Association and dissociation kinetics of colicin E3 and immunity protein 3: convergence of theory and experiment

The rapid binding of cytotoxic colicin E3 by its cognate immunity protein Im3 is essential in safeguarding the producing cell. The X-ray structure of the E3/Im3 complex shows that the Im3 molecule interfaces with both the C-terminal ribonuclease (RNase) domain and the N-terminal translocation domain of E3. The association and dissociation rates of the RNase domain and Im3 show drastically different sensitivities to ionic strength, as previously rationalized for electrostatically enhanced diffusion-limited protein-protein associations. Relative to binding to the RNase domain, binding to full-length E3 shows a comparable association rate but a significantly lower dissociation rate. This outcome is just what was anticipated by a theory for the binding of two linked domains to a protein. The E3/Im3 system thus provides a powerful paradigm for the interplay of theory and experiment.     

Release of immunity protein requires functional endonuclease colicin import machinery

Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.     

Global structural rearrangement of the cell penetrating ribonuclease colicin E3 on interaction with phospholipid membranes

Nuclease type colicins and related bacteriocins possess the unprecedented ability to translocate an enzymatic polypeptide chain across the Gram-negative cell envelope. Here we use the rRNase domain of the cytotoxic ribonuclease colicin E3 to examine the structural changes on its interaction with the membrane. Using phospholipid vesicles as model membranes we show that anionic membranes destabilize the nuclease domain of the rRNase type colicin E3. Intrinsic tryptophan fluorescence and circular dichroism show that vesicles consisting of pure DOPA act as a powerful protein denaturant toward the rRNase domain, although this interaction can be entirely prevented by the addition of salt. Binding of E3 rRNase to DOPA vesicles is an endothermic process (DeltaH=24 kcal mol-1), reflecting unfolding of the protein. Consistent with this, binding of a highly destabilized mutant of the E3 rRNase to DOPA vesicles is exothermic. With mixed vesicles containing anionic and neutral phospholipids at a ratio of 1:3, set to mimic the charge of the Escherichia coli inner membrane, destabilization of E3 rRNase is lessened, although the melting temperature of the protein at pH 7.0 is greatly reduced from 50 degrees C to 30 degrees C. The interaction of E3 rRNase with 1:3 DOPA:DOPC vesicles is also highly dependent on both ionic strength and temperature. We discuss these results in terms of the likely interaction of the E3 rRNase and the related E9 DNase domains with the E. coli inner membrane and their subsequent translocation to the cell cytoplasm.     

Highly discriminating protein-protein interaction specificities in the context of a conserved binding energy hotspot

We explore the thermodynamic basis for high affinity binding and specificity in conserved protein complexes using colicin endonuclease-immunity protein complexes as our model system. We investigated the ability of each colicin-specific immunity protein (Im2, Im7, Im8 and Im9) to bind the endonuclease (DNase) domains of colicins E2, E7 and E8 in vitro and compared these to the previously studied colicin E9. We find that high affinity binding (Kd < or = 10(-14) M) is a common feature of cognate colicin DNase-Im protein complexes as are non-cognate protein-protein associations, which are generally 10(6)-10(8)-fold weaker. Comparative alanine scanning of Im2 and Im9 residues involved in binding the E2 DNase revealed similar behaviour to that of the two proteins binding the E9 DNase; helix III forms a conserved binding energy hotspot with specificity residues from helix II only contributing favourably in a cognate interaction, a combination we have termed as "dual recognition". Significant differences are seen, however, in the number and side-chain chemistries of specificity sites that contribute to cognate binding. In Im2, Asp33 from helix II dominates colicin E2 specificity, whereas in Im9 several hydrophobic residues, including position 33 (leucine), help define its colicin specificity. A similar distribution of specificity sites was seen using phage display where, with Im2 as the template, a library of randomised sequences was generated in helix II and the library panned against either the E2 or E9 DNase. Position 33 was the dominant specificity site recovered in all E2 DNase-selected clones, whereas a number of Im9 specificity sites were recovered in E9 DNase-selected clones, including position 33. In order to probe the relationship between biological specificity and in vitro binding affinity we compared the degree of protection afforded to bacteria against colicin E9 toxicity by a set of immunity proteins whose affinities for the E9 DNase differed by up to ten orders of magnitude. This analysis indicated that the Kd required for complete biological protection is <10(-10)M and that the "affinity window" over which the selection of novel immunity protein specificities likely evolves is 10(-6)-10(-10)M. This comprehensive survey of colicin DNase-immunity protein complexes illustrates how high affinity protein-protein interactions can be very discriminating even though binding is dominated by a conserved hotspot, with single or multiple specificity sites modulating the overall binding free energy. We discuss these results in the context of other conserved protein complexes and suggest that they point to a generic specificity mechanism in divergently evolved protein-protein interactions.     

