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• W2080766424 abstract "The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-Å crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg452 at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon. The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-Å crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg452 at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon. Protein transport across membranes in organelles and bacteria is known to involve multiprotein complexes (1Mokranjac D. Neupert W. Biochem. Soc. Trans. 2005; 33: 1019-1023Crossref PubMed Scopus (97) Google Scholar, 2Pfanner N. Wiedemann N. Meisinger C. Lithgow T. Nat. Struct. Mol. Biol. 2004; 11: 1044-1048Crossref PubMed Scopus (182) Google Scholar). Colicin import across the outer membrane of Escherichia coli has also been inferred to involve such a translocon (3Lazdunski C.J. Mol. Microbiol. 1995; 16: 1059-1066Crossref PubMed Scopus (60) Google Scholar, 4Lazzaroni J.C. Dubuisson J.F. Vianney A. Biochimie (Paris). 2002; 84: 391-397Crossref PubMed Scopus (94) Google Scholar, 5Braun V. Patzer S.I. Hantke K. Biochimie (Paris). 2002; 84: 365-380Crossref PubMed Scopus (144) Google Scholar, 6Zakharov S.D. Cramer W.A. Front. Biosci. 2004; 9: 1311-1317Crossref PubMed Scopus (39) Google Scholar, 7Kurisu G. Zakharov S.D. Zhalnina M.V. Bano S. Eroukova V.Y. Rokitskaya T.I. Antonenko Y.N. Wiener M.C. Cramer W.A. Nat. Struct. Biol. 2003; 10: 948-954Crossref PubMed Scopus (125) Google Scholar, 8Zakharov S.D. Zhalnina M. Sharma O. Cramer W.A. Biochemistry. 2006; 45: 10199-10207Crossref PubMed Scopus (29) Google Scholar). An experimentally useful attribute of colicin uptake for studies on protein transport across membranes is that the end result is cytotoxicity. Colicins are plasmid-encoded bactericidal proteins that are released in response to stress, enter the bacterial cell by appropriating its outer membrane nutrient-uptake machinery, and provide an advantage to colicin-resistant cells in the competition for nutrition (9Riley M.A. Gordon D.M. Trends Microbiol. 1999; 7: 129-133Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Colicins are produced in complex with a small immunity (∼10 kDa) protein that binds to, and prevents, the colicin from killing the producing cell (10Bowman C.M. Sidikaro J. Nomura M. Nat. New Biol. 1971; 234: 133-137Crossref PubMed Scopus (130) Google Scholar, 11Jakes K.S. Zinder N.D. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3380-3384Crossref PubMed Scopus (84) Google Scholar). Nuclease colicins consist of three domains: an N-terminal T (translocation)-domain that functions in the import of the colicin across the outer membrane, a C-terminal C (catalysis or channel)domain that contains the cytotoxic activity, and a central R (receptor-binding)-domain that functions in irreversible attachment to an outer membrane receptor. Colicins have been divided into two groups, A and B, based on the intracellular protein translocation network that is utilized. “Group A” colicins utilize the Tol proteins TolA, TolB, TolQ, TolR, and Pal (4Lazzaroni J.C. Dubuisson J.F. Vianney A. Biochimie (Paris). 2002; 84: 391-397Crossref PubMed Scopus (94) Google Scholar, 12Davies J.K. Reeves P. J. Bacteriol. 1975; 123: 102-117Crossref PubMed Google Scholar, 13Nagel de Zwaig R. Luria S.E. J. Bacteriol. 1967; 94: 1112-1123Crossref PubMed Google Scholar), whereas “Group B” colicins utilize the Ton proteins, TonB, ExbB, and ExbD (5Braun V. Patzer S.I. Hantke K. Biochimie (Paris). 2002; 84: 365-380Crossref PubMed Scopus (144) Google Scholar, 14Davies J.K. Reeves P. J. Bacteriol. 