Directed epitope delivery across the Escherichia coli ... · Directed epitope delivery across the...

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Directed epitope delivery across the Escherichia coli outer membrane through the porin OmpF Nicholas G. Housden a , Justyna A. Wojdyla a , Justyna Korczynska b , Irina Grishkovskaya a,1 , Nadine Kirkpatrick a , A. Marek Brzozowski b , and Colin Kleanthous a,2 a Department of Biology (Area 10), and b York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved October 19, 2010 (received for review July 26, 2010) The porins OmpF and OmpC are trimeric β-barrel proteins with narrow channels running through each monomer that exclude molecules >600 Da while mediating the passive diffusion of small nutrients and metabolites across the Gram-negative outer mem- brane (OM). Here, we elucidate the mechanism by which an entire soluble protein domain (>6 kDa) is delivered through the lumen of such porins. Following high-affinity binding to the vitamin B 12 receptor in Escherichia coli, the bacteriocin ColE9 recruits OmpF or OmpC using an 83-residue intrinsically unstructured transloca- tion domain (IUTD) to deliver a 16-residue TolB-binding epitope (TBE) in the center of the IUTD to the periplasm where it triggers toxin entry. We demonstrate that the IUTD houses two OmpF- binding sites, OBS1 (residues 218) and OBS2 (residues 5463), which flank the TBE and bind with K d s of 2 and 24 μM, respectively, at pH 6.5 and 25 ºC. We show the two OBSs share the same binding site on OmpF and that the colicin must house at least one of them for antibioticactivity. Finally, we report the structure of the OmpF- OBS1 complex that shows the colicin bound within the porin lumen spanning the membrane bilayer. Our study explains how colicins exploit porins to deliver epitope signals to the bacterial periplasm and, more broadly, how the inherent flexibility and narrow cross- sectional area of an IUP domain can endow it with the ability to traverse a biological membrane via the constricted lumen of a β-barrel membrane protein. colicin translocation intrinsically unstructured protein proteinprotein interaction transmembrane signalling isothermal titration calorimetry L igand-mediated signalling across a biological membrane typi- cally occurs when the ligand binds a specific membrane recep- tor causing it to change conformation or oligomeric status (1). Both outcomes communicate to the cell that a receptor-binding event has taken place on the other side of the membrane. The present work describes an alternative transmembrane signalling mechanism in which the signal itself, in the form of an intrinsically unstructured polypeptide epitope, is transferred through a porin in order to meet its cellular target. Intrinsically unstructured proteins (IUPs) are found in all kingdoms of life (2) but are particularly abundant in eukaryotes, where they are tightly regulated, subject to a wide variety of posttranslational modifications, and key to processes as diverse as cell cycle progression, endocytosis, intracellular signalling, and transcription activation (36). Although less abundant in pro- karyotes, IUPs are nonetheless important in their lifestyles (2, 7). An example of this is the role IUPs play in the import of bacter- iocins in Gram-negative bacteria (8). Bacteriocins are protein antibiotics that translocate into cells following receptor binding at the outer membrane (OM) (911). They are particularly abun- dant in the enterobacteriaciae, where they are the agents of com- petition between populations in structured environments such as the mammalian colon (12). Bacteriocins typically have a narrow killing spectrum dictated by their associations with particular OM and periplasmic proteins; hence, colicins are specific for Escherichia coli, pyocins target Pseudomonas aeruginosa, and pes- ticins kill Yersinia pestis. A single bacteriocin molecule is capable of killing a bacterial cell so understanding their modes of entry may uncover novel strategies for the creation of species-specific antibiotics as well as providing insight into mechanisms of intra- cellular targeting in microbes. Here we focus on the import mechanism of enzymatic E colicins (ColE2-E9) that are dependent on an IUTD for transport into E. coli. Enzymatic E colicins are cosynthesized with a high- affinity immunity protein that binds and inactivates the nuclease in the producing host (13). The immunity protein is lost during colicin translocation to the cytoplasm (14), with cell death result- ing through one of three nuclease activities (8). ColE3, ColE4, and ColE6 are rRNases that inhibit protein synthesis through site-specific cleavage of a single phosphodiester bond within the A-site of 30S ribosomal RNA (15, 16). ColE5 is a tRNase that blocks protein synthesis through the cleavage of the antico- don loops of a subset of tRNAs (17). ColE2 and ColE7-E9 are metal-dependent, nonspecific enzymes that degrade the bacterial genome (18). While the C-terminal nuclease domains of these approximately 60-kDa toxins are unrelated to each other their mechanism of import is the same, dictated by conserved domains involved in receptor binding and membrane translocation. Firstly, a central coiled-coil receptor-binding (R-) domain binds the vitamin B 12 receptor BtuB with high affinity (K d 12 nM), which localizes the colicin to the cell surface (9, 19). From here the colicin recruits a porin via its 83-residue IUTD (19), which is part of a larger 37 kDa translocation (T-) domain. Colicin delivery and hence cytotoxicity is dependent upon interactions with components of the Tol (group A colicins) or Ton (group B colicins) systems in the bacterial inner membrane and periplasm. The Tol system is a five-protein assembly comprising TolA, TolB, TolQ, TolR, and Pal. These proteins are ubiquitous in Gram-negative bacteria and are required for virulence by some pathogens (20). The Tol assembly is recruited to the septation site during cell division, where it is involved in stabilizing the OM (8). The Ton system composed of TonB, ExbB, and ExbD plays an essential role in the uptake of scarce nutrients across the OM (21). Both Tol and Ton systems are coupled to the proton-motive force of the inner membrane. How colicin IUTDs translocate to the bacterial periplasm to bind their specific targets remains an unresolved problem in colicin biology. In the case of ColIa, a group B colicin that binds Author contributions: N.G.H. and C.K. designed research; N.G.H., J.A.W., and J.K. performed research; N.G.H., J.A.W., A.M.B., and C.K. analyzed data; I.G. and N.K. contributed new reagents/analytic tools; and N.G.H., J.A.W., and C.K. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. Data deposition: The atomic coordinates and structural amplitudes have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3O0E). 1 Present address: CharitéUniversitätsmedizin Berlin, Institut für Biochemie, AG Proteinstrukturforschung, Monbijoustr.2a, 10117 Berlin. 2 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/ doi:10.1073/pnas.1010780107/-/DCSupplemental. 2141221417 PNAS December 14, 2010 vol. 107 no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1010780107 Downloaded by guest on February 19, 2021

