Unfolding pathway of the colicin E1 channel protein on a membrane surface

The channel-forming domain of colicin E1 is composed of a soluble helical bundle which, upon membrane binding, unfolds to form an extended, two-dimensional helical net in the membrane interfacial layer. To characterize the pathway of unfolding of the protein and the structure of the surface-bound intermediate, the time-course of intra-protein distance changes and unfolding on a millisecond time-scale were determined from the kinetics of changes in the efficiency of fluorescence resonance energy transfer, and of the donor-acceptor overlap integral, between each of six individual tryptophan residues and a Cys-conjugated energy transfer acceptor (C509-AEDANS). Comparison of the rate constants revealed the following order of events associated with unfolding of the protein at the membrane surface: (A) movement of the hydrophobic core helices VIII-IX, coincident with a small change in Trp-Cys509 distances of the outer helices; (B) unfolding of surface helices in the helical bundle in the order: helix I, helices III, IV, VI, VII, and helix V; (C) a slow (time-scale, seconds) condensation of the surface-bound helices. The rate of protein unfolding events increased with increasing anionic lipid content. Unfolding did not occur below the lipid thermal phase transition, indicating that unfolding requires mobility in the interfacial layer. The structure of the two-dimensional membrane-bound intermediate in the steady-state was inferred to consist of a quasi-circular arrangement of eight helices embedded in the membrane interfacial layer and anchored by the hydrophobic helical hairpin. The pathway of unfolding of the colicin channel at the membrane surface, catalyzed by electrostatic and hydrophobic forces, is the first described for a membrane-active protein. It is proposed that the pathway and principles described for the colicin protein are relevant to membrane protein import.     

Initial steps of colicin E1 import across the outer membrane of Escherichia coli

Data suggest a two-receptor model for colicin E1 (ColE1) translocation across the outer membrane of Escherichia coli. ColE1 initially binds to the vitamin B(12) receptor BtuB and then translocates through the TolC channel-tunnel, presumably in a mostly unfolded state. Here, we studied the early events in the import of ColE1. Using in vivo approaches, we show that ColE1 is cleaved when added to whole cells. This cleavage requires the presence of the receptor BtuB and the protease OmpT, but not that of TolC. Strains expressing OmpT cleaved ColE1 at K84 and K95 in the N-terminal translocation domain, leading to the removal of the TolQA box, which is essential for ColE1's cytotoxicity. Supported by additional in vivo data, this suggests that a function of OmpT is to degrade colicin at the cell surface and thus protect sensitive E. coli cells from infection by E colicins. A genetic strategy for isolating tolC mutations that confer resistance to ColE1, without affecting other TolC functions, is also described. We provide further in vivo evidence of the multistep interaction between TolC and ColE1 by using cross-linking followed by copurification via histidine-tagged TolC. First, secondary binding of ColE1 to TolC is dependent on primary binding to BtuB. Second, alterations to a residue in the TolC channel interfere with the translocation of ColE1 across the TolC pore rather than with the binding of ColE1 to TolC. In contrast, a substitution at a residue exposed on the cell surface abolishes both binding and translocation of ColE1.     

Determination of membrane protein topology by red-edge excitation shift analysis: application to the membrane-bound colicin E1 channel peptide

A new approach for the determination of the bilayer location of Trp residues in proteins has been applied to the study of the membrane topology of the channel-forming bacteriocin, colicin E1. This method, red-edge excitation shift (REES) analysis, was initially applied to the study of 12 single Trp-containing channel peptides of colicin E1 in the soluble state in aqueous medium. Notably, REES was observed for most of the channel peptides in aqueous solution upon low pH activation. The extent of REES was subsequently characterized using a model membrane system composed of the tripeptide, Lys-Trp-Lys, bound to dimyristoyl-sn-glycerol-3-phosphatidylserine liposomes. Subsequently, data accrued from the model peptide-lipid system was used to interpret information obtained on the channel peptides when bound to dioleoyl-sn-glycerol-3-phosphatidylcholine/dioleoyl-sn-glycerol-3-phosphatidylglycerol membrane vesicles. The single Trp mutant peptides were divided into three categories based on the change in the REES values observed for the Trp residues when the peptides were bound to liposomes as compared to the REES values measured for the soluble peptides. F-404W, F-413W, F-443W, F-484W, and W-495 peptides exhibited small and/or insignificant REES changes (ΔREES) whereas W-424, F-431W, and Y-507W channel peptides possessed modest REES changes (3 nm≤ΔREES≤7 nm). In contrast, wild-type, Y-367W, W-460, Y-478W, and I-499W channel peptides showed large ΔREES values upon membrane binding (7 nm<ΔREES≤12 nm). The REES data for the membrane-bound structure of the colicin E1 channel peptide proved consistent with previous data for the topology of the closed channel state, which lends further credence to the currently proposed channel model. In conclusion, the REES method provides another source of topological data for assignment of the bilayer location for Trp residues within membrane-associated proteins; however, it also requires careful interpretation of spectral data in combination with structural information on the proteins being investigated.     

