negatively charged, are conducted by this channel at very slow rates. We have examined the permeability of colicin E1 channels to anionic probes having a variety of sizes, shapes, and charge distributions. In contrast to the behavior of cations, polyvalent as well as monovalent organic anions were found to permeate the colicin E1 channel. Inorganic sulfate was able to permeate the channel only when the pH was 4 or less, conditions under which the colicin E1 protein is predominantly in an anion-preferring conformational state. The less selective state(s) of the colicin E1 channel, observed when the pH was 5 or greater, was not permeable to inorganic sulfate. The sulfate salt of the impermeant cation Bis-T6 (N,N,N,N-tetramethyl-1,6-hexanediamine) had no effect on the single channel conductance of colicin E1 channels exposed to solutions containing 1 m NaCl at pH 5. The complete lack of blocking activity by either of these two impermeant ions indicates that both are excluded from the channel lumen. These results are consistent with our hypothesis that there is but a single location in the lumen of the colicin E1 channel where positively charged groups can be effectively hydrated. This site may coincide with the location of the energetic barrier which impedes the movement of anions.The authors wish to thank Dr. F.S. Cohen for making available unpublished data and for helpful comments. This work was supported by National Institutes of Health grant GM 37396 and by the Howard Hughes Medical Institute Undergraduate Biological Sciences Education Initiative (E.R.K.)     

The cytotoxic domain of colicin E9 is a channel-forming endonuclease

Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.     

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.     

The pore-forming domain of colicin A fused to a signal peptide: a tool for studying pore-formation and inhibition

Pore-forming colicins are plasmid-encoded bacteriocins that kill Escherichia coli and closely related bacteria. They bind to receptors in the outer membrane and are translocated across the cell envelope to the inner membrane where they form voltage-dependent ion-channels. Colicins are composed of three domains, with the C-terminal domain responsible for pore-formation. Isolated C-terminal pore-forming domains produced in the cytoplasm of E. coli are inactive due to the polarity of the transmembrane electrochemical potential, which is the opposite of that required. However, the pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of E. coli from the periplasmic side, forming a functional channel. Sp-pfColA is specifically inhibited by the colicin A immunity protein (Cai). This construct has been used to investigate colicin A channel formation in vivo and to characterise the interaction of pfColA with Cai within the inner membrane. These points will be developed further in this review.     

The channel domain of colicin A is inhibited by its immunity protein through direct interaction in the Escherichia coli inner membrane.

A bacterial signal sequence was fused to the colicin A pore-forming domain: the exported pore-forming domain was highly cytotoxic. We thus introduced a cysteine-residue pair in the fusion protein which has been shown to form a disulfide bond in the natural colicin A pore-forming domain between alpha-helices 5 and 6. Formation of the disulfide bond prevented the cytotoxic activity of the fusion protein, presumably by preventing the membrane insertion of helices 5 and 6. However, the cytotoxicity of the disulfide-linked pore-forming domain was reactivated by adding dithiothreitol into the culture medium. We were then able to co-produce the immunity protein with the disulfide linked pore-forming domain, by using a co-immunoprecipitation procedure, in order to show that they interact. We showed both proteins to be co-localized in the Escherichia coli inner membrane and subsequently co-immunoprecipitated them. The interaction required a functional immunity protein. The immunity protein also interacted with a mutant form of the pore-forming domain carrying a mutation located in the voltage-gated region: this mutant was devoid of pore-forming activity but still inserted into the membrane. Our results indicate that the immunity protein interacts with the membrane-anchored channel domain; the interaction requires a functional membrane-inserted immunity protein but does not require the channel to be in the open state.     

pH-dependent membrane fusion is promoted by various colicins.

The ability of colicin A, a bacteriocin produced by some Enterobacteriaceae, to fuse phospholipid vesicles at acidic pH, was demonstrated by electron microscopy and resonance energy transfer. The fusion depends on protein concentration and on the nature of the phospholipids. Vesicles, prepared from Escherichia coli phospholipids, fused one or more rounds at pH 4.5 upon addition of stoichiometric amounts of colicin A. Fusion was not only induced by pore-forming colicins (E1, K) but also by colicins that contain nuclease activities (E2, E3). By recombinant DNA technology it is shown that the first glycine-rich 70 NH2-terminal amino acids and, most probably, the extreme COOH-terminal end of colicin A are involved in the fusion activity of the protein. The physiological relevance of this property of colicins is discussed.