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Page 1: Directed epitope delivery across the Escherichia coli ... · Directed epitope delivery across the Escherichia coli outer membrane through the porin OmpF Nicholas G. Housdena, Justyna

Directed epitope delivery across the Escherichia coliouter membrane through the porin OmpFNicholas G. Housdena, Justyna A. Wojdylaa, Justyna Korczynskab, Irina Grishkovskayaa,1, Nadine Kirkpatricka,A. Marek Brzozowskib, and Colin Kleanthousa,2

aDepartment of Biology (Area 10), and bYork Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD,United Kingdom

Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved October 19, 2010 (received for review July 26, 2010)

The porins OmpF and OmpC are trimeric β-barrel proteins withnarrow channels running through each monomer that excludemolecules >600 Da while mediating the passive diffusion of smallnutrients and metabolites across the Gram-negative outer mem-brane (OM). Here, we elucidate the mechanism by which an entiresoluble protein domain (>6 kDa) is delivered through the lumen ofsuch porins. Following high-affinity binding to the vitamin B12

receptor in Escherichia coli, the bacteriocin ColE9 recruits OmpFor OmpC using an 83-residue intrinsically unstructured transloca-tion domain (IUTD) to deliver a 16-residue TolB-binding epitope(TBE) in the center of the IUTD to the periplasm where it triggerstoxin entry. We demonstrate that the IUTD houses two OmpF-binding sites, OBS1 (residues 2–18) and OBS2 (residues 54–63),which flank the TBE and bindwith Kds of 2 and 24 μM, respectively,at pH 6.5 and 25 ºC. We show the two OBSs share the same bindingsite on OmpF and that the colicin must house at least one of themfor antibiotic activity. Finally, we report the structure of the OmpF-OBS1 complex that shows the colicin boundwithin the porin lumenspanning the membrane bilayer. Our study explains how colicinsexploit porins to deliver epitope signals to the bacterial periplasmand, more broadly, how the inherent flexibility and narrow cross-sectional area of an IUP domain can endow it with the abilityto traverse a biological membrane via the constricted lumen ofa β-barrel membrane protein.

colicin translocation ∣ intrinsically unstructured protein ∣ protein–proteininteraction ∣ transmembrane signalling ∣ isothermal titration calorimetry

Ligand-mediated signalling across a biological membrane typi-cally occurs when the ligand binds a specific membrane recep-

tor causing it to change conformation or oligomeric status (1).Both outcomes communicate to the cell that a receptor-bindingevent has taken place on the other side of the membrane. Thepresent work describes an alternative transmembrane signallingmechanism in which the signal itself, in the form of an intrinsicallyunstructured polypeptide epitope, is transferred through a porinin order to meet its cellular target.

Intrinsically unstructured proteins (IUPs) are found in allkingdoms of life (2) but are particularly abundant in eukaryotes,where they are tightly regulated, subject to a wide variety ofposttranslational modifications, and key to processes as diverseas cell cycle progression, endocytosis, intracellular signalling,and transcription activation (3–6). Although less abundant in pro-karyotes, IUPs are nonetheless important in their lifestyles (2, 7).An example of this is the role IUPs play in the import of bacter-iocins in Gram-negative bacteria (8). Bacteriocins are proteinantibiotics that translocate into cells following receptor bindingat the outer membrane (OM) (9–11). They are particularly abun-dant in the enterobacteriaciae, where they are the agents of com-petition between populations in structured environments such asthe mammalian colon (12). Bacteriocins typically have a narrowkilling spectrum dictated by their associations with particularOM and periplasmic proteins; hence, colicins are specific forEscherichia coli, pyocins target Pseudomonas aeruginosa, and pes-ticins kill Yersinia pestis. A single bacteriocin molecule is capable

of killing a bacterial cell so understanding their modes of entrymay uncover novel strategies for the creation of species-specificantibiotics as well as providing insight into mechanisms of intra-cellular targeting in microbes.