1975; 123: 96-101Crossref PubMed Google Scholar), to enter the bacterial cytoplasmic compartment and/or insert into the cytoplasmic membrane. Colicins E2 (a DNase) and E3 (a 16 S rRNase), both nuclease E colicins and members of Group A, utilize the same outer membrane receptor, BtuB, to bind to and enter the cell. BtuB, a 22-stranded antiparallel β-barrel integral outer membrane protein, functions physiologically to transport cobalamin (vitamin B12) across the outer membrane (15Chimento D.P. Kadner R.J. Wiener M.C. J. Mol. Biol. 2003; 332: 999-1014Crossref PubMed Scopus (66) Google Scholar). The interaction of nuclease E colicins with BtuB is neither TonB-nor energy-dependent. The central pore of the BtuB-barrel is occluded by an N-terminal 132-residue “plug” domain, which, in the absence of an energy input, would prevent the passage of any colicin. The crystal structure of a complex of the R-domain (E3R135) 4The abbreviations used are: E2R135 and E3R135, 135-residue receptor-binding domain of colicins E2 and E3, respectively; MES, 2-(N-morpholino)ethanesulfonic acid; LDAO, N,N-dimethyl-dodecylamine-N-oxide; OMP, outer membrane protein. of colicin E3 in complex with BtuB, solved to a resolution of 2.75 Å, provides several clues to the mechanism of colicin import through the outer membrane (7Kurisu G. Zakharov S.D. Zhalnina M.V. Bano S. Eroukova V.Y. Rokitskaya T.I. Antonenko Y.N. Wiener M.C. Cramer W.A. Nat. Struct. Biol. 2003; 10: 948-954Crossref PubMed Scopus (125) Google Scholar). The peripheral and oblique binding of E3 R-domain to BtuB, the complete absence of any colicin-induced current through BtuB incorporated into planar bilayers (16Zakharov S.D. Eroukova V.Y. Rokitskaya T.I. Zhalnina M.V. Sharma O. Loll P.J. Zgurskaya H.I. Antonenko Y.N. Cramer W.A. Biophys. J. 2004; 87: 3901-3911Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and the fact that the plug domain of BtuB was stable with little or no conformational change upon interaction with the colicin R-domain (7Kurisu G. Zakharov S.D. Zhalnina M.V. Bano S. Eroukova V.Y. Rokitskaya T.I. Antonenko Y.N. Wiener M.C. Cramer W.A. Nat. Struct. Biol. 2003; 10: 948-954Crossref PubMed Scopus (125) Google Scholar), implied that the transfer of any domain of colicin E3 through the BtuB structure was unlikely. At least one additional embedded outer membrane protein participates in an outer membrane translocon for colicin import through the outer membrane. It was hypothesized that the unusual elongate receptor-binding domain, with a length of ∼100 Å for rRNase colicin E3 (17Soelaiman S. Jakes K. Wu N. Li C. Shoham M. Mol. Cell. 2001; 8: 1053-1062Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), which is also inferred for structures of colicins E2, E6, E7, and E9 (18Sharma O. Zakharov S.D. Cramer W.A. Handbook of Biologically Active Peptides. 2006; (Kastin, A. J., ed) 1st Ed., pp. , Academic Press, San Diego: 115-123Crossref Scopus (5) Google Scholar) on the basis of sequence similarity, and ∼160 Å for colicin Ia (19Wiener M. Freymann D. Ghosh P. Stroud R.M. Nature. 1997; 385 (– 464): 461Crossref PubMed Scopus (228) Google Scholar), is associated with a “fishing rod” mechanism (16Zakharov S.D. Eroukova V.Y. Rokitskaya T.I. Zhalnina M.V. Sharma O. Loll P.J. Zgurskaya H.I. Antonenko Y.N. Cramer W.A. Biophys. J. 2004; 87: 3901-3911Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) for recruitment of an outer membrane protein in addition to BtuB to form a translocon. The additional protein utilized to form a translocon for the import of nuclease E colicins is inferred to be the OmpF porin in most studies on colicins E2, E3, E7, E9, and A (7Kurisu G. Zakharov S.D. Zhalnina M.V. Bano S. Eroukova V.Y. Rokitskaya T.I. Antonenko Y.N. Wiener M.C. Cramer W.A. Nat. Struct. Biol. 2003; 10: 948-954Crossref PubMed Scopus (125) Google Scholar, 16Zakharov S.D. Eroukova V.Y. Rokitskaya T.I. Zhalnina M.V. Sharma O. Loll P.J. Zgurskaya H.I. Antonenko Y.N. Cramer W.A. Biophys. J. 2004; 87: 3901-3911Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 20Benedetti H. Frenette M. Baty D. Lloubes R. Geli V. Lazdunski C. J. Gen. Microbiol. 