Determination of membrane protein topology by red-edge excitation shift analysis: application to the membrane-bound colicin E1 channel peptide

A new approach for the determination of the bilayer location of Trp residues in proteins has been applied to the study of the membrane topology of the channel-forming bacteriocin, colicin E1. This method, red-edge excitation shift (REES) analysis, was initially applied to the study of 12 single Trp-containing channel peptides of colicin E1 in the soluble state in aqueous medium. Notably, REES was observed for most of the channel peptides in aqueous solution upon low pH activation. The extent of REES was subsequently characterized using a model membrane system composed of the tripeptide, Lys-Trp-Lys, bound to dimyristoyl-sn-glycerol-3-phosphatidylserine liposomes. Subsequently, data accrued from the model peptide-lipid system was used to interpret information obtained on the channel peptides when bound to dioleoyl-sn-glycerol-3-phosphatidylcholine/dioleoyl-sn-glycerol-3-phosphatidylglycerol membrane vesicles. The single Trp mutant peptides were divided into three categories based on the change in the REES values observed for the Trp residues when the peptides were bound to liposomes as compared to the REES values measured for the soluble peptides. F-404 W, F-413 W, F-443 W, F-484 W, and W-495 peptides exhibited small and/or insignificant REES changes (Delta REES) whereas W-424, F-431 W, and Y-507 W channel peptides possessed modest REES changes (3 nm< or = Delta REES< or = 7 nm). In contrast, wild-type, Y-367 W, W-460, Y-478 W, and I-499 W channel peptides showed large Delta REES values upon membrane binding (7 nm< Delta REES< or =12 nm). The REES data for the membrane-bound structure of the colicin E1 channel peptide proved consistent with previous data for the topology of the closed channel state, which lends further credence to the currently proposed channel model. In conclusion, the REES method provides another source of topological data for assignment of the bilayer location for Trp residues within membrane-associated proteins; however, it also requires careful interpretation of spectral data in combination with structural information on the proteins being investigated.     

Voltage-dependent,monomeric channel activity of colicin E1 in artificial membrane vesicles

Summary The dependence of colicin channel activity on membrane potential and peptide concentration was studied in large unilamellar vesicles using colicin E1, its COOH-terminal thermolytic peptide and other channel-forming colicins. Channel activity was assayed by release of vesicle-entrapped chloride, and could be detected at a peptide: lipid molar ratio as low as 10^–7. The channel activity was dependent on the magnitude of atrans-negative potassium diffusion potential, with larger potentials yielding faster rates of solute efflux. For membrane potentials greater than –60mV (K _in ⁺ /K _out ⁺ 10), addition of valinomycin resulted in a 10-fold increase in the rate of Cl^– efflux. A delay in Cl^– efflux observed when the peptide was added to vesicles in the presence of a membrane potential implied a potential-independent binding-insertion mechanism. The initial rate of Cl^– efflux was about 1% of the single-channel conductance, implying that only a small fraction of channels were initially open, due to the delay or latency of channel formation known to occur in planar bilayers.The amount of Cl^– released as a function of added peptide increased monotonically to a concentration of 0.7 ng peptide/ml, corresponding to release of 75% of the entrapped chloride. It was estimated from this high activity and consideration of vesicle number that 50–100% of the peptide molecules were active. The dependence of the initial rate of Cl^– efflux on peptide concentration was linear to approximately the same concentration, implying that the active channel consists of a monomeric unit.     

