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Structural and Functional Alterations of a Colicin-Resistant Mutant of Ompf

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Structural and Functional Alterations of a Colicin-Resistant Mutant of Ompf Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10675-10679, October 1994 Biophysics Structural and functional alterations of a colicin-resistant mutant of OmpF porin from Escherichia coli (bacteroi sentlty/coe N/porin channel/x-ray analysis) DENIS JEANTEUR*, TILMAN SCHIRMERt, DIDIER FOUREL0, VALERIE SIMONETt, GABRIELE RUMMEL§, CHRISTINE WIDMER§, JURG P. ROSENBUSCH§, FRANC PATTUS*¶, AND JEAN-MARIE PAGtSfII *European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, D-69012 Heidelberg, Germany; Departments of tStructural Biology and *Microbiology, Biozentrum, University of Basel, CH4056, Basel, Switzerland; and *Unit6 Propre de Recherche 9027, Centre de Biochimie et de Biologie Moleculaire, Centre National de la Recherche Scientifique, 31 Chemin Joseph-Aiguier, B.P. 71, Marseille Cedex 20, France Communicated by Eugene P. Kennedy, July 7, 1994 ABSTRACT A strain of Escherichia coli, selected on the residues that protrude from the barrel wall near the threefold basis of Its resistance to colicin N, reveals distinct structural molecular axis, faces two acidic side chains located on L3. and functional alterations in unspecific OmpF porin. A single This establishes a strong electrostatic field parallel to the mutation [Gly-119 -- Asp (G119D)] was identified in the membrane plane (11). Of the four colicin N-resistant point internal loop L3 that contributes critically to the formation of mutations that have recently been isolated and characterized the constriction inside the lumen of the pore. X-ray structure in OmpF porin (7), three are located on the external loops, analysis to a resolution of 3.0 A reveals a locally altered presumably impairing binding ofthe toxin. The fourth is aGly peptide backbone, with the side chain of residue Asp-119 Asp substitution at position 119 (G119D), located in loop protruding into the channe, causing the area of the constric- L3, far from the external surface of the molecule. Interest- tion (7 x 11 A in the wild type) to be subdivided into two ingly, Gly-119 belongs to the sequence motif PEFG119G that intercommunicating subcompartments of 3-4 A in diameter. is found in several enterobacterial porins (12). It is evident The functional consequences of this structural modification from the x-ray structure of the wild-type protein that in this consist of a reduction of the channel conductance by about position, no side chain can be accommodated without per- one-third, of altered ion selectivity and voltage gating, and of turbing the backbone structure. Here, we describe the struc- a decrease of permeation rates of various sugars by factors of tural and functional properties ofthis mutated porin.** While 2-12. The structural modification of the mutant protein the results cannot explain unambiguously the effect on colicin affects neither the «-barrel structure nor those regions of the N entry, the distinct structural alterations do explain the molecule that are exposed at the cell surface. Considering the pronounced functional changes observed in the mutant at the colicin resistance of the mutant, it is inferred that in vivo, atomic level. colicin N traverses the outer membrane through the porin channel or that the dynamics ofthe exposed loops are affected MATERIALS AND in the mutant such that these may impede the binding of the METHODS toxin. Bacterial Strains, Plasmids, Media, and Mutagenesis of ompF Gene. A "porin-deficient" strain, BZB 1107 (E. coli The unspecific porin, encoded by the ompF gene, is one of BE, ompF::TnS), was derived from the wild-type E. coli BE the major outer membrane proteins expressed in wild-type (BZB 3000BE) from the Biozentrum collection. Plasmids Escherichia coli K-12 cells growing under standard labora- pLG361 and pFD119 have been described (7, 13) and encode tory conditions (1). The protein forms three large water-filled wild-type or mutant (G119D) OmpF porin, respectively. Cells channels per trimer, allowing the diffusion of small hydro- were routinely grown in Luria-Bertani (LB) broth, at 37C philic molecules across the outer bacterial membrane (2-4). with gentle shaking. Kanamycin and tetracyclin were added It also serves as a cell-surface-exposed receptor for many as required. Mutagenesis and isolation of the G119D OmpF phages and colicins (1, 5). The role ofthe OmpF porin during have been described (7). Briefly, plasmid pLG361 encoding entry of colicin N (and A) in the binding step to the bacterial OmpF (13) was incubated overnight with 1 M hydroxylamine surface and in translocation across the outer membrane has in 0.5 M sodium phosphate (pH 6) at 37TC. DNA was purified recently been investigated (6, 7). Several antibiotics, includ- and used to transform BZB1107 cells. The cells were plated ing (3-lactams, use the porin pathway to cross the outer and incubated with colicin N. From the resistant clones on