1989; 135: 3413-3420PubMed Google Scholar, 21Housden N.G. Loftus S.R. Moore G.R. James R. Kleanthous C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13849-13854Crossref PubMed Scopus (81) Google Scholar, 22Law C.J. Penfold C.N. Walker D.C. Moore G.R. James R. Kleanthous C. FEBS Lett. 2003; 545: 127-132Crossref PubMed Scopus (15) Google Scholar, 23Cao Z. Klebba P.E. Biochimie (Paris). 2002; 84 (– 412): 399Crossref PubMed Scopus (75) Google Scholar). A dependence of the activities of nuclease E colicins on OmpC has also been noted (24Mock M. Pugsley A.P. J. Bacteriol. 1982; 150: 1069-1076Crossref PubMed Google Scholar, 25Pan Y.H. Liao C.C. Kuo C.C. Duan K.J. Liang P.H. Yuan H.S. Hu S.T. Chak K.F. J. Biol. Chem. 2006; 281: 13083-13091Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Colicin E1 does not require OmpF, but it needs TolC (13Nagel de Zwaig R. Luria S.E. J. Bacteriol. 1967; 94: 1112-1123Crossref PubMed Google Scholar, 26Otsuji N. Soejima T. Maki S. Shinagawa H. Mol. Gen. Genet. 1982; 187: 30-36Crossref PubMed Scopus (11) Google Scholar) to form a translocon for import. The nuclease E colicins utilize OmpF or OmpC to cross the outer membrane and enter the periplasm, where they can interact with the periplasmic protein TolB (27Loftus S.R. Walker D. Mate M.J. Bonsor D.A. James R. Moore G.R. Kleanthous C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12353-12358Crossref PubMed Scopus (61) Google Scholar, 28Bouveret E. Rigal A. Lazdunski C. Benedetti H. Mol. Microbiol. 1997; 23: 909-920Crossref PubMed Scopus (56) Google Scholar, 29Hands S.L. Holland L.E. Vankemmelbeke M. Fraser L. Macdonald C.J. Moore G.R. James R. Penfold C.N. J. Bacteriol. 2005; 187 (– 6741): 6733Crossref PubMed Scopus (23) Google Scholar) to accomplish the first phase of protein import. Nuclease E colicins have highly conserved T- and R-domains, with the major difference in their amino acid sequences residing in their C-domains and at the junction of the R- and C-domain, suggesting that they utilize a common pathway for entry into the bacterial cell. A question was posed as to whether the structure of the E3R135-BtuB complex could be applied to other nuclease E colicins. Alignment of the R-domain of colicin E3 and E2 revealed a difference of 12 of 135 residues, with all of the mismatches concentrated in the C terminus of the R-domain (Fig. 1). The structure of the E2R135-BtuB complex, obtained in the present study at a resolution of 3.5 Å, was found to be virtually identical to the corresponding structure of the E3R135-BtuB complex solved previously (7Kurisu G. Zakharov S.D. Zhalnina M.V. Bano S. Eroukova V.Y. Rokitskaya T.I. Antonenko Y.N. Wiener M.C. Cramer W.A. Nat. Struct. Biol. 2003; 10: 948-954Crossref PubMed Scopus (125) Google Scholar). Studies that bear on the import mechanism show that, under normal growth conditions, among the tested porins only OmpF or OmpC is necessary for colicin cytotoxicity and that the site of protease cleavage of the imported colicin E2 is similar or identical to that studied previously in colicin E7 (30Shi Z. Chak K.F. Yuan H.S. J. Biol. Chem. 2005; 280: 