Toward elucidating the membrane topology of helix two of the colicin E1 channel domain

The membrane-bound closed state of the colicin E1 channel domain was investigated by site-directed fluorescence labeling using a bimane fluorophore attached to each single cysteine residue within helix 2 of each mutant protein. The fluorescence properties of the bimane fluorophore were measured for the membrane-associated form of the closed channel and included fluorescence emission maximum, fluorescence anisotropy, apparent polarity, surface accessibility, and membrane bilayer penetration depth. The fluorescence data show that helix 2 is an amphipathic alpha-helix that is situated parallel to the membrane surface, but it is less deeply embedded within the bilayer interfacial region than is helix 1 in the closed channel. A least squares fit of the various data sets to a harmonic wave function indicated that the periodicity and angular frequency for helix 2 in the membrane-bound state are typical for an amphipathic alpha-helix (3.8 +/- 0.1 residues per turn and 94 +/- 4 degrees, respectively) that is located at an interfacial region of a membrane bilayer. Dual quencher analysis also revealed that helix 2 is peripherally membrane associated, with one face of the helix dipping into the interfacial region of the lipid bilayer and the other face projecting outwardly into the aqueous solvent. Finally, our data show that helices 1 and 2 remain independent helices upon membrane association with a short connector link (Tyr(363)-Gly(364)) and that short amphipathic alpha-helices participate in the formation of a lipid-dependent, toroidal pore for this colicin.     

Dynamic properties of the colicin E1 ion channel

Abstract The mechanism of channel formation and action of channel-forming colicins is a paradigm for the study of dynamic aspects of membrane-protein interactions. The following experimental results concerning interaction of the colicin E1 channel domain with target membranes, in vitro and in vivo, are discussed: (1) the nature of the translocation-competent state of the channel-forming domain; (2) unfolding of the colicin channel peptide during in vitro binding and anchoring of the channel to liposome membranes at acidic pH; (3) reversal of channel peptide binding to liposomes by an alkaline-directed pH shift; (4) voltage-driven translocation and gating of the ion channel, discussed in the context of a four-helix model for a monomeric channel; (5) rescue of colicin-treated cells by high levels of external K⁺; (6) trypsin rescue of cells depolarized by the colicin ion channel; and (7) interaction of the channel domain with its immunity protein.     

Structure and dynamics of the colicin E1 channel

The toxin-like and bactericidal colicin E1 molecule is of interest for problems of toxin action, polypeptide translocation across membranes, voltage-gated channels, and receptor function. Colicin E1 binds to a receptor in the outer membrane and is translocated across the cell envelope to the inner membrane. Import of the colicin channel-forming domain into the inner membrane involves a translocation-competent intermediate state and a membrane potential-dependent movement of one third to one half of the channel peptide into the membrane bilayer. The voltage-gated channel has a conductance sufficiently large to depolarize the Escherichia coli cytoplasmic membrane. Amino acid residues that affect the channel ion selectivity have been identified by site-directed mutagenesis. The colicin E1 channel is one of a few membrane proteins whose secondary structures in the membrane, predominantly alpha-helix, have been determined by physico-chemical techniques. Hypothesis for the identity of the trans-membrane helices, and the mechanism of binding to the membrane, are influenced by the solved crystal structure of the soluble colicin A channel peptide. The protective action of immunity protein is a unique aspect of the colicin problem, and information has been obtained, by genetic techniques, about the probable membrane topography of the imm gene product.     

Adventures in membrane protein topology. A study of the membrane-bound state of colicin E1.

The molecular aggregate size of the closed state of the colicin E1 channel was determined by fluorescence resonance energy transfer experiments involving a fluorescence donor (three tryptophans, wild-type protein) and a fluorescence acceptor (5-(((acetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (AEDANS), Trp-deficient protein). There was no evidence of energy transfer between the donor and acceptor species when bound to membrane large unilamellar vesicles. These experiments led to the conclusion that the colicin E1 channel is monomeric in the membrane-bound closed channel state. Experiments were also conducted to study the membrane topology of the closed colicin channel in membrane large unilamellar vesicles using acrylamide as the membrane-impermeant, nonionic quencher of tryptophan fluorescence in a battery of single tryptophan mutant proteins. Furthermore, additional fluorescence parameters, including fluorescence emission maximum, fluorescence quantum yield, and fluorescence decay times, were used to assist in mapping the topology of the closed channel. Results suggest that the closed channel comprises most of the polypeptide of the channel domain and that the hydrophobic anchor domain does not transverse the membrane bilayer but nonetheless is deeply embedded within the hydrocarbon core of the membrane. Finally, a model is proposed which features at least two states that are in rapid equilibrium with each other and in which one state is more heavily populated than the other.     

