Proc. Nati. Acad. Sci. USAVol. 91, pp. 10675-10679, October 1994Biophysics

Structural and functional alterations of a colicin-resistant mutant ofOmpF 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 BiologieMoleculaire, 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 thebasis of Its resistance to colicin N, reveals distinct structuraland functional alterations in unspecific OmpF porin. A singlemutation [Gly-119 -- Asp (G119D)] was identified in theinternal loop L3 that contributes critically to the formation ofthe constriction inside the lumen of the pore. X-ray structureanalysis to a resolution of 3.0 A reveals a locally alteredpeptide backbone, with the side chain of residue Asp-119protruding into the channe, causing the area of the constric-tion (7 x 11 A in the wild type) to be subdivided into twointercommunicating subcompartments of 3-4 A in diameter.The functional consequences of this structural modificationconsist of a reduction of the channel conductance by aboutone-third, of altered ion selectivity and voltage gating, and ofa decrease of permeation rates of various sugars by factors of2-12. The structural modification of the mutant proteinaffects neither the «-barrel structure nor those regions of themolecule that are exposed at the cell surface. Considering thecolicin resistance of the mutant, it is inferred that in vivo,colicin N traverses the outer membrane through the porinchannel or that the dynamics of the exposed loops are affectedin the mutant such that these may impede the binding of thetoxin.

The unspecific porin, encoded by the ompF gene, is one ofthe major outer membrane proteins expressed in wild-typeEscherichia coli K-12 cells growing under standard labora-tory conditions (1). The protein forms three large water-filledchannels per trimer, allowing the diffusion of small hydro-philic molecules across the outer bacterial membrane (2-4).It also serves as a cell-surface-exposed receptor for manyphages and colicins (1, 5). The role of the OmpF porin duringentry of colicin N (and A) in the binding step to the bacterialsurface and in translocation across the outer membrane hasrecently been investigated (6, 7). Several antibiotics, includ-ing (3-lactams, use the porin pathway to cross the outermembrane and to find their targets (8). Deletion and substi-tution mutations in the lumen of the porin channel dramati-cally modify cell growth conditions and outer membranepermeability to hydrophobic antibiotics (9).The three-dimensional structure of the OmpF porin has

been solved at 2.4-A resolution (10). Each monomer consistsof a (3-barrel (16 antiparallel (-strands) that contains thechannel. Six loops (each 11-17 residues long) are exposed tothe surface ofthe cell, and one is involved in subunit contact.The longest loop (L3 with 34 residues) is bent into the channelat a height corresponding to the center of the membrane,forming the constriction site or selectivity gate (10). In thisnarrow region, a positively charged cluster, formed by basic

residues that protrude from the barrel wall near the threefoldmolecular axis, faces two acidic side chains located on L3.This establishes a strong electrostatic field parallel to themembrane plane (11). Of the four colicin N-resistant pointmutations that have recently been isolated and characterizedin OmpF porin (7), three are located on the external loops,presumably impairing binding ofthe toxin. The fourth is aGly

Asp substitution at position 119 (G119D), located in loopL3, far from the external surface of the molecule. Interest-ingly, Gly-119 belongs to the sequence motif PEFG119G thatis found in several enterobacterial porins (12). It is evidentfrom the x-ray structure of the wild-type protein that in thisposition, no side chain can be accommodated without per-turbing the backbone structure. Here, we describe the struc-tural and functional properties of this mutated porin.** Whilethe results cannot explain unambiguously the effect on colicinN entry, the distinct structural alterations do explain thepronounced functional changes observed in the mutant at theatomic level.

MATERIALS AND METHODSBacterial Strains, Plasmids, Media, and Mutagenesis of

ompF Gene. A "porin-deficient" strain, BZB 1107 (E. coliBE, ompF::TnS), was derived from the wild-type E. coli BE(BZB 3000BE) from the Biozentrum collection. PlasmidspLG361 and pFD119 have been described (7, 13) and encodewild-type or mutant (G119D) OmpF porin, respectively. Cellswere routinely grown in Luria-Bertani (LB) broth, at 37Cwith gentle shaking. Kanamycin and tetracyclin were addedas required. Mutagenesis and isolation of the G119D OmpFhave been described (7). Briefly, plasmid pLG361 encodingOmpF (13) was incubated overnight with 1 M hydroxylaminein 0.5 M sodium phosphate (pH 6) at 37TC. DNA was purifiedand used to transform BZB1107 cells. The cells were platedand incubated with colicin N. From the resistant clones onantibiotic plates, cells expressing OmpF were selected. Fourtypes of substitutions were identified on the ompF gene bysequencing 34 selected clones (7). In addition to resistance tocolicin N, the mutant G119D also exhibited resistance tocolicin A.

Purification and Characterization of the Mutated OmpFPorin. The level of mutated OmpF synthesized was deter-mined by immunoblot analysis and was similar to the wild-type OmpF (data not shown). Extraction and purificationwere performed as described (14). The cells were broken

Present address: Ecole Superieure de Biotechnologie de Stras-bourg, Pole Universitaire Illkirch, rue Sebastien Brant, 67400Illkirch, France.ItTo whom reprint requests should be addressed.**The atomic coordinates and structure factors have been deposited

in the Protein Data Bank, Chemistry Department, BrookhavenNational Laboratory, Upton, NY 11973 (I.D. code 1MPF).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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using a French press, and envelopes were recovered bycentrifugation. Porins were then treated by preextractigeight times with a buffer containing 0.5% octyl-polyoxyeth-ylene, which eliminated the majority of contaminants. Fiveextraction steps with a buffer containing 3% octyl-polyoxyethylene allowed solubilization of integral membraneproteins. The porin obtained after this last step was purifiedby ion-exchange chromatography (DEAE-cellulose, Merck)followed by chromatofocusing (PBE94, Pharmacia) and fi-nally by gel filtration on Sephadex G-150 (Pharmacia). Puritywas checked by SDS/PAGE and isoelectrofocusing.

Bacteriodn Sensitiity. The