24663-24668Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Bacterial Strains—The E. coli XL1 Blue strain was used as the host strain for cloning of E2R135. Cloning was done in the pET41b vector such that there was a His8 tag at the C terminus of the protein. E. coli BL21(DE3) was the host strain for expressing the protein. In the pET41b vector, protein expression is under the control of a strong isopropyl-1-thio-β-d-galactopyranoside-inducible T7 RNA polymerase promoter. All cultures were grown in LB media or on LB agar plates supplemented with antibiotic when required. Purification of E2R135—The E2R135 construct was created by cloning residues Thr313–Asp447,a 135-residue-long peptide, in the expression vector pET41b. The cloning was done using standard protocols. E2R135 was first amplified using upstream (Nde primer) and downstream (Xho primer) primers and then cloned between the NdeI-XhoI sites of pET41b in such a way that there was a His8 tag at the C terminus joined by a linker consisting of residues Lys and Glu, bringing the total length of the peptide to 145 residues. Purification of the over-expressed E2R135 construct was accomplished using a nickel-charged IDA-agarose column. Purification of BtuB—BtuB was expressed in strain TNE012 (pJC3) and purified using a previously published protocol (31Taylor R. Burgner J.W. Clifton J. Cramer W.A. J. Biol. Chem. 1998; 273: 31113-31118Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). BtuB was extracted from the outer membrane with 1.5% (w/v) β-d-octyl-glucoside, 50 mm Tris-HCl, 5 mm EDTA, phenylmethylsulfonyl fluoride/tosylphenylalanyl chloromethyl ketone (PMSF/TPCK), pH 8.0, and purified by ion-exchange chromatography in 0.1% (w/v) LDAO using a 0–0.8 m LiCl gradient in a DEAE fast protein liquid chromatography column. BtuB-enriched fractions were concentrated and further purified on a Superdex 200HR size exclusion column in 10 mm Tris-HCl, 0.1 m NaCl, 0.1% (w/v) LDAO, pH 8.0. BtuB concentrations were determined from its UV extinction coefficient: [ɛ (280 nm) = 137 cm–1mm–1]. Knock-out Strains—Strains having single, double, and triple porin knock-outs were used to study the role of these porins in the colicin import. Knock-out strains were produced by using a previously published method (32Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97 (– 6645): 6640Crossref PubMed Scopus (11304) Google Scholar). PCR primers were used to provide homology to the targeted gene(s). The chromosomal sequence was replaced with a kanamycin resistance gene generated by PCR by using primers with 36-nucleotide homology extensions. This is accomplished by Red-mediated recombination in these flanking homologies. The Red helper plasmid, a temperature sensitive replicon, was cured by growth at 37 °C (32Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97 (– 6645): 6640Crossref PubMed Scopus (11304) Google Scholar). Cytotoxicity Assays—190 μl of LB broth with or without 1 nm colicin E3 was inoculated with 10 μl of overnight cultures of porin knock-out strains in a 96-well plate. The cells were allowed to grow over a period of 5 h, and growth was monitored by measuring optical density at 410 nm at 30-min intervals. The cytotoxicity of wild type E2 and E2 R452A was assayed using a “spot test.” Spot tests involve plating colicin-sensitive pT7-7 cells (Ampr cells) onto a LB Amp plate to which different concentrations of colicin are applied in 20-μl drops. Lysis of the bacterial cells is seen as clear spots in a lawn of bacteria. The lowest inhibitory concentration was defined as the smallest concentration that would generate a clear zone of