A DNA segment within the colicin E1 structural gene on ColE1 affecting immunity to colicin

Summary Insertion of DNA at the EcoRI site of ColE1 results in increase of immunity to colicin killing in E. coli harboring such recombinant ColE1 plasmid as compared to E. coli (ColE1). This effect is neither due to cis or trans interactions originating from the inserted foreign DNA fragment, nor to changes in plasmid copy number. This defect in the immunity mechanism is not trans complemented for by wild type ColE1. Increase in immunity can also be obtained by deleting a DNA segment from the ColE1 genome. This segment is 120 bp left to the EcoRI site within the colicin structural gene. It is concluded that the structure of DNA per se, around the EcoRI site, within colicin structural gene, is the structure which affects immunity expression.     

Synthesis and functioning of the colicin E1 lysis protein: comparison with the colicin A lysis protein.

The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either [35S]cysteine or [3H]lysine. This 3-kDa protein was acylated, as shown by [2-3H]glycerol labeling, and seemed to correspond to the mature CelA protein. The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein. In contrast to Cal, no intermediate form was detected for CelA, no signal peptide accumulated, and no modified precursor form was observed after globomycin treatment. Thus, the rate of synthesis would not be specific to lysis proteins. Solubilization in sodium dodecyl sulfate of the mature forms of both CelA and Cal varied similarly at the time of colicin release, indicating a change in lysis protein structure. This particular property would play a role in the mechanism of colicin export. The accumulation of the signal peptide seems to be a factor determining the toxicity of the lysis proteins since CelA provoked less cell damage than Cal. Quasi-lysis and killing due to CelA were higher in degP mutants than in wild-type cells. They were minimal in pldA mutants.     

Identification of a voltage-responsive segment of the potential-gated colicin E1 ion channel.

The voltage dependence of channel activity of the bactericidal protein colicin E1 was found to be correlated with insertion into the membrane bilayer of a specific segment of the 178-residue COOH-terminal thermolytic colicin channel peptide. The insertion into the bilayer was detected by an increase in labeling by one of two different lipophilic photoaffinity probes or by a decrease in iodination of peptide tyrosines from the external solution. Imposition of a potassium diffusion potential of -100 mV resulted in an increase of 35-60% in the labeling of the peptide by the lipophilic probe in the bilayer and a concomitant decrease in labeling of Tyr residues in the peptide by the iodination reagent in the external solution. The change in labeling decreased upon dissipation of the membrane potential with a half-time of about 1 min. The labeling change was localized to a 36-residue peptide segment bounded by alanine-425 and by tryptophan-460. This segment containing seven positively charged residues at low pH is a voltage-sensitive region that inserts into the membrane bilayer when the channel is turned on by the potential and is extruded from it when the voltage is removed and the channel is turned off.     

Mechanism of export of colicin E1 and colicin E3.

The mechanism of export of colicins E1 and E3 was examined. Neither colicin E1, colicin E3, Nor colicin E3 immunity protein appears to be synthesized as a precursor protein with an amino-terminal extension. Instead, the colicins, as well as the colicin E3 immunity protein, appear to leave the cells where they are made, long after their synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells. Induction of ColE3-containing cells with mitomycin C leads to actual lysis of those cells, as some time after synthesis of the colicin E3 and its immunity protein has been completed. Induction of ColE1-containing cells results in increased permeability of the cells, but not in actual lysis, and most of the colicin E1 produced never leaves the producing cells. Intracellular proteins such as elongation factor G can be found outside of colicinogenic cells after mitomycin C induction, along with the colicin. Until substantial increases in permeability occur, most of the colicin remains cell associated, in the soluble cytosol, rather than in a membrane-associated form.     

Maturation and localization of the TolB protein required for colicin import.

The tolB gene has been shown previously to encode two proteins of 47.5 kDa (TolB) and 43 kDa (TolB*). To explain the presence of these two forms, two hypotheses have been proposed: TolB might be posttranslationally processed to TolB*, or an internal in-frame translation initiation resulting in TolB* may occur (S. K. Levengood and R. E. Webster, J. Bacteriol. 171:6600-6609, 1989). To address this question, TolB was tagged by inserting in its C-terminal region an epitope recognized by monoclonal antibody 1C11 without altering the function of TolB. It was then demonstrated that the functional protein corresponded to TolB*, the mature periplasmic protein, and that TolB was its precursor form, which was observed only when the protein was overexpressed. These two forms were purified by immunoprecipitation, and their N-terminal sequences were determined. An antibody directed against TolB was raised, which confirmed the results obtained with the tagged TolB.     

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.     

