The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding.

Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the "TolC box," was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. IMPORTANCE: The Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.

Daring to be different: colicin N finds another way.

The mechanisms by which colicins, protein toxins produced by Escherichia coli, kill other E. coli, have become much better understood in recent years. Most colicins initially bind to an outer membrane protein receptor, and then search for a separate nearby outer membrane protein translocator that serves as a pathway into target cells. Many colicins use the outer membrane porin, OmpF, as that translocator, while using a different primary receptor. Colicin N is unique among known colicins in that only OmpF had been identified as being required for uptake of the colicin and it was presumed to somehow serve as both receptor and translocator. Genetic screens also identified a number of genes required for lipopolysaccharide (LPS) synthesis as uniquely required for killing by colicin N, but not by other colicins. Johnson et al. show that the receptor-binding domain of colicin N binds to LPS, and does not require OmpF for that binding. LPS of a minimal length is required for binding, explaining the requirement for specific elements of the LPS biosynthetic pathway. For colicin N, the receptor-binding domain does not recognize a protein, but rather the most abundant component of the outer membrane itself, LPS.

Translocation trumps receptor binding in colicin entry into Escherichia coli.

Of the steps involved in the killing of Escherichia coli by colicins, binding to a specific outer-membrane receptor was the best understood and earliest characterized. Receptor binding was believed to be an indispensable step in colicin intoxication, coming before the less well-understood step of translocation across the outer membrane to present the killing domain to its target. In the process of identifying the translocator for colicin Ia, I created chimaeric colicins, as well as a deletion missing the entire receptor-binding domain of colicin Ia. The normal pathway for colicin Ia killing was shown to require two copies of Cir: one that serves as the primary receptor and a second copy that serves as translocator. The novel Ia colicins retain the ability to kill E. coli, even in the absence of receptor binding, as long as they can translocate via their Cir translocator. Experiments to determine whether colicin M uses a second copy of its receptor, FhuA, as its translocator were hampered by precipitation of colicin M chimaeras in inclusion bodies. Nevertheless, I show that receptor binding can be bypassed for killing, as long as a translocation pathway is maintained for colicin M. These experiments suggest that colicin M, unlike colicin Ia, may normally use a single copy of FhuA as both its receptor and its translocator. Colicin E1 can kill in the absence of receptor binding, using translocation through TolC.

Border crossings: colicins and transporters.

Colicins are protein toxins produced by Escherichia coli to kill related bacteria. They must cross the target cell outer membrane (OM), and some must also cross the inner membrane (IM). To accomplish cellular import, colicins have parasitized E. coli nutrient transporters as well as IM and periplasmic proteins normally used to maintain cell wall integrity or provide energy for nutrient uptake through transporters. Colicins have evolved to use both transporters and other membrane proteins through mechanisms different from those employed in physiological substrate uptake. Extended receptor-binding domains allow some colicins to search by lateral diffusion for binding sites on their OM translocators while bound to their primary OM receptor. Transport across the OM is initiated by entry of the unstructured N-terminal translocation domain into the translocator. Periplasmic and IM networks subsequently accomplish insertion of the colicin cytotoxic domain into or across the IM.

TonB-dependent transporter FhuA in planar lipid bilayers: partial exit of its plug from the barrel.

TonB-dependent transporters (TBDTs), which transport iron-chelating siderophores and vitamin B(12) across the outer membrane of Gram-negative bacteria, share a conserved architecture of a 22-stranded ß-barrel with an amino-terminal plug domain occluding the barrel. We previously reported that we could induce TBDTs to reversibly open in planar lipid bilayers via the use of urea and that these channels were responsive to physiological concentrations of ligands. Here we report that in the presence of urea, trypsin can cleave the amino-terminal 67 residues of the plug of the TonB-dependent transporter FhuA, as assessed by gel shift and mass spectrometry assays. On the bilayer, trypsin treatment in the presence of urea resulted in the induced conductance no longer being reversed upon removal of urea, suggesting that urea opens intact FhuA channels by pulling the plug at least partly out of the barrel and that removal of the urea then allows reinsertion of the plug into the barrel. When expressed separately, the FhuA plug domain was found to be a mostly unfolded structure that was able to occlude isolated FhuA ß-barrels inserted into the membrane. Thus, although folded in the barrel, the plug need not be folded upon exiting the barrel. The rate of insertion of the ß-barrels into the membrane was tremendously increased in the presence of an osmotic gradient provided by either urea or glycerol. Negative staining electron microscopy showed that FhuA in a detergent solution formed vesicles, thus explaining why an osmotic gradient promoted the insertion of FhuA into membranes.

Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins.

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA(63))(7), in the host cell's endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA(63))(7) incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ∼100 Å-long, 15 Å-wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LF(N) (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LF(N) adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LF(N) in the normal direction but also can, at least partially, translocate LF(N) in the opposite direction.

The colicin Ia receptor, Cir, is also the translocator for colicin Ia.