Here we focus on the import mechanism of enzymatic Ecolicins (ColE2-E9) that are dependent on an IUTD for transportinto E. coli. Enzymatic E colicins are cosynthesized with a high-affinity immunity protein that binds and inactivates the nucleasein the producing host (13). The immunity protein is lost duringcolicin translocation to the cytoplasm (14), with cell death result-ing through one of three nuclease activities (8). ColE3, ColE4,and ColE6 are rRNases that inhibit protein synthesis throughsite-specific cleavage of a single phosphodiester bond withinthe A-site of 30S ribosomal RNA (15, 16). ColE5 is a tRNasethat blocks protein synthesis through the cleavage of the antico-don loops of a subset of tRNAs (17). ColE2 and ColE7-E9 aremetal-dependent, nonspecific enzymes that degrade the bacterialgenome (18). While the C-terminal nuclease domains of theseapproximately 60-kDa toxins are unrelated to each other theirmechanism of import is the same, dictated by conserved domainsinvolved in receptor binding and membrane translocation. Firstly,a central coiled-coil receptor-binding (R-) domain binds thevitamin B12 receptor BtuB with high affinity (Kd ∼ 1–2 nM),which localizes the colicin to the cell surface (9, 19). From herethe colicin recruits a porin via its 83-residue IUTD (19), which ispart of a larger 37 kDa translocation (T-) domain.

Colicin delivery and hence cytotoxicity is dependent uponinteractions with components of the Tol (group A colicins) or Ton(group B colicins) systems in the bacterial inner membrane andperiplasm. The Tol system is a five-protein assembly comprisingTolA, TolB, TolQ, TolR, and Pal. These proteins are ubiquitous inGram-negative bacteria and are required for virulence by somepathogens (20). The Tol assembly is recruited to the septation siteduring cell division, where it is involved in stabilizing the OM (8).The Ton system composed of TonB, ExbB, and ExbD plays anessential role in the uptake of scarce nutrients across the OM(21). Both Tol and Ton systems are coupled to the proton-motiveforce of the inner membrane.

How colicin IUTDs translocate to the bacterial periplasmto bind their specific targets remains an unresolved problem incolicin biology. In the case of ColIa, a group B colicin that binds

Author contributions: N.G.H. and C.K. designed research; N.G.H., J.A.W., and J.K.performed research; N.G.H., J.A.W., A.M.B., and C.K. analyzed data; I.G. and N.K.contributed new reagents/analytic tools; and N.G.H., J.A.W., and C.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The atomic coordinates and structural amplitudes have been deposited inthe Protein Data Bank, www.pdb.org (PDB ID code 3O0E).1Present address: Charité—Universitätsmedizin Berlin, Institut für Biochemie, AGProteinstrukturforschung, Monbijoustr.2a, 10117 Berlin.

2To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1010780107/-/DCSupplemental.

21412–21417 ∣ PNAS ∣ December 14, 2010 ∣ vol. 107 ∣ no. 50 www.pnas.org/cgi/doi/10.1073/pnas.1010780107

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TonB, recent evidence points to the IUTD recruiting anothercopy of its primary receptor Cir, an iron-siderophore transporter(22). Although the mechanism by which the ColIa IUTD pene-trates the periplasm is not known, it is thought the IUTD maymimic a siderophore ligand in order to trigger translocationthrough the receptor in a TonB-dependent step (22, 23). GroupA colicins tend to use porins such as OmpF or OmpC to reach Tolproteins in the periplasm. Lakey and coworkers have argued,based on biochemical and EM studies, that the IUTD of ColN,which uses OmpF both as a receptor and translocator, traversesdown the sides of the porin at the interface with the LPS (24). Inthe case of the ColE3 IUTD, which is essentially identical to thatof ColE9 in the present work, Cramer and coworkers have arguedfor a lumenal translocation route through OmpF based on theocclusion of OmpF voltage-gated channels by the IUTD in planarlipid bilayer experiments and the presence of as yet unassignedelectron density in crystals of the IUTD in complex with OmpF(25, 26).

Following OmpF-mediated translocation of the IUTD, enzy-matic colicins such as ColE3 and ColE9 bind the periplasmic pro-tein TolB through a 16-residue TBE within the IUTD (residues32–47) (27). Binding of the colicin TBE to TolB promotes inter-action between TolB and the inner membrane protein TolA,which through its association with TolQ and TolR is coupled tothe proton-motive force. Colicin-directed contact between TolBand TolA promotes release of the high-affinity immunity proteinat the cell surface in a pmf-dependent step that initiates translo-cation of ColE9 across the OM (14, 28).

The question we address in the present paper is how OmpF isbound by the IUTD and how this establishes the translocationroute of the TBE to the periplasm. Using biochemical, biophysi-cal and in vivo experiments we demonstrate that recruitment ofOmpF by the ColE9 IUTD occurs through tandem binding sitesthat flank the TBE. We also report the crystal structure of E. coliOmpF in complex with one of these sites revealing the route ofpassage of the ColE9 IUTD across the OM. Cumulatively, ourresults suggest the colicin’s TBE is translocated and displayedin the periplasm by a sequential OmpF-binding mechanismand introduces a new mode of transmembrane signalling in whicha peptide signal is posited directly through a membrane-embedded protein pore.