On the nature of the structural change of the colicin E1 channel peptide necessary for its translocation-competent state

Acidic pH conditions required in vitro for membrane binding and activity of the channel-forming colicin E1 resulted in an increased susceptibility to proteases of the 178-residue thermolytic channel peptide, an increased accessibility to acrylamide of a fluorescence probe linked to cysteine-505 of the peptide, and an increased partition into nonionic detergent. The structural change in the peptide sensed by the fluorescence probe caused by a transition from pH 6.0 to 3.5 occurred in less than 1 s. The presence of low concentrations of detergents (0.001% SDS or 0.44% octyl beta-D-glucoside) or urea (0.2 M) at pH 6 or 4 also increased the susceptibility of the channel peptide to proteases. The increase in protease susceptibility and acrylamide accessibility at low pH, as well as partition of the peptide into nonionic detergent, suggested that acidic pH or the detergents might cause peptide unfolding. However, the hydrodynamic radius of the channel peptide at pH 6, 21-23 A, was not changed at pH 3.5 or by detergents or urea under conditions that increased the susceptibility of the peptide to protease. The activity of the channel peptide at pH 6 measured with liposomes and planar bilayers, which was a factor of 10(3)-10(4) smaller than that at pH 4, was increased by 2-4 orders of magnitude by 0.001% SDS or 0.44% octyl beta-D-glucoside, with an additional small increment of activity on planar bilayers caused by 0.01% SDS. A small increase in Stokes radius of the peptide in the presence of SDS could be detected that was approximately correlated with increased activity.     

Multistep assembly of the protein import channel of the mitochondrial outer membrane

Proteins targeted to mitochondria are transported into the organelle through a high molecular weight complex called the translocase of the outer mitochondrial membrane (TOM). At the core of this machinery is a multisubunit general import pore (GIP) of 400 kDa. Here we report the assembly of the yeast GIP that involves two successive intermediates of 250 kDa and 100 kDa. The precursor of the channel-lining Tom40 is first targeted to the membrane via the receptor proteins Tom20 and Tom22; it then assembles with Tom5 to form the 250 kDa intermediate exposed to the intermembrane space. The 250 kDa intermediate is followed by the formation of the 100 kDa intermediate that associates with Tom6. Maturation to the 400 kDa complex occurs by association of Tom7 and Tom22. Tom7 functions by promoting both the dissociation of the 400 kDa complex and the transition from the 100 kDa intermediate to the mature complex. These results indicate that the dynamic conversion between the 400 kDa complex and the 100 kDa late intermediate allows integration of new precursor subunits into pre-existing complexes.     

Folded state of the integral membrane colicin E1 immunity protein in solvents of mixed polarity

The colicin E1 immunity protein (ImmE1), a 13.2-kDa hydrophobic integral membrane protein localized in the Escherichia coli cytoplasmic membrane, protects the cell from the lethal, channel-forming activity of the bacteriocin, colicin E1. Utilizing its solubility in organic solvents, ImmE1 was purified by 1-butanol extraction of isolated membranes, followed by gel filtration and ion-exchange chromatography in a chloroform/methanol/H(2)O (4:4:1) solvent system. Circular dichroism analysis indicated that the alpha-helical content of ImmE1 is approximately 80% in 1-butanol or 2,2,2-trifluoroethanol, consistent with a previous membrane-folding model with three extended hydrophobic transmembrane helical domains, H1-H3. Each of these extended hydrophobic domains contains a centrally located single Cys residue that could be used as a probe of protein structure. The presence of tertiary structure of purified ImmE1 in a solvent of mixed polarity, chloroform/methanol/H(2)O (4:4:1) was demonstrated by (i) the constraints on Tyr residues shown by the amplitude of near-UV circular dichroism spectra in the wavelength interval, 270-285 nm; (ii) the correlation between the near-UV Tyr CD spectrum of single and double Cys-to-X mutants of the Imm protein and their in vivo activity; (iii) the upfield shift of methyl groups in a 1D NMR spectrum, a 2D- HSQC NMR spectrum of ImmE1 in the mixed polarity solvent mixture, and a broadening and disappearance of the indole (1)H proton resonance from Trp94 in H3 by a spin label attached to Cys16 in the H2 hydrophobic domain; (iv) near-UV circular dichroism spectra with a prominent ellipticity band centered at 290 nm from a single Trp inserted into the extended hydrophobic domains. It was concluded that the colicin E1 immunity protein adopts a folded conformation in chloroform/methanol/H(2)O (4:4:1) that is stabilized by helix-helix interactions. Analysis of the probable membrane folding topology indicated that several Tyr residues in the bilayer region of the three transmembrane helices could contribute to the near-UV CD spectrum through helix-helix interactions.