inhibition. Crystallization—Crystallization screening was done in a 96-well plate utilizing a Genomic Solutions robot. E2R135 and BtuB were mixed at a ratio of 1.6:1 (mol:mol) and allowed to react for ∼30 min. The protein was dissolved in 20 mm Tris-HCl, pH 8.0, 0.1 m NaCl, and 0.1% LDAO buffer. The reservoir solution contained 10% dioxane, 0.1 m MES, and 1.6 m ammonium sulfate. Crystals of the E2R135-BtuB complex were obtained by hanging drop vapor diffusion, mixing 0.7 μlofthe protein solution with 0.7 μl of reservoir buffer at 20 °C. Crystals grew within 1 week. Structure Determination and Refinement—X-ray diffraction data at 3.5-Å resolution were collected at 100 K at the SBC 19-ID beamline at the Advanced Photon Source (APS), Argonne National Laboratory. Data reduction and scaling were processed using the program HKL2000 (33Otwinowski Z. Minor W. Method Enzymol. 1997; 276: 307-326Crossref Scopus (38617) Google Scholar). The crystals belong to the orthorhombic space group P212121, with a = 76.2 Å, b = 80.7 Å, c = 235.9 Å. The unit cell of the E2R135-BtuB crystal was similar to that of E3R135-BtuB (a = 76.9 Å, b = 80.1 Å, c = 233.6 Å; Protein Data Bank accession code 1UJW). The structure was determined by rigid body refinement with the program REFMAC5 in CCP4 (34Bailey S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (42) Google Scholar) using the published structure of the E3R135-BtuB complex (1UJW) as an initial model. Structure refinement and model construction used the programs REFMAC5 and O (35Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). A summary of the crystallographic data and refinement statistics is presented in Table 1. The programs Molscript (36Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), Povscript (37Fenn T.D. Ringe D. Petsko G.A. J. Appl. Crystallogr. 2003; 36: 944-947Crossref Scopus (299) Google Scholar), and Raster3D (38Merritt E.A. Bacon D.J. Method Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3878) Google Scholar) were used for molecular graphics presentations. The program SURFACE in CCP4 was used to calculate surface areas.TABLE 1Intensity data and refinement statisticsData collectionX-ray sourceAPSSBC-19IDWavelength (Å)0.97934Resolution (Å)3.50 (3.63-3.50)Measured reflections127,245 (10,186)Unique reflections18,963 (1,787)Redundancy6.7 (5.7)I/σ(I)16.3 (3.7)Completeness (%)99.3 (95.5)RmergeaRmerge = ΣhΣi|Ii(h) — 〈I(h) 〉|/ΣhΣ| iIi(h) where Ii is the ith measurement of reflection h and 〈I(h) 〉 is a weighted mean of all measurements of h. (%)10.8 (37.7)RefinementRcrystbr = Σh∥Fobs(h)| — |Fcalc(h)∥| /Σh|Fobs|.Rcryst and Rfree were calculated from the working and test reflection sets, respectively. The test set comprised 5% of the total reflections not used in refinement.0.250Rfreebr = Σh∥Fobs(h)| — |Fcalc(h)∥| /Σh|Fobs|.Rcryst and Rfree were calculated from the working and test reflection sets, respectively. The test set comprised 5% of the total reflections not used in refinement.0.315r.m.s.cr.m.s., root mean square. deviation from idealBond lengths (Å)0.01Bond angles (°)1.02Ramachandran plotFavored (%)86.7Allowed (%)12.8Generously allowed (%)0.5Disallowed (%)0.0Mean B-value (Å2)77.2a Rmerge = ΣhΣi|Ii(h) — 〈I(h) 〉|/ΣhΣ| iIi(h) where Ii is the ith measurement of reflection h and 〈I(h) 〉 is a weighted mean of all measurements of h.b r = Σh∥Fobs(h)| — |Fcalc(h)∥| /Σh|Fobs|.Rcryst and Rfree were calculated from the working and test reflection sets, respectively. The test set comprised 5% of the total reflections not used in refinement.c r.m.s., root mean square. Open table in a new tab Occlusion of OmpF—The occlusion by colicin E2 of OmpF channels incorporated into planar bilayers was studied as described previously (16Zakharov S.D. Eroukova V.Y. Rokitskaya T.I. Zhalnina M.V. Sharma O. Loll P.J. Zgurskaya H.I. Antonenko Y.N. Cramer W.A. Biophys. J. 2004; 87: 3901-3911Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The synthetic phospholipids DOPC and DOPE were mixed in a 1:1 molar (total concentration, 10 mg/ml) ratio, dissolved in n-decane, and then used to form planar bilayers (39Benz R. Schmid A. Hancock R.E. J. Bacteriol. 