ResultsThe ColE9 Principal OmpF-Binding Site Spans Residues 2–18 of theIUTD. Recruitment of OmpF to the BtuB-bound ColE9 complexin vivo has previously been shown to require the colicin IUTD,with the main interaction site localized to the N-terminal halfof the disordered domain (19). Preliminary size-exclusion chro-matography experiments (see SI Text) indicated that a binaryColE9–OmpF complex could be formed in vitro without priorbinding of colicin to BtuB and that binding was mediated entirelyby the colicin T-domain (Fig. S1 A and B). Having verified thata binary complex could be formed in vitro we identified theprincipal OmpF-binding site for ColE9 using fusion proteins incombination with deletion analysis, detecting binding throughisothermal titration calorimetry (ITC) (see Fig. S2 and Table S1for details). We narrowed down the main OmpF-binding site toa 17-residue epitope from the N terminus of the ColE9 IUTD(residues 2–18), for which a peptide encompassing this sequencebound OmpF with a Kd of approximately 2 μM and a stoichio-metry of one colicin peptide/OmpF monomer (Fig. 1A). Methio-nine 1 of the colicin is often missing and so was omitted fromthe final peptide sequence. We denote this IUTD binding epitopeas OmpF-Binding Site 1 (OBS1). The interaction of the OBS1peptide with OmpF is characterized by a large and favorableenthalpy (ΔH ¼ −19.1 kcal∕mol) and an unfavorable entropychange (ΔS ¼ −37.8 cal∕mol∕K), consistent with the disorderedpeptide becoming ordered upon complex formation. We alsofound that LPS had no effect on OBS1 binding (Table S1), sug-gesting the epitope makes little or no contact with regions ofOmpF involved in binding LPS.

Deconstructing the Role of OBS1 Residues in OmpF-Binding and ColicinTranslocation. The OBS1 sequence has eleven side-chain-bearingamino acids and six glycines, the latter interspersed throughoutthe epitope. An N-terminal GST fusion to the ColE9 IUTD com-pletely abolished binding to OmpF in ITC experiments, whereasC-terminal fusions had no effect (Fig. S2). Cramer and colleagueshave shown previously that the ability of ColE3 to occlude OmpFvoltage-gated channels is dependent on a free N terminus (29).Together these data suggest that the N terminus of an enzymaticcolicin IUTD is important for binding OmpF either directly orindirectly. With the exception of Ala13, we next mutated each

Fig. 1. Delineation of the two OmpF-binding sitesin the ColE9 IUTD. Figure shows ITC data for ColE9IUTD peptide sequences binding OmpF in 20 mMpotassium phosphate buffer pH 6.5 containing 1 %(w∕v) OG at 25 ºC. (A) Titration of 1060 μMOBS1 pep-tide (NH2-2SGGDGRGHNTGAHSTSG18-CONH2) into19 μMOmpF trimer measured on a VP-ITC. Data werecorrected for heats of dilution obtained by titrationof OBS1 into buffer (control data offset by þ1 μcal∕sec in top panel). Fitted parameters for a single sitebinding model from three independent titrationswere: ΔH, −19.1� 2.5 kcal∕mol; ΔS, −37.8� 8.2 cal∕mol∕K; Kd , 1.8� 0.1 μM and N, 3.2� 0.1∕OmpFtrimer. (B) Titration of 2 mM OBS2 peptide(CH3CONH-54IHWGGGSGR63-CONH2) into 50 μMOmpF trimer (closed circles), measured on aniTC200. Fitted parameters for a single site bindingmodel from duplicate independent titrations were:ΔH ¼ −16.8� 0.2 kcal∕mol; ΔS ¼ −35.2� 0.5 cal∕mol∕K; Kd ¼ 23.9� 1.6 μM and N ¼ 2.60� 0.01∕OmpF trimer. Data for the titration of 2 mM OBS2peptide into 50 μMOmpF∕1 mM OBS1 complex areshown as open circles with the raw data offset byþ0.5 μcal∕ sec in the top panel. We note that forboth OBS1 and OBS2 in these experiments three mo-lecules bind per OmpF trimer reflecting independentbinding of the peptides to each subunit. Thermody-namic parameters for all T-domain constructs used toidentify OBS1 and OBS2 are shown in Table S1.

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of the side-chain-bearing amino acids to alanine and assayed theireffects in in vivo cell-killing assays (using intact ColE9 mutants;Fig. S3) and in binding OmpF in vitro by ITC (using ColE9 IUTDfusions; Fig. S4). While many of the alanine mutations boundsubstantially weaker (D5A, R7A, H9A, T11A, and H14A allhad ΔΔGs > 1 kcal∕mol; Fig. S4D and Table S2) none abolishedOmpF binding. Consistent with these residues stabilizing thecomplex with OmpF, Yamashita et al. have reported that a ColE3D5A/R7A double mutant abolishes the ability of the colicin toocclude OmpF channels (30).