Immunity protein protects colicin E2 from OmpT protease

The endonuclease colicin E2 (ColE2), a bacteriocidal protein, and the associated cognate immunity protein (Im2) are released from producing Escherichia coli cells. ColE2 interaction with the target cell outer membrane BtuB protein and Tol import machinery allows the dissociation of Im2 from its colicin at the outer membrane surface. Here, we use in vivo approaches to show that a small amount of ColE2-Im2 protein complex bound to sensitive cells is susceptible to proteolytic cleavage by the outer membrane protease, OmpT. The presence of BtuB is required for ColE-Im2 cleavage by OmpT. The amount of colicin cleaved by OmpT is greatly enhanced when ColE2 is dissociated from Im2. We further demonstrate that OmpT cleaves the C-terminal DNase domain of the toxin. As expected, strains that over-produce OmpT are less susceptible to infection by ColE2 than by ColE2-Im2. Our findings reveal an additional function for the immunity protein beside protection of producing cells against their own colicin in the cytoplasm. Im2 protects ColE2 against OmpT-mediated proteolytic attack.     

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.     

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.     

A Force-Activated Trip Switch Triggers Rapid Dissociation of a Colicin from Its Immunity Protein

Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (K_d≈fM, lifetime ≈4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (<20 pN) increases the rate of complex dissociation 10⁶-fold, to a timescale (lifetime ≈10 ms) compatible with intoxication. We term this catastrophic force-triggered increase in off-rate a trip bond. Using mutational analysis, we elucidate the mechanism of this switch in affinity. We show that the N-terminal region of E9, which has sparse contacts with the hydrophobic core, is linked to an allosteric activator region in E9 (residues 21–30) whose remodelling triggers immunity protein release. Diversion of the force transduction pathway by the introduction of appropriately positioned disulfide bridges yields a force resistant complex with a lifetime identical to that measured by ensemble techniques. A trip switch within E9 is ideal for its function as it allows bipartite complex affinity, whereby the stable colicin:immunity protein complex required for host protection can be readily converted to a kinetically unstable complex whose dissociation is necessary for cellular invasion and competitor death. More generally, the observation of two force phenotypes for the E9:Im9 complex demonstrates that force can re-sculpt the underlying energy landscape, providing new opportunities to modulate biological reactions in vivo; this rationalises the commonly observed discrepancy between off-rates measured by dynamic force spectroscopy and ensemble methods.     

Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes

Bacteria producing endonuclease colicins are protected against their cytotoxic activity by virtue of a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. DNase binding by the immunity protein occurs through a "dual recognition" mechanism in which conserved residues from helix III act as the binding-site anchor, while variable residues from helix II define specificity. We now report the 1.7 A crystal structure of the 24.5 kDa complex formed between the endonuclease domain of colicin E9 and its cognate immunity protein Im9, which provides a molecular rationale for this mechanism. Conserved residues of Im9 form a binding-energy hotspot through a combination of backbone hydrogen bonds to the endonuclease, many via buried solvent molecules, and hydrophobic interactions at the core of the interface, while the specificity-determining residues interact with corresponding specificity side-chains on the enzyme. Comparison between the present structure and that reported recently for the colicin E7 endonuclease domain in complex with Im7 highlights how specificity is achieved by very different interactions in the two complexes, predominantly hydrophobic in nature in the E9-Im9 complex but charged in the E7-Im7 complex. A key feature of both complexes is the contact between a conserved tyrosine residue from the immunity proteins (Im9 Tyr54) with a specificity residue on the endonuclease directing it toward the specificity sites of the immunity protein. Remarkably, this tyrosine residue and its neighbour (Im9 Tyr55) are the pivots of a 19 degrees rigid-body rotation that relates the positions of Im7 and Im9 in the two complexes. This rotation does not affect conserved immunity protein interactions with the endonuclease but results in different regions of the specificity helix being presented to the enzyme.     

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.     