1985; 162: 722-727Crossref PubMed Google Scholar, 40Song J. Minetti C.A. Blake M.S. Colombini M. Biochim. Biophys. Acta. 1998; 1370: 289-298Crossref PubMed Scopus (31) Google Scholar). Bilayers were formed by painting lipids using a “brush technique” (41Mueller P. Rudin D.O. Tien H.T. Wescott W.C. Nature. 1962; 194: 979-980Crossref PubMed Scopus (1042) Google Scholar) on a 0.2-mm aperture in a partition separating two 4-ml chambers containing 10 mm HEPES, pH 7.0, 0.1 m KCl, 23 °C. Occlusion was studied by measuring the current across this aperture with Ag/AgCl electrodes immersed into the two chambers, using a Warner BC-525C amplifier (Hamden, CT). To form channels, OmpF (1 pg/ml-1 ng/ml) was added to the cis-side of the membrane, and the solution was stirred until channels appeared. A negative transmembrane potential was applied to the cis-side, with the trans-side at electrical ground. Colicin (0.5–1 μg) was added to the trans-side of the aperture. Structure of the E2R135-BtuB Complex—Crystals of the E2R135-BtuB complex diffracted to 3.5 Å. The structure was solved as described under “Materials and Methods” using the E3R135-BtuB structure as a reference (Protein Data Bank code 1UJW). The structure reveals 123 residues of E2R135 (residues 321–443), in a coiled-coil conformation, bound to BtuB (Fig. 2A). A total of 12 residues distal to the BtuB binding site, eight N-terminal and four C-terminal, are disordered and not resolved in the structure. This extent of disorder differs slightly from the total of 19 residues (10 N-terminal and 9 C-terminal) of E3R135 that are disordered in the E3R135-BtuB structure. Otherwise, the crystal structure of E2R135-BtuB is very similar to that of E3R135-BtuB (Fig. 2B (7Kurisu G. Zakharov S.D. Zhalnina M.V. Bano S. Eroukova V.Y. Rokitskaya T.I. Antonenko Y.N. Wiener M.C. Cramer W.A. Nat. Struct. Biol. 2003; 10: 948-954Crossref PubMed Scopus (125) Google Scholar)). In a recently published structure of Cir and the receptor-binding domain of colicin Ia, a single C-terminal residue is disordered and not resolved (42Buchanan S.K. Lukacik P. Grizot S. Ghirlando R. Ali M.M. Barnard T.J. Jakes K.S. Kienker P.K. Esser L. EMBO J. 2007; 26: 2594-2604Crossref PubMed Scopus (81) Google Scholar). The BtuB-R135 interaction surface, defined by a 4.5-Å van der Waals distance of closest approach, is similar for the E2 and E3 structures: (i) in all, 28 residues of E2R135 (and E3R135) interact with 29 residues of BtuB; (ii) Met383 and Pro382, respectively, of E2R135 and E3R135 are in contact with six residues of the plug domain (Thr55, Gln56, Asn57, Leu63, Ser64, and Ser65); (iii) for both colicins, the buried area of E2R135 is 1262 Å2, whereas the corresponding figure for E3R135 is 1287 Å2; (iv) the principle axis of the R-peptide resides at an identical angle of ∼44° relative to the membrane surface for both colicins; (v) the number of hydrogen bonds between the plug and the barrel of BtuB is 61, 56, and 65 for E2R135-BtuB, E3R135-BtuB, and apo-BtuB crystallized in surfo (Protein Data Bank code 1NQE) structure, respectively; (vi) the accessible surface area of the plug domain is also essentially