None of the ColE9 OBS1 alanine mutants had a discerniblephenotype in cell-killing assays (Fig. S3), implying the assay isnot sensitive to the weakening of OmpF binding. This was borneout by the effects of more drastic mutations. When Gly6 wassubstituted for proline, for example, all OmpF binding in ITC ex-periments was lost (Fig. S2) and the mutant exhibited cell-killingkinetics typical of an OBS1 deletion (Fig. S3 and Fig. 2, respec-tively). In contrast, when Gly6 was substituted for alanine, bind-ing was reduced 50-fold (Fig. S4D), and cell killing was wild type.The Gly-to-Pro effect, presumably due to kinking of the OBS1polypeptide chain, prompted an investigation of the other gly-cines. We found that substitution of any of the glycines for prolineresulted in ColE9 toxicity equivalent to that of an OBS1 deletion(Fig. S3) showing that proline is not tolerated anywhere alongOBS1. Prolines are, however, found within other regions of theColE9 IUTD that translocate to the periplasm, a point we returnto inDiscussion. In summary, while multiple OBS1 side chains areundoubtedly involved in stabilizing the complex with OmpF, themost important factors in colicin translocation are the flexibilityof the polypeptide backbone and the unconstrained nature of theN terminus.

A Second Porin Binding Site within the IUTD Binds OmpF with LowerAffinity. Truncation of the ColE9 N terminus up to residue 53 haspreviously been shown to markedly reduce the ability of thecolicin to recruit OmpF at the E. coli cell surface but not abolishit (19). Recruitment of OmpF is only fully lost when more than53 amino acids are deleted from the IUTD. Moreover, work ofSharma et al. (31) on ColE3 showed that deletions downstreamof residue 65 (Δ65-73 ColE3 and Δ72-80 ColE3) retained wild-type colicin activity. Taken together, these experiments suggestedthe presence of a second OmpF-binding site between residues 53and 64. Consistent with this hypothesis, ITC measurements ona fusion construct containing ColE9 residues 53–83 at the N ter-minus revealed weak binding to OmpF (Kd ∼ 130 μM), whichwas lost when residues 53–64 were deleted (Fig. S2). Delineationof OBS2 was confirmed through ITC experiments using a syn-thetic peptide of residues 54 to 63 of the ColE9 IUTD binding

to OmpF (Fig. 1B). Although OBS2 binds 10-fold weaker toOmpF than OBS1 (Kd ¼ 24 μM) the thermodynamic parametersof complex formation are similar to those of OBS1 (Table S1).Importantly, when OmpF was incubated with an excess of theOBS1 peptide, OBS2 was no longer able to bind OmpF, suggest-ing the two ColE9 IUTD OmpF-binding sites associate with thesame region on the porin (Fig. 1B).

The Presence of at Least One OBS Is Vital for Cytotoxicity. Enzymaticgroup A colicins such as ColE3 and ColE9 show no cytotoxicactivity against porin deficient cells (32). Hence, we next soughtto test the importance of OBS1 and OBS2 in colicin-mediatedkilling of E. coli JM83 cells using liquid growth assays and acombination of multiple residue mutations and deletion analysis.Interestingly, we found that mutations had subtly different effectsto deletions at each of the porin binding sites (Fig. S5). Mutatingthe majority of the OBS1 side chains in a single construct (ColE9D5S R7G H9S N10G T11G H14G) yielded less efficient cellkilling than its deletion (ColE9 Δ1-30), suggesting mutation ofthis region affects translocation as well as diminishing binding.Conversely, deletion of residues within OBS2 (ColE9 Δ54-62)had a more profound effect than their combined mutation(ColE9 I54G H55G W56S R62G), highlighting the importanceof polypeptide length in this region of the IUTD, in agreementwith previous observations (31). Despite a 13-fold difference inOmpF-binding affinities, the effect on colicin-mediated cellkilling of losing OBS1 by deletion or OBS2 through mutation iscomparable (Fig. 2A). Only upon combining deletion of OBS1and mutation of OBS2 (ColE9 Δ1-30 I54G H55GW56S R62G)is colicin cytotoxicity against JM83 cells abolished (Fig. 2A).These data show that while neither OmpF-binding site is essentialfor ColE9 activity, the toxin must house at least one of them tokill E. coli cells.

The OM of JM83 cells contains OmpF and OmpC the propor-tions of which vary with growth conditions (33), with both porinsable to mediate colicin toxicity (32). To assess the impact ofOBS1 and OBS2 on OmpF- and OmpC-specific colicin translo-cation, killing assays were performed using JW0912 (Fig. 2B) andJW2203 (Fig. 2C) E. coli, which are ompF− and ompC−, respec-tively (34). As observed previously (32), OmpF is more efficientthan OmpC in enzymatic colicin translocation, with a shorterdelay before the onset of cytotoxicity. The loss of either porinbinding site is readily tolerated in the killing of JW2203 cellswhere translocation occurs via OmpF, with ColE9 Δ1-30 andColE9 I54G H55GW56S R62G showing near wild-type toxicity.Moreover, a very low residual level of killing is seen with thesecells when both OBSs are removed (ColE9 Δ1-30 I54G H55GW56S R62G), indicating that even in the absence of any

Fig. 2. The involvement of OBS1 and OBS2 in ColE9 cytotoxicity in the context of mixed porin, OmpC-specific, and OmpF-specific membrane environments.The figure shows ColE9 induced cell death of 50 ml shake flask cultures grown at 37 ºC upon addition of wild-type (ColE9·Im9, ○), Δ1-30 (▾), I54G H55G W56SR62G (△), orΔ1-30 I54G H55GW56S R62G ColE9·Im9 (▪) to final concentrations of 80 nM (time of colicin addition indicated by arrow), compared to a no colicincontrol (•). Growth of cultures was monitored at 30 min intervals through measurement of OD600 nm. (A) JM83 (ompF+, ompC+), (B) JW0912 (ompF−, ompC+),and (C) JW2203 (ompF+, ompC−) E. coli cultures.