Crystal structure of colicin E3 immunity protein: an inhibitor of a ribosome-inactivating RNase

BACKGROUND: Colicins are antibiotic-like proteins of Escherichia coli that kill related strains. Colicin E3 acts as an RNase that specifically cleaves 16S rRNA, thereby inactivating the ribosomes in the infected cell. The producing organism is protected against colicin E3 by a specific inhibitor, the immunity protein Im3, which forms a tight 1:1 complex with colicin E3 and renders it inactive. Crystallographic studies on colicin E3 and Im3 have been undertaken to unravel the structural basis for the ribonucleolytic activity and its inhibition. RESULTS: The crystal structure of Im3 has been determined to a resolution of 1.8 A. The structure consists of a four-standard antiparallel beta sheet flanked by three alpha helices on one side of the sheet. Thr7, Phe9, Phe16 and Phe74 form a hydrophobic cluster on the surface of the protein in the vicinity of Cys47. This cluster is part of a putative binding pocket which also includes nine polar residues. CONCLUSIONS: The putative binding pocket of Im3 is the probable site of interaction with colicin E3. The six acidic residues in the pocket may interact with some of the numerous basic residues of colicin E3. The involvement of hydrophobic moieties in the binding is consistent with the observation that the tight complex can only be dissociated by denaturation. The structure of Im3 resembles those of certain nucleic acid binding proteins, in particular domain II of topoisomerase I and RNA-binding proteins that contain the ribonucleoprotein (RNP) sequence motif. This observation suggests that Im3 has a nucleic acid binding function in addition to binding colicin E3.     

Structure of the ultra-high-affinity colicin E2 DNase--Im2 complex

How proteins achieve high-affinity binding to a specific protein partner while simultaneously excluding all others is a major biological problem that has important implications for protein design. We report the crystal structure of the ultra-high-affinity protein-protein complex between the endonuclease domain of colicin E2 and its cognate immunity (Im) protein, Im2 (K(d)～10(-)(15)?M), which, by comparison to previous structural and biophysical data, provides unprecedented insight into how high affinity and selectivity are achieved in this model family of protein complexes. Our study pinpoints the role of structured water molecules in conjoining hotspot residues that govern stability with residues that control selectivity. A key finding is that a single residue, which in a noncognate context massively destabilizes the complex through frustration, does not participate in specificity directly but rather acts as an organizing center for a multitude of specificity interactions across the interface, many of which are water mediated.     

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.     

Molecular basis of inhibition of the ribonuclease activity in colicin E5 by its cognate immunity protein

Colicin E5 is a tRNA-specific ribonuclease that recognizes and cleaves four tRNAs in Escherichia coli that contain the hypermodified nucleoside queuosine (Q) at the wobble position. Cells that produce colicin E5 also synthesize the cognate immunity protein (Im5) that rapidly and tightly associates with colicin E5 to prevent it from cleaving its own tRNAs to avoid suicide. We report here the crystal structure of Im5 in a complex with the activity domain of colicin E5 (E5-CRD) at 1.15A resolution. The structure reveals an extruded domain from Im5 that docks into the recessed RNA binding cleft in E5-CRD, resulting in extensive interactions between the two proteins. The interactions are primarily hydrophilic, with an interface that contains complementary surface charges between the two proteins. Detailed interactions in three separate regions of the interface account for specific recognition of colicin E5 by Im5. Furthermore, single-site mutational studies of Im5 confirmed the important role of particular residues in recognition and binding of colicin E5. Structural comparison of the complex reported here with E5-CRD alone, as well as with a docking model of RNA-E5-CRD, indicates that Im5 achieves its inhibition by physically blocking the cleft in colicin E5 that engages the RNA substrate.     

Structural aspects of the inhibition of DNase and rRNase colicins by their immunity proteins

Nuclease E colicins that exert their cytotoxic activity through either non-specific DNase or specific rRNase action are inhibited by immunity proteins in a high affinity interaction that gives complete protection to the producing host cell from the deleterious effects of the toxin. Previous X-ray crystallographic analysis of these systems has revealed that in both cases, the immunity protein inhibitor forms its highly stable complex with the enzyme by binding as an exosite inhibitor-adjacent to, but not obscuring, the enzyme active site. The structures of the free E9 DNase domain and its complex with an ssDNA substrate now show that inhibition is achieved without deformation of the enzyme and by occlusion of a limited number of residues of the enzyme critical in recognition and binding of the substrate that are 3' to the cleaved scissile phosphodiester. No sequence or structural similarity is evident between the two classes of cytotoxic domain, and the heterodimer interfaces are also dissimilar. Thus, whilst these structures suggest the basis for specificity in each case, they give few indications as to the basis for the remarkably strong binding that is observed. Structural analyses of complexes bearing single site mutations in the immunity protein at the heterodimer interface reveal further differences. For the DNases, a largely plastic interface is suggested, where optimal binding may be achieved in part by rigid body adjustment in the relative positions of inhibitor and enzyme. For the rRNases, a large solvent-filled cavity is found at the immunity-enzyme interface, suggesting that other considerations, such as that arising from the entropy contribution from bound water molecules, may have greater significance in the determination of rRNase complex affinity than for the DNases.     