Colicin Ia, a channel-forming bactericidal protein, uses the outer membrane protein, Cir, as its primary receptor. To kill Escherichia coli, it must cross this membrane. The crystal structure of Ia receptor-binding domain bound to Cir, a 22-stranded plugged beta-barrel protein, suggests that the plug does not move. Therefore, another pathway is needed for the colicin to cross the outer membrane, but no 'second receptor' has ever been identified for TonB-dependent colicins, such as Ia. We show that if the receptor-binding domain of colicin Ia is replaced by that of colicin E3, this chimera effectively kills cells, provided they have the E3 receptor (BtuB), Cir, and TonB. This is consistent with wild-type Ia using one Cir as its primary receptor (BtuB in the chimera) and a second Cir as the translocation pathway for its N-terminal translocation (T) domain and its channel-forming C-terminal domain. Deletion of colicin Ia's receptor-binding domain results in a protein that kills E. coli, albeit less effectively, provided they have Cir and TonB. We show that purified T domain competes with Ia and protects E. coli from being killed by it. Thus, in addition to binding to colicin Ia's receptor-binding domain, Cir also binds weakly to its translocation domain.

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes.

Micronutrients such as siderophore-bound iron and vitamin B(12) cross the outer membrane of gram-negative bacteria through a group of 22-stranded beta-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the beta-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ion-conducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if the cis solution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to the trans, extracellular-facing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to the trans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs.

Identification of channel-lining amino acid residues in the hydrophobic segment of colicin Ia.

Colicin Ia is a bactericidal protein of 626 amino acid residues that kills its target cell by forming a channel in the inner membrane; it can also form voltage-dependent channels in planar lipid bilayer membranes. The channel-forming activity resides in the carboxy-terminal domain of approximately 177 residues. In the crystal structure of the water-soluble conformation, this domain consists of a bundle of 10 alpha-helices, with eight mostly amphipathic helices surrounding a hydrophobic helical hairpin (helices H8-H9). We wish to know how this structure changes to form a channel in a lipid bilayer. Although there is evidence that the open channel has four transmembrane segments (H8, H9, and parts of H1 and H6-H7), their arrangement relative to the pore is largely unknown. Given the lack of a detailed structural model, it is imperative to better characterize the channel-lining protein segments. Here, we focus on a segment of 44 residues (573-616), which in the crystal structure comprises the H8-H9 hairpin and flanking regions. We mutated each of these residues to a unique cysteine, added the mutant colicins to the cis side of planar bilayers to form channels, and determined whether sulfhydryl-specific methanethiosulfonate reagents could alter the conduction of ions through the open channel. We found a pattern of reactivity consistent with parts of H8 and H9 lining the channel as alpha-helices, albeit rather short ones for spanning a lipid bilayer (12 residues). The effects of the reactions on channel conductance and selectivity tend to be greater for residues near the amino terminus of H8 and the carboxy terminus of H9, with particularly large effects for G577C, T581C, and G609C, suggesting that these residues may occupy a relatively constricted region near the cis end of the channel.

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.

Protein translocation by bacterial toxin channels: a comparison of diphtheria toxin and colicin Ia.

Regions of both colicin Ia and diphtheria toxin N-terminal to the channel-forming domains can be translocated across planar phospholipid bilayer membranes. In this article we show that the translocation pathway of diphtheria toxin allows much larger molecules to be translocated than does the translocation pathway of colicin Ia. In particular, the folded A chain of diphtheria toxin is readily translocated by that toxin but is not translocated by colicin Ia. This difference cannot be attributed to specific recognition of the A chain by diphtheria toxin's translocation pathway because the translocation pathway also accommodates folded myoglobin.

Sizing the protein translocation pathway of colicin Ia channels.

The bacterial toxin colicin Ia forms voltage-gated channels in planar lipid bilayers. The toxin consists of three domains, with the carboxy-terminal domain (C-domain) responsible for channel formation. The C-domain contributes four membrane-spanning segments and a 68-residue translocated segment to the open channel, whereas the upstream domains and the amino-terminal end of the C-domain stay on the cis side of the membrane. The isolated C-domain, lacking the two upstream domains, also forms channels; however, the amino terminus and one of the normally membrane-spanning segments can move across the membrane. (This can be observed as a drop in single-channel conductance.) In longer carboxy-terminal fragments of colicin Ia that include /=90 mV, even a 26-A stopper is translocated. Upon reduction of their disulfide bonds, all of the stoppers are easily translocated, indicating that it is the folded structure, rather than some aspect of the primary sequence, that slows translocation of the stoppers. Thus, the pathway for translocation is >/=26 A in diameter, or can stretch to this value. This is large enough for an alpha-helical hairpin to fit through.

On the interaction of colicin E3 with the ribosome.

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone

Conformational changes of colicin Ia channel-forming domain upon membrane binding: a solid-state NMR study.

Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected (13)C and (15)N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val C(alpha) signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the alpha-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by (2)H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane-water interface upon membrane binding.

Translocation of a functional protein by a voltage-dependent ion channel.

The voltage-dependent gating of the colicin channel involves a substantial structural rearrangement that results in the transfer of about 35% of the 200 residues in its pore-forming domain across the membrane. This transfer appears to represent an unusual type of protein translocation that does not depend on a large, multimeric, protein pore. To investigate the ability of this system to transport arbitrary proteins, we made use of a pair of strongly interacting proteins, either of which could serve as a translocated cargo or as a probe to detect the other. Here we show that both an 86-residue and a 134-residue hydrophilic protein inserted into the translocated segment of colicin A are themselves translocated and are functional on the trans side of the bilayer. The disparate features of these proteins suggest that the colicin channel has a general protein translocation mechanism.