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OmpF-binding sites OmpF can still facilitate translocation of theIUTD across the OM, albeit very poorly. Conversely, the role ofeach OBS is amplified in JW0912 cells where translocation occursvia OmpC. The loss of either site significantly impairs colicin toxi-city, with the combined loss of both sites rendering the colicininactive. These data highlight that OmpC and OmpF do notbehave identically in nuclease colicin-mediated cell killing, theirdifferences likely reflecting subtle differences in the structures ofthe porins (such as pore size and the sequence and flexibility ofexternal loops) and hence the kinetics of porin recruitment by thecolicin IUTD. We conclude that the ColE9 OBSs are required toexpedite passage of the IUTD across the OM via a porin in orderto present the TBE correctly in the periplasm.

The OBS1 Peptide Spans the Aqueous Channel Running Through OmpF.The previously reported X-ray diffraction data for crystals ofthe OmpF-ColE3 IUTD complex (26) revealed some electrondensity within the OmpF lumen, estimated as approximatelyseven residues of the colicin, although their identity was notestablished. The present work shows the colicin IUTD has twodistinct OmpF-binding sites that flank the TBE and likely sharethe same binding site on the porin (Fig. 1B). In order to definethis common binding site we crystallized OmpF in the presence ofthe higher affinity OBS1 peptide, the resulting crystals diffractingto 3.0-Å resolution. The monoclinic asymmetric unit containedtwo OmpF trimers and the electron density maps revealed elec-tron density in the lumen of all six OmpF molecules, indicatingthe presence of the OBS1 peptide. Fifteen (pdb chain L) or ele-ven (pdb chains M–Q) residues were built into this electrondensity (Fig. 3A), and the whole structure refined at 3.0 Å to finalR value of 20.8% (Rfree ¼ 26.0%). Although the orientation ofthe OBS1 peptide cannot be unambiguously assigned, it hasbeen modeled with its N terminus facing the periplasm. Thisconformation was based on the positions of several bulky sidechains (Ser2, Arg7 and Asn10) that are clearly visible in the elec-tron density (Fig. 3 B and C and Fig. S6). The OBS1 peptideadopts a crescent-shaped, extended conformation that spansthe aqueous channel running through OmpF, from its extracellu-lar surface through the constriction zone at its center all theway to the periplasm (Fig. 3D). The location of OBS1 within

the lumen of OmpF, where it causes no conformational changesto the porin, is consistent with LPS having no effect on its bindingbecause the colicin only makes contact with the aqueous pore.Superposition of our structure with the model of Yamashita etal. for the OmpF-ColE3 IUTD complex (26) shows that residues7 to 13 of OBS1 occupy the same position as the density observedin this complex (Fig. 3D). Because OBS1 blocks OBS2 binding toOmpF (Fig. 1B) we conclude that OBS2 must also bind in thelumen of the porin.

DiscussionSequential OmpF Binding by OBS1 and OBS2 Ensures Anchored Deploy-ment of the Colicin TBE in the Periplasm Following Receptor Binding.Enzymatic E colicins become localized to the surface of E. colifollowing high-affinity binding of the long (∼100 Å) coiled-coilR-domain to the vitamin B12 receptor BtuB, present at approxi-mately 100–200 copies per cell in the OM. The unusual orienta-tion of the colicin, which sits at an angle 45° with respect to themembrane (9), projects the N-terminal T-domain above themembrane and away from its initial docking site. From here,the conformational space accessible to the colicin is considerablebecause the 83-residue IUTD can access a radius of >300 Å2 inits random two-dimensional search for a porin. It is estimated thatOmpF/OmpC are present at approximately 105 copies/cell; con-sequently, a BtuB-bound colicin will invariably find itself close toa translocator OmpF or OmpC with which its IUTD can makecontact. The present work explains how the IUTD binds such por-ins and suggests a mechanism by which the TBE epitope could bedelivered through the porin to the periplasm.