In vivo and in vitro characterization of overproduced colicin E9 immunity protein.

We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.     

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

Background: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition.Results: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 Å resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel α/β fold and contains a Zn²⁺ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (α1¹–loop12–α2¹; where ¹ represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23–α3¹–loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7.Conclusions: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.     

Structural and mechanistic basis of immunity toward endonuclease colicins

The crystal structure of the cytotoxic endonuclease domain from the bacterial toxin colicin E9 in complex with its cognate immunity protein Im9 reveals that the inhibitor does not bind at the active site, the core of which comprises the HNH motif found in intron-encoded homing endonucleases, but rather at an adjacent position leaving the active site exposed yet unable to bind DNA because of steric and electrostatic clashes with incoming substrate. Although its mode of action is unorthodox, Im9 is a remarkably effective inhibitor since it folds within milliseconds and then associates with its target endonuclease at the rate of diffusion to form an inactive complex with sub-femtomolar binding affinity. This hyperefficient mechanism of inhibition could be well suited to other toxic enzyme systems, particularly where the substrate is a polymer extending beyond the boundaries of the active site.     

Energy-dependent Immunity Protein Release during tol-dependent Nuclease Colicin Translocation

Nuclease colicins bind their target receptor in the outer membrane of sensitive cells in the form of a high affinity complex with their cognate immunity proteins. Upon cell entry the immunity protein is lost from the complex by means that are poorly understood. We have developed a sensitive fluorescence assay that has enabled us to study the molecular requirements for immunity protein release. Nuclease colicins use members of the tol operon for their translocation across the outer membrane. We have demonstrated that the amino-terminal 80 residues of the colicin E9 molecule, which is the region that interacts with TolB, are essential for immunity protein release. Using tol deletion strains we analyzed the cellular components necessary for immunity protein release and found that in addition to a requirement for tolB, the tolA deletion strain was most affected. Complementation studies showed that the mutation H22A, within the transmembrane segment of TolA, abolishes immunity protein release. Investigation of the energy requirements demonstrated that the proton motive force of the cytoplasmic membrane is critical. Taken together these results demonstrate for the first time a clear energy requirement for the uptake of a nuclease colicin complex and suggest that energy transduced from the cytoplasmic membrane to the outer membrane by TolA could be the driving force for immunity protein release and concomitant translocation of the nuclease domain.Membrane translocation is a formidable challenge for folded proteins. Eukaryotes have an array of dedicated translocation machineries to accomplish this feat, for example during mitochondrial import of cytosolic precursor proteins for which it has recently become clear that there is a surprising diversity in targeting signals, import routes, and translocation complexes (1, 2). It is now widely accepted that the mitochondrial genome originated from within the (eu)bacterial domain of life, so it should perhaps not come as a surprise that certain features of mitochondrial import have evolved from these ancestors.Gram-negative bacteria possess two membranes to protect them from the external world, separated by a layer of peptidoglycan and the periplasmic space. Their outer membrane, with its asymmetrical composition of lipopolysaccharide (LPS)² and phospholipids, forms an impressive barrier to most substances with the exception of small hydrophilic nutrients that can diffuse through the resident porins (3). Processes that require an energy input at the outer membrane, such as iron siderophore uptake, therefore often rely on energy generated by ion gradients at the cytoplasmic membrane (4). Energy-transducing systems such as the ton and tol systems in Escherichia coli harvest energy generated at the cytoplasmic membrane and transduce it to the outer membrane. These two systems have a number of features in common, and cross-complementation between the two systems has been observed (5).The energy transducing capacity of the ton system is somewhat better defined and is accomplished by three proteins: the cytoplasmic membrane proteins ExbB and ExbD, which form a heteromultimeric complex that interacts with TonB (4). As a result, TonB undergoes a conformational change in response to the PMF of the cytoplasmic membrane, which allows it to traverse the periplasm and make contact with nutrient-loaded outer membrane receptors, thereby facilitating active import (6). The homology between ExbB/D, TolQ/R, and the PMF-responsive flagellar motor proteins MotA and MotB is well established, and the cumulative evidence now suggests that they act as energy-harvesting complexes (7–9). Evidence of an evolutionary relationship between TolA and TonB comes from work demonstrating structural similarities between the Pseudomonas aeruginosa TolAIII globular domain and the carboxyl-terminal domain of E. coli TonB despite the very low sequence conservation (10). The activities of TonB and TolA are also critically dependent on a conserved SHLS motif in their transmembrane region, the mutation of which affects the interaction with their respective energy-harvesting complexes (11, 12). The cellular function of the tol system in E. coli is, however, less clear. It is thought that the Tol proteins play a role in maintaining cell envelope integrity through a network of interactions spanning the cytoplasmic membrane, periplasm, and outer membrane (13).Both energy-transducing systems have been parasitized by the colicins, plasmid-encoded antibacterial proteins produced by E. coli, and phages for their translocation into the cell, but the energy requirements for these processes are not unequivocal (14). Group A colicins use the tol system and group B colicins the ton system in a process whereby interactions of their amino-terminal translocation domains with the Tol or Ton proteins in the periplasm ultimately lead to the entry of their carboxyl-terminal cytotoxic domain into the cell (15, 16). In common with most colicins, the DNase-type colicin E9 consists of three functional domains: the killing activity is contained in its carboxyl-terminal DNase domain; the central section contains the receptor-binding domain, which binds the vitamin B₁₂ receptor, BtuB, in the outer membrane; and the amino-terminal translocation domain is needed for the entry of the cytotoxic domain into the target cell. The first 83 residues of this translocation domain, commonly referred to as the NDR, contain the OmpF and TolB binding sites (17, 18). Upon synthesis colicin E9 forms a high affinity interaction with its cognate immunity protein, Im9, also encoded by the colicin operon. This heterodimeric complex formation protects colicin-producing cells against DNA damage and potential suicide prior to release of the complex in the environment. The nature of the complex formation between colicin E9 and Im9 and other colicin-immunity complexes has been well characterized, and in the case of colicin E9-Im9 the interaction is strong, as reflected by its dissociation constant on the order of 10⁻¹⁴ m under physiological conditions (19). Despite the high avidity of this interaction, the DNase domain of colicin E9 appears to have only a marginally stabilizing effect on Im9 (20).Currently much progress is being made to unravel the early events that take place after receptor binding, where it has been shown that the colicin E9 NDR enters the periplasm through the OmpF lumen where it interacts with TolB, possibly displacing it from its interaction with Pal (18, 21–24). It was also recently demonstrated that the receptor binding and translocation domains remain in contact with their binding partners in the outer membrane and the periplasm, respectively, when the DNase domain gains access to the cytoplasm (25). In contrast, the molecular mechanisms that govern the loss of the immunity protein from the colicin complex and the cell entry of the DNase domain are less well documented. Because of the strength of the interaction between the colicin and its cognate immunity protein, it has been proposed that removal of the immunity protein from the complex would require a cellular energy source. One recent report investigating immunity protein loss from the colicin E2-Im2 complex qualitatively concluded that receptor binding alone does not lead to immunity protein release and that a functional tol translocation complex is required to establish immunity protein release (26).Here we have presented data that for the first time demonstrate a role for the individual Tol proteins and address the issue of energy requirements for immunity protein release. We observed, by using a previously described disulfide-“locked” colicin construct and domain deletion mutants thereof, that entry of the amino-terminal 80 residues of the colicin translocation domain and its interaction with TolB are essential factors for immunity protein release. We have also demonstrated a crucial role for TolA and its transmembrane region in this process, showing that immunity protein release from the colicin complex is an energy-dependent process governed by the cytoplasmic membrane PMF. Finally we have provided a rationale for how an energized Tol system might lead to immunity protein loss and concomitant colicin uptake in sensitive cells.     

Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import

The interaction of colicins with target cells is a paradigm for protein import. To enter cells, bactericidal colicins parasitize Escherichia coli outer membrane receptors whose physiological purpose is the import of essential metabolites. Colicins E1 and E3 initially bind to the BtuB receptor, whose beta-barrel pore is occluded by an N-terminal globular "plug". The x-ray structure of a complex of BtuB with the coiled-coil BtuB-binding domain of colicin E3 did not reveal displacement of the BtuB plug that would allow passage of the colicin (Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova, T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer. 2003. Nat. Struct. Biol. 10:948-954). This correlates with the inability of BtuB to form ion channels in planar bilayers, shown in this work, suggesting that an additional outer membrane protein(s) is required for colicin import across the outer membrane. The identity and interaction properties of this OMP were analyzed in planar bilayer experiments.OmpF and TolC channels in planar bilayers were occluded by colicins E3 and E1, respectively, from the trans-side of the membrane. Occlusion was dependent upon a cis-negative transmembrane potential. A positive potential reversibly opened OmpF and TolC channels. Colicin N, which uses only OmpF for entry, occludes OmpF in planar bilayers with the same orientation constraints as colicins E1 and E3. The OmpF recognition sites of colicins E3 and N, and the TolC recognition site of colicin E1, were found to reside in the N-terminal translocation domains. These data are considered in the context of a two-receptor translocon model for colicin entry into cells.