membrane and to find their targets (8). Deletion and substi- antibiotic plates, cells expressing OmpF were selected. Four tution mutations in the lumen of the porin channel dramati- types of substitutions were identified on the ompF gene by cally modify cell growth conditions and outer membrane sequencing 34 selected clones (7). In addition to resistance to permeability to hydrophobic antibiotics (9). colicin N, the mutant G119D also exhibited resistance to The three-dimensional structure of the OmpF porin has colicin A. been solved at 2.4-A resolution (10). Each monomer consists Purification and Characterization of the Mutated OmpF of a (3-barrel (16 antiparallel (-strands) that contains the Porin. The level of mutated OmpF synthesized was deter- channel. Six loops (each 11-17 residues long) are exposed to mined by immunoblot analysis and was similar to the wild- the surface ofthe cell, and one is involved in subunit contact. type OmpF (data not shown). Extraction and purification The longest loop (L3 with 34 residues) is bent into the channel were performed as described (14). The cells were broken at a height corresponding to the center of the membrane, forming the constriction site or selectivity gate (10). In this Present address: Ecole Superieure de Biotechnologie de Stras- narrow region, a positively charged cluster, formed by basic bourg, Pole Universitaire Illkirch, rue Sebastien Brant, 67400 Illkirch, France. ItTo whom reprint requests should be addressed. The publication costs ofthis article were defrayed in part by page charge **The atomic coordinates and structure factors have been deposited payment. This article must therefore be hereby marked "advertisement" in the Protein Data Bank, Chemistry Department, Brookhaven in accordance with 18 U.S.C. §1734 solely to indicate this fact. National Laboratory, Upton, NY 11973 (I.D. code 1MPF). 10675 Downloaded by guest on September 24, 2021 10676 Biophysics: Jeanteur et al. Proc. Natl. Acad. Sci. USA 91 (1994) using a French press, and envelopes were recovered by FAST area detector and processed by the program MADNES centrifugation. Porins were then treated by preextractig (24). The crystals of space group P321 have cell constants a eight times with a buffer containing 0.5% octyl-polyoxyeth- = b = 117.9 A, c = 52.8 A, a = 13 = 90°, and fy = 1200. The ylene, which eliminated the majority of contaminants. Five agreement between symmetry related reflections was R8y. = extraction steps with a buffer containing 3% octyl- 9.4% (34% in the highest resolution shell). Data reduction and polyoxyethylene allowed solubilization ofintegral membrane map calculations were performed by programs from the proteins. The porin obtained after this last step was purified CCP4 package (25). The wild-type OmpF model including by ion-exchange chromatography (DEAE-cellulose, Merck) ordered water molecules and detergent fragments (10) served followed by chromatofocusing (PBE94, Pharmacia) and fi- as the starting model. The crystallographic R factor was nally by gel filtration on Sephadex G-150 (Pharmacia). Purity 23.9% (20-3.0 A). After remodeling of the site of mutation was checked by SDS/PAGE and isoelectrofocusing. and conventional positional refinement using the program Bacteriodn Sensitiity. The colicin survival tests and the XPLOR (26), the final model had an R factor of16.6% and good various bacteriocins (6, 15) were tested with cells grown in stereochemistry (rms deviations of bond lengths and angles LB medium (0.1 ml of suspension at OD600 = 0.5 unit). from ideal values are 0.014 A and 1.8°, respectively). Tem- Various dilutions (101 to 106) of colicins A or N were added perature factors were taken from the wild-type structure to the cells in 0.15 M NaCl/3 mM KCl/1 mM potassium without further refinement. The negative difference electron phosphate/10 mM sodium phosphate, pH 7, and incubated density at residue Asp-119 disappeared after assigning a for 20 min at 37"C. The cell suspension was then diluted with temperature factor of 45 A2 to its atoms. The final difference 15 vol of fresh LB medium. In the direct test, the percentage electron density has a rms deviation of0.034 electron perA3 of surviving cells with or without bacteriocin treatment was and extreme values of ±0.16 electron per Ak. monitored after 2 hr at 37"C by determining the ratio of the optical densities at 600 nm. When the normal pathway was RESULTS bypassed (6, 16) by treatment at low ionic strength ("bypass" experiments), cells were washed twice in 10 mM sodium High-Resolution Structure of G119D OmpF Porn. Trigonal phosphate (pH 6.8) and resuspended in the same buffer at the crystals (space group P321) of the mutant porin were readily initial density (ODWO = 0.5 unit). Portions (0.1 ml) of the cell obtained using the established conditions for the wild-type suspension were incubated with various dilutions of bacte- protein (23). The structure was solved by the difference riocins (101 to 106) in this buffer and treated as in the direct Fourier method at a 3.0-A resolution.
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