The N-terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step

Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli : the Tol system for group A colicins and the TonB system for group B colicins. The N-terminal domain of colicins is involved in the translocation step. To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N-terminal domains of colicin E3 (group A) and colicin B (group B). Producing cells became specifically tolerant to entire extracellular colicins of the same group. The periplasmic N-terminal domains therefore compete with entire colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane. In vivo cross-linking and co-immunoprecipitation experiments in cells producing the colicin E3 N-terminal domain demonstrated the existence of a 120 kDa complex containing the colicin domain and TolB. After in vitro cross-linking experiments with these two purified proteins, a 120 kDa complex was also obtained. This suggests that the complex obtained in vivo contains exclusively TolB and the colicin E3 domain. The N-terminal domain of a translocation-defective colicin E3 mutant was found to no longer interact with TolB. Hence, this interaction must play an important role in colicin E3 translocation.     

Clusters in an intrinsically disordered protein create a protein-binding site: the TolB-binding region of colicin E9

The 61-kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins and kills them by hydrolyzing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies [Collins et al. (2002) J. Mol. Biol. 318, 787-904; MacDonald et al. (2004), J. Biomol. NMR 30, 81-96] have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is intrinsically disordered and contains clusters of interacting side chains. To further define the properties of this region of colicin E9, we have investigated the effects on the dynamical and TolB-binding properties of three mutations of colicin E9 that inactivate it as a toxin. The mutations were contained in a fusion protein consisting of residues 1-61 of colicin E9 connected to the N terminus of the E9 DNase by an eight-residue linking sequence. The NMR data reveals that the mutations cause major alterations to the properties of some of the clusters, consistent with some form of association between them and other more distant parts of the amino acid sequence, particularly toward the N terminus of the protein. However, (15)N T(2) measurements indicates that residues 5-13 of the fusion protein bound to the 43-kDa TolB remain as flexible as they are in the free protein. The NMR data point to considerable dynamic ordering within the intrinsically disordered translocation domain of the colicin that is important for creating the TolB-binding site. Furthermore, amino acid sequence considerations suggest that the clusters of amino acids occur because of the size and polarities of the side chains forming them influenced by the propensities of the residues within the clusters and those immediately surrounding them in sequence space to form beta turns.     

Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli

The product of the malE—lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Echerichia coli membrane. The fusion product also inhibited colicin E1 export. Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells. Since colicin E1 contains the internal signal-like sequence [Proc. Natl. Acad. Sci. USA (1982) 79, 2827–2831], these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.     

Processing of DNase domain during translocation of colicin E7 across the membrane of Escherichia coli

Translocation of colicin across the membrane of sensitive cells has been studied extensively. However, processing of the toxicity domain of colicin during translocation has been the subject of much controversy. To investigate the final translocation product of colicin across the membrane of Escherichia coli, an endogenously expressed His-tagged Im7 protein was constructed to detect any translocation product containing the DNase domain traversed the inner membrane into cytoplasm of the E. coli cells. As a result, a final processed DNase domain of ColE7 was identified in the intracellular space of the cells treated with Col-Im complex. In the presence of periplasmic extracts, in vitro processing of DNase domain of ColE7 was also observed. These results suggest that the processing of ColE7 has occurred for translocation of the DNase-type colicin across the membrane and the process is probably taking place in the periplasmic space of the membrane.     

Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9

The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies (Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D (1)H-(13)C-(15)N NMR experiments, and the backbone dynamics of these regions investigated through measurement of (1)H-(15)N relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R(2) relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain. Abbreviations : Im9, the immunity protein for colicin E9; E9 DNase, the endonuclease domain of colicin E9; HSQC, heteronuclear single quantum coherence; ppm, parts per million; DSS, 2,2-(dimethylsilyl)propanesulfonic acid; TSP, sodium 3-trimethylsilypropionate; T(1 - 61)-DNase fusion protein, residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight residue thrombin cleavage sequence.     

Intracellular Immunization of Prokaryotic Cells against a Bacteriotoxin

Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E. coli. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.     

Minimum length requirement of the flexible N-terminal translocation subdomain of colicin E3

The 315-residue N-terminal T domain of colicin E3 functions in translocation of the colicin across the outer membrane through its interaction with outer membrane proteins including the OmpF porin. The first 83 residues of the T domain are known from structure studies to be disordered. This flexible translocation subdomain contains the TolB box (residues 34 to 46) that must cross the outer membrane in an early translocation event, allowing the colicin to bind to the TolB protein in the periplasm. In the present study, it was found that cytotoxicity of the colicin requires a minimum length of 19 to 23 residues between the C terminus (residue 46) of the TolB box and the end of the flexible subdomain (residue 83). Colicin E3 molecules of sufficient length display normal binding to TolB and occlusion of OmpF channels in vitro. The length of the N-terminal subdomain is critical because it allows the TolB box to cross the outer membrane and interact with TolB. It is proposed that the length constraint is a consequence of ordered structure in the downstream segment of the T domain (residues 84 to 315) that prevents its insertion through the outer membrane via a translocation pore that includes OmpF.     

Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system.

Colicins are antibiotic proteins that kill sensitive Escherichia coli cells. Their mode of action involves three steps: binding to specific receptors located in the outer membrane, translocation across this membrane, and action on their targets. A specific colicin domain can be assigned to each of these steps. Colicins have been subdivided into two groups (A and B) depending on the proteins required for them to cross the external membrane. Plasmids were constructed which led to an overproduction of the Tol proteins involved in the import of group A colicins. In vitro binding of overexpressed Tol proteins to either Tol-dependent (group A) or TonB-dependent (group B) colicins was analyzed. The Tol dependent colicins A and E1 were able to interact with TolA but the TonB dependent colicin B was not. The C-terminal region of TolA, which is necessary for colicin uptake, was also found to be necessary for colicin A and E1 binding to occur. Furthermore, only the isolated N-terminal domain of colicin A, which is involved in the translocation step, was found to bind to TolA. These results demonstrate the existence of a correlation between the ability of group A colicins to translocate and their in vitro binding to TolA protein, suggesting that these interactions might be part of the colicin import process.     

OmpF enhances the ability of BtuB to protect susceptible Escherichia coli cells from colicin E9 cytotoxicity

The outer membrane (OM) vitamin B(12) receptor, BtuB, is the primary receptor for E group colicin adsorption to Escherichia coli. Cell death by this family of toxins requires the OM porin OmpF but its role remains elusive. We show that OmpF enhances the ability of purified BtuB to protect bacteria against the endonuclease colicin E9, demonstrating either that the two OM proteins form the functional receptor or that OmpF is recruited for subsequent translocation of the bacteriocin. While stable binary colicin E9-BtuB complexes could be readily shown in vitro, OmpF-containing complexes could not be detected, implying that OmpF association with the BtuB-colicin complex, while necessary, must be weak and/or transient in nature.     

Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.     

The structure of TolB, an essential component of the tol-dependent translocation system, and its protein-protein interaction with the translocation domain of colicin E9

BACKGROUND: E colicin proteins have three functional domains, each of which is implicated in one of the stages of killing Escherichia coli cells: receptor binding, translocation and cytotoxicity. The central (R) domain is responsible for receptor-binding activity whereas the N-terminal (T) domain mediates translocation, the process by which the C-terminal cytotoxic domain is transported from the receptor to the site of its cytotoxicity. The translocation of enzymatic E colicins like colicin E9 is dependent upon TolB but the details of the process are not known. RESULTS: We have demonstrated a protein-protein interaction between the T domain of colicin E9 and TolB, an essential component of the tol-dependent translocation system in E. coli, using the yeast two-hybrid system. The crystal structure of TolB, a procaryotic tryptophan-aspartate (WD) repeat protein, reveals an N-terminal alpha + beta domain based on a five-stranded mixed beta sheet and a C-terminal six-bladed beta-propeller domain. CONCLUSIONS: The results suggest that the TolB-box residues of the T domain of colicin E9 interact with the beta-propeller domain of TolB. The protein-protein interactions of other beta-propeller-containing proteins, the yeast yPrp4 protein and G proteins, are mediated by the loops or outer sheets of the propeller blades. The determination of the three-dimensional structure of the T domain-TolB complex and the isolation of mutations in TolB that abolish the interaction with the T domain will reveal fine details of the protein-protein interaction of TolB and the T domain of E colicins.     

Delineation of the translocation of colicin E7 across the inner membrane of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The lysis protein of the colicinogenic operon is essential for colicin release and its main function is to activate the outer membrane phospholipase A (OMPLA) for the traverse of colicin across the cell envelope. However, little is known about the involvement of the lysis protein in the translocation of colicin across the inner membrane into the periplasm. The introduction of specific point mutations into the lipobox or sorting signal sequence of the lysE7 gene resulted in the production of various forms of lysis proteins. Our experimental results indicated that cells with wild-type mature LysE7 protein exhibited higher efficiency of colicin E7 translocation across the inner membrane into the periplasm than those with premature LysE7 protein. Moreover, the degree of permeability of the inner membrane induced by the mature LysE7 protein was significantly increased as compared to the unmodified LysE7 precursor. These results suggest that the efficiency of colicin movement into the periplasm is correlated with the increase in inner membrane permeability induced by the LysE7 protein. Thus, we propose that mature LysE7 protein has two critical roles: firstly mediating the translocation of colicin E7 across the inner membrane into the periplasm, and secondly activating the OMPLA to allow colicin release.     

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.     