identical, as the area for the apo-BtuB (1NQE), for E2R135-BtuB (2YSU) and E3R135-BtuB (1UJW) is 2768, 2900, and 2904 Å2, respectively (the above calculation includes the surface area of the BtuB plug domain/R-domain interface for the E2R135-BtuB and E3R135-BtuB structures); (vii) R-domain residues involved in crystal contacts (Leu330, Asn331, Asn334, Ala338, Gln341, Gln342, Ala345, Arg432, and Glu436 of E2R135) are identical in the case of both structures; (viii) all of the crystal contacts of the R-domain for both the E2- and E3R135 complexes are contained in extracellular loops 5–6 and 7–8 of BtuB. It is inferred that the entry of the colicin does not occur through the pore of BtuB because; (i) there is no detectable distortion of the plug domain in the E2R135 and E3R135 structures relative to that of apo-BtuB (Fig. 2C); (ii) no ionic current (>1.0 × 10–12 A) was induced by the addition of colicin E3 to BtuB incorporated into planar bilayers (16Zakharov S.D. Eroukova V.Y. Rokitskaya T.I. Zhalnina M.V. Sharma O. Loll P.J. Zgurskaya H.I. Antonenko Y.N. Cramer W.A. Biophys. J. 2004; 87: 3901-3911Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). As was the case for E3R135-BtuB, binding of E2R135 affects the loops of BtuB in the E2R135-BtuB structure when compared with both the in surfo (Fig. 2D) (15Chimento D.P. Kadner R.J. Wiener M.C. J. Mol. Biol. 2003; 332: 999-1014Crossref PubMed Scopus (66) Google Scholar) and in meso (Fig. 3) structures of apo-BtuB (43Cherezov V. Yamashita E. Liu W. Zhalnina M. Cramer W.A. Caffrey M. J. Mol. Biol. 2006; 364: 716-734Crossref PubMed Scopus (89) Google Scholar). Loops 5–6 and 7–8, which are disordered in the in surfo apo-BtuB structure, are ordered in the E2R135-BtuB structure (Fig. 2D). This can be compared with the Cir-colicin Ia structure in which there is a large outward movement of loops 7 and 8 (42Buchanan S.K. Lukacik P. Grizot S. Ghirlando R. Ali M.M. Barnard T.J. Jakes K.S. Kienker P.K. Esser L. EMBO J. 2007; 26: 2594-2604Crossref PubMed Scopus (81) Google Scholar). In E2R135-BtuB there is a change in the position of loop 19–20, and the location of the partial order in loop 3–4 is altered. When compared with the in meso BtuB structure, the disordered loops 5–6 and 21–22 become ordered upon binding of E2R135 (Fig. 3). There is also a difference in the position of the loops 9–10, 13–14, 15–16, and 19–20, and the location of the partial order in loop3–4 is altered. In addition, unlike the Cir-Ia complex structure (42Buchanan S.K. Lukacik P. Grizot S. Ghirlando R. Ali M.M. Barnard T.J. Jakes K.S. Kienker P.K. Esser L. EMBO J. 2007; 26: 2594-2604Crossref PubMed Scopus (81) Google Scholar), where the binding of the R-domain of colicin Ia does not elicit a change in the N-terminal tonB box, binding of the R-domain of both colicins E2 and E3 causes ordering of residues Ser4 and Pro5 in the tonB box on the periplasmic side of BtuB. There are some differences between the R-domain of the two structures, with most of these differences located at the termini of the coiled-coil. The major difference is that the E2R135 structure is longer by two residues at the N terminus and by five residues at the C terminus. Also, compared with the corresponding residues of E3R135-BtuB structure (7Kurisu G. Zakharov S.D. Zhalnina M.V. Bano S. Eroukova V.Y. Rokitskaya T.I. Antonenko Y.N. Wiener M.C. Cramer W.A. Nat. Struct. Biol. 2003; 10: 948-954Crossref PubMed Scopus (125) Google Scholar), the C-α positions of the N-terminal seven residues 323–329 of E2R135 show a gradual increase in displacement as a function of increasing distance from BtuB along the