Recruitment of OmpF by the ColE9 IUTD is through twolinear epitopes, OBS1 and OBS2, which are separated by 35amino acids and flank the intervening TBE. ITC data indicatethe two OBSs bind to the same site on OmpF, with OBS1 havingan approximately 10-fold higher affinity than OBS2 (Table S1). Athird of OBS1 residues and a half in OBS2 are glycine, whichemphasizes the intrinsically disordered nature of these epitopes.While the two epitopes do not share significant sequence identitythey both contain a GGxGRG sequence motif, at the N terminusof OBS1 and C terminus of OBS2, which does not appearanywhere else in the ColE9 IUTD. The OmpF-OBS1 crystal

Fig. 3. Structure of the OmpF·ColE9 OBS1 peptide complex. (A) OmpF monomer shown in green ribbon with OBS1 peptide (chain L; residues 2-16) modeledinto the electron density map (2Fo − Fc shown at 1σ sigma cutoff) within the lumen. (B and C) Hydrogen bond interactions between OBS1 peptide (chain L, red)and OmpF monomer (chain A, green) (dashed lines); electron density map (2Fo − Fc) is shown at 1σ sigma cutoff, length of hydrogen bonds is shown in Å.(D) Superposition of colicin peptide structures from the complex of ColE3 IUTD (26) and the present structure of ColE9 OBS1 bound to OmpF, shown as amolecular surface cutaway to reveal the lumen of the porin. OBS1 peptide is shown in red and fragment from the ColE3 IUTD in yellow. Figures were preparedwith the program ccp4mg (48).

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structure indicates that this epitope is located toward the peri-plasmic surface of the porin. The only other common featureof the two binding sites is their overall basic nature, distinguishingthem from the TBE in the center of the IUTD, which is negativelycharged and does not bind to OmpF in ITC experiments(Table S1). The positively charged nature of the OBSs is likelyto be important for their binding to the OmpF pore, which ismildly cation-selective and implicated previously in the importof cationic antimicrobial peptides into the bacterial periplasm(35). While the low resolution nature of the OmpF·OBS1 peptidecrystal structure and the absence of several OBS1 side chainsprecludes detailed analysis, the side chains of Arg7 and Asn10of OBS1 are within hydrogen bonding distance of Asp107 andArg82 of OmpF, respectively (Fig. 3 B and C), in addition tohydrogen bonding between OmpF and the OBS1 backbone.Mutation of Asn10 to Ala had little impact on OmpF binding,whereas the R7A mutation weakened binding 10-fold. OtherOBS1 residues not visible in the structure had even greater im-pact on binding (Asp5, His9, His14) although none was essential.

Our data are consistent with the ColE9 IUTD translocatingthrough the OmpF pore by sequential binding of the two OBSs(Fig. 4). We suggest the following sequence of events after ColE9binds BtuB. First, OBS1 engages OmpF (or OmpC) through anelectrostatically driven interaction. OBS1 is the most likely initialdocking site because it carries greater positive charge (þ2) thanOBS2 (þ1) and binds with a higher affinity (Fig. 4A). This wouldalso be consistent with our experiments indicating the importanceof the colicin having a free N terminus, allowing the epitope toenter N terminus first and form a weak complex with the OmpFlumen. Second, following unbinding of peptide residues from thewalls of the lumen and aided by the inherent flexibility of theIUTD, OBS1 is directed to the periplasm by the overhangingT-domain, kept in place above OmpF by the colicin’s preformedcomplex with BtuB. Third, the next 30 residues containing the

negatively charged TBE and two proline residues (Pro24, Pro45)pass through the porin without interaction (Fig. 4B). Fourth,OBS2 docks in the lumen anchoring the TBE on the undersideof the OM (Fig. 4C) allowing the colicin to capture TolB. Bindingof the ColE9 TBE promotes contact between TolB and TolA inthe inner membrane, which is the initiating, pmf-dependent stepfor cellular import.

Lumenal Translocation of an IUP—a Novel Mechanism for Transmem-brane Signalling. IUP associations with soluble proteins, nucleicacids, and membranes are well documented (36–38). Thereare few reports, however, of IUPs recruiting membrane proteinsdirectly (39) and none that define the mechanism by which suchbinding occurs. As well as Gram-negative bacteria, porins areabundant in eukaryotes where they mediate metabolite diffusionacross the mitochondrial OM. They also play a central role insignalling apoptosis to cells. We speculate that the deploymentof IUP epitopes through porins in order to engage in transmem-brane signalling could therefore be relevant in the biology ofeukaryotic porins.

A number of reports have highlighted how the involvement ofIUPs in protein–protein interactions brings distinct advantages tobiological systems (3, 40–42), such as the capacity to arraymultiple epitopes in relatively small proteins, the formation ofcomplexes of low affinity but high specificity, binding through“fly casting,” and the moulding of the same IUP sequence to dif-ferent binding partners. We can now add a further advantageIUPs have over their globular counterparts that centers on theirflexibility and lack of structure, characteristics that allow them totraverse a membrane through the narrow channels of a proteinpore in order to deliver a signal directly into a cell. The key to thistransfer is the intrinsically unstructured nature of an IUP and itsnarrow cross-sectional area, properties that allow a macromole-cule as big as 6,000 Da to pass through a protein pore that has amolecular weight cutoff filter of 600 Da.

Materials and MethodsProtein Purification. The construction of plasmids for the expression ofmutated and truncated ColE9 and fusion proteins and the purification ofOmpF from E. coli BE3000 cells and ColE9 constructs from pET21a BL21(DE3) cultures were all carried out as detailed in SI Text.

Synthetic peptides were purchased from Activotec Ltd. with purities inexcess of 90% as determined through HPLC analysis and their predictedmolecular weights confirmed by electrospray mass spectrometry.

Protein concentrations were determined through A280 nm measure-ments using theoretical molar extinction coefficients calculated from aminoacid sequence for all colicin constructs and an extinction coefficient of177742 M−1·cm−1 derived through amino acid analysis (Alta Bioscience)for the OmpF trimer. Concentrations of the OBS1 peptide ðNH2Þ-SGGDGRGHNTGAHSTSG-ðCONH2Þ, devoid of aromatic amino acids were de-termined using the Fluoraldehyde assay (PIERCE) calibrated by amino acidanalysis. Concentrations of the OBS2 peptide ðCH3CONHÞ-IHWGGGSGRG-ðCONH2Þ were determined through A280 nm using an extinction coefficientof 5;500 M−1·cm−1.

Isothermal Titration Calorimetry (ITC). ITC measurements were performedusing either a MicroCal VP-ITC or a MicroCal ITC200 thermostated at 25 °C,with all protein samples prepared in 20 mM potassium phosphate bufferpH 6.5 in the presence of 1% (w∕v) OG. OmpF was present in the sample cellat a concentration of 18–50 μM with the ligand concentration in the syringevarying from 600 to 2,500 μM depending on the affinity of the binding inter-action. Binding isotherms were analyzed using the manufacturer’s software.

In Vivo Cytotoxicity Assay. The ability of ColE9 mutants to translocate into andkill E. coli cells was assessed through liquid growth cell-killing assays. Typically,50 ml LB-Amp shake flask cultures were inoculated 1∶100 with JM83 pTrc99aovernight cultures (the pTrc99a plasmid included merely for antibiotic resis-tance). Cultures were grown at 37 °C with shaking, monitoring growththrough OD600 nm measurements at 30 min intervals. ColE9 was added 2 hafter inoculation to a final concentration of 80 nM, and growth was moni-tored for a further 3 h. Cell killing mediated specifically though either OmpC

Fig. 4. Schematic model of the sequential binding mechanism that under-pins directed epitope delivery across the OM by the ColE9 IUTD. OmpF isshown in cyan, OBS1 in red, TBE in green, OBS2 in orange, and TolB in purple.The two OBSs are positively charged, whereas the TBE is negatively charged.* denotes positions of prolines in the IUTD. (A) Positively charged OBS1 isguided initially into the lumen of OmpF through an electrostatically drivenassociation after docking of the colicin’s R-domain to its primary receptor,BtuB. (B) Following unbinding of OBS1 residues from the porin lumen (wherebreaking many of the side-chain-bearing amino acid interactions weakensbinding by 10-100-fold; Fig. S4D), diffusion of the epitope is directed throughthe pore by virtue of the extracellular T-domain, held in place above OmpF bythe BtuB-bound colicin. Sequences containing proline and the negativelycharged TBE are unable to bind in the pore and so translocate directly tothe periplasm. (C) OBS2 passes into the pore and binds, anchoring the IUTDin the OM displaying the TBE sequence in the periplasm. Binding of TolB tothe ColE9 TBE initiates entry of the colicin into the cell.

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or OmpF was investigated using JW0912 and JW2203 cultures, devoid ofOmpF and OmpC, respectively.

Crystal Structure Determination. OmpF in 20 mM MES pH 6.5, 1% (w∕v) OG at90 μM was mixed with excess of OBS1 (1350 μM). Crystallization trials wereperformed using sitting drop vapor diffusionmethod. Crystals were obtainedfrom 25% (w∕v) PEG 3350, 0.2 M Li2SO4, 0.1 M NaCacodylate 6.5, 0.2% (v∕v)BDTM PuraMatrix (BD Bioscience). OmpF·OBS1 complex crystals belonged tospace group P21 with unit cell dimensions a ¼ 101.49 Å, b ¼ 101.62 Å, andc ¼ 162.14 Å. A single crystal was transferred into Paratone-N (HamptonResearch) and flash-cooled in liquid nitrogen. The single wavelength X-raydiffraction data was collected from a single crystal at 100 K on the Diamondbeamline i04 using ADSC Q315 CCD detector. Data were measured withcrystal-to-detector distance of 434 mm, using an oscillation range of 0.5°.Two hundred forty seven images were collected to a maximum resolutionof 3.0 Å. Recorded data were indexed and scaled using HKL2000 (43). The

structure was determined by molecular replacement using the program Mol-Rep (44). The solution contained six molecules in the asymmetric unit. Refine-ment was carried out using the program REFMAC5 (45) using local NCS andmap sharpening options. The structure was visualized andmanually modifiedusing program Coot (46). The stereochemistry of the model was evaluatedwith the program MolProbity (47). Data collection and refinement statisticsare shown in Table S2.

ACKNOWLEDGMENTS. We are indebted to Garib Murshudov (York StructuralBiology Laboratory) for help with refinement of the OmpF·OBS1 complexstructure. We are also grateful to Kayleigh Gilmore and Matthew Wiseman(York) for early contributions on T-domain fusions and our collaboratorsRichard James (Nottingham) and Geoff Moore (Norwich) for helpful discus-sions. We thank the Kleanthous lab for helpful comments on the manuscript.This work was funded by the Wellcome Trust and the Biotechnology andBiological Sciences Research Council.

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