Macromolecular import into Escherichia coli: the TolA C-terminal domain changes conformation when interacting with the colicin A toxin

Various macromolecules such as bacteriotoxins and phage DNA parasitize some envelope proteins of Escherichia coli to infect the bacteria. A two-step import mechanism involves the primary interaction with an outer membrane receptor or with a pilus followed by the translocation across the outer membrane. However, this second step is poorly understood. It was shown that the TolA, TolQ, and TolR proteins play a critical role in the translocation of group A colicins and filamentous bacteriophage minor coat proteins (g3p). Translocation of these proteins requires the interaction of their N-terminal domain with the C-terminal domain of TolA (TolAIII). In this work, short soluble TolAIII domains were overproduced in the cytoplasm and in the periplasm of E. coli. In TolAIII, the two cysteine residues were found to be reduced in the cytoplasmic form and oxidized in the periplasmic form. The interaction of TolAIII with the N-terminal domain of colicin A (ATh) is observed in the presence and in the absence of the disulfide bridge. The complex formation of TolAIII and ATh was found to be independent of the ionic strength. An NMR study of TolAIII, both free and bound, shows a significant structural change when interacting with ATh, in the presence or absence of the disulfide bridge. In contrast, such a structural modification was not observed when TolAIII interacts with g3p N1. These results suggest that bacteriotoxins and Ff bacteriophages parasitize E. coli using different interactions between TolA and the translocation domain of the colicin and g3p protein, respectively.     

The structure of BtuB with bound colicin E3 R-domain implies a translocon

Cellular import of colicin E3 is initiated by the Escherichia coli outer membrane cobalamin transporter, BtuB. The 135-residue 100-A coiled-coil receptor-binding domain (R135) of colicin E3 forms a 1:1 complex with BtuB whose structure at a resolution of 2.75 A is reported. Binding of R135 to the BtuB extracellular surface (DeltaG(o) = -12 kcal mol(-1)) is mediated by 27 residues of R135 near the coiled-coil apex. Formation of the R135-BtuB complex results in unfolding of R135 N- and C-terminal ends, inferred to be important for unfolding of the colicin T-domain. Small conformational changes occur in the BtuB cork and barrel domains but are insufficient to form a translocation channel. The absence of a channel and the peripheral binding of R135 imply that BtuB serves to bind the colicin, and that the coiled-coil delivers the colicin to a neighboring outer membrane protein for translocation, thus forming a colicin translocon. The translocator was concluded to be OmpF from the occlusion of OmpF channels by colicin E3.     

Kinetic basis for the competitive recruitment of TolB by the intrinsically disordered translocation domain of colicin E9

TolB and Pal are members of the Tol-Pal system that spans the cell envelope of Gram-negative bacteria and contributes to the stability and integrity of the bacterial outer membrane (OM). Lipoylated Pal is tethered to the OM and binds the β-propeller domain of periplasmic TolB, which, as recent evidence suggests, disengages TolB from its interaction with other components of the Tol system in the inner membrane. Antibacterial nuclease colicins such as colicin E9 (ColE9) also bind the β-propeller domain of TolB in order to catalyze their translocation across the bacterial OM. In contrast to Pal, however, colicin binding to TolB promotes its interaction with other components of the Tol system. Here, through a series of pre-steady-state kinetic experiments utilizing fluorescence resonance energy transfer pairs within the individual protein-protein complexes, we establish the kinetic basis for such 'competitive recruitment' by the TolB-binding epitope (TBE) of ColE9. Surprisingly, the 16-residue disordered ColE9 TBE associates more rapidly with TolB than Pal, a folded 13-kDa protein. Moreover, we demonstrate that calcium ions, which bind within the confines of the TolB β-propeller domain tunnel and are known to increase the affinity of the TolB-ColE9 complex, do not exert their influence through long-range electrostatic effects, as had been predicted, but through short-range effects that slow the dissociation rate of ColE9 TBE from its complex with TolB. Our study demonstrates that an intrinsically disordered protein undergoing binding-induced folding can compete effectively with a globular protein for a common target by associating more rapidly than the globular protein.     

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.     

A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12-dependent Escherichia coli 113/3 cells

The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing. Using N- and C-terminal deletions of the N-terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor-binding activity consisted of residues 343-418. The results of the in vivo receptor-binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1-497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitamin B12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB. The sequence of the 76-residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase-type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13. The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity. The minimum receptor-binding domain polypeptide inhibited the growth of the vitamin B12-dependent E. coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive.     

Dynamic aspects of colicin N translocation through the Escherichia coli outer membrane.

Colicin N is a bacteriocin that kills sensitive Escherichia coli cells. After binding to the cell surface-exposed receptor, a short period exists when a significant number of the cell-associated colicin N molecules are sensitive to external enzymes. Two colicin N populations are discriminated by proteases: the susceptible pool bound to OmpF porin on the cell surface and another population corresponding to protease-inaccessible colicin N. During translocation, colicin N reaches the periplasmic space and proteolytic cleavage of the colicin occurs only when the outer membrane barrier is permeabilized.     

大肠杆菌周质和外膜蛋白的定位

大肠杆菌周质和外膜蛋白发挥功能必须首先到达其特定亚细胞分区.大肠杆菌通过一系列与蛋白质分泌有关的蛋白（Sec蛋白）将周质和外膜蛋白转运至内膜.在切除了信号肽后,与周质蛋白的定位不同的是,外膜蛋白的最终定位还需要其他因子的协助.外膜蛋白的定位近来认为是以周质作为中介的.