coiled-coil, with the N-terminal residue, Tyr323, having a maximum displacement of 5.2 Å. Occlusion of OmpF Channels by E2—OmpF channels incorporated into planar bilayer membranes were occluded by colicin E2 (4 μg/ml) added to the trans-side in the presence of a cis-negative potential (data not shown) in a manner similar to the occlusion by colicin E3 (7Kurisu G. Zakharov S.D. Zhalnina M.V. Bano S. Eroukova V.Y. Rokitskaya T.I. Antonenko Y.N. Wiener M.C. Cramer W.A. Nat. Struct. Biol. 2003; 10: 948-954Crossref PubMed Scopus (125) Google Scholar, 16Zakharov S.D. Eroukova V.Y. Rokitskaya T.I. Zhalnina M.V. Sharma O. Loll P.J. Zgurskaya H.I. Antonenko Y.N. Cramer W.A. Biophys. J. 2004; 87: 3901-3911Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This is consistent with the 100% identity of the N-terminal 83 residues of colicin E3 and E2. In Vivo Cytotoxicity Assays—A library of indicator knock-out strains each missing one or more porins (44Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Syst. Biol. 2006. 2006; 2: 0008Google Scholar) was tested for colicin sensitivity. No single porin knockout was resistant to colicin E3 (Fig. 4A). However, the ΔOmpR strain, in which the OmpF and OmpC positive regulator is knocked out and thus is missing both OmpF and OmpC, was resistant to E3 (Fig. 4B). This implies that either OmpF or OmpC, but no other OMP, can function in the translocon for the nuclease" @default.
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• W2080766424 title "Structure of the Complex of the Colicin E2 R-domain and Its BtuB Receptor" @default.
• W2080766424 cites W1503309822 @default.
• W2080766424 cites W1539796472 @default.
• W2080766424 cites W1554612905 @default.
• W2080766424 cites W1701782453 @default.
• W2080766424 cites W1729750927 @default.
• W2080766424 cites W177419946 @default.
• W2080766424 cites W1944771822 @default.
• W2080766424 cites W1971385382 @default.
• W2080766424 cites W1975246808 @default.
• W2080766424 cites W1978845112 @default.
• W2080766424 cites W1979155909 @default.
• W2080766424 cites W1990991452 @default.
• W2080766424 cites W1991932967 @default.
• W2080766424 cites W2002952229 @default.
• W2080766424 cites W2006349794 @default.
• W2080766424 cites W2010143056 @default.
• W2080766424 cites W2013083986 @default.
• W2080766424 cites W2017238222 @default.
• W2080766424 cites W2018808204 @default.
• W2080766424 cites W2027790619 @default.
• W2080766424 cites W2028231353 @default.
• W2080766424 cites W2037767151 @default.
• W2080766424 cites W2039647109 @default.
• W2080766424 cites W2040117880 @default.
• W2080766424 cites W2048708888 @default.
• W2080766424 cites W2063177628 @default.
• W2080766424 cites W2064389237 @default.
• W2080766424 cites W2069519711 @default.
• W2080766424 cites W2071064456 @default.
• W2080766424 cites W2077610206 @default.
• W2080766424 cites W2080528351 @default.
• W2080766424 cites W2080627708 @default.
• W2080766424 cites W2085381747 @default.
• W2080766424 cites W2098601281 @default.
• W2080766424 cites W2110633542 @default.
• W2080766424 cites W2116047897 @default.
• W2080766424 cites W2116137883 @default.
• W2080766424 cites W2116573011 @default.
• W2080766424 cites W2125651557 @default.
• W2080766424 cites W2126131817 @default.
• W2080766424 cites W2142230661 @default.
• W2080766424 cites W2145007018 @default.
• W2080766424 cites W2151635778 @default.
• W2080766424 cites W2152967503 @default.
• W2080766424 cites W2163388241 @default.
• W2080766424 cites W2166650607 @default.
• W2080766424 cites W4243392427 @default.
• W2080766424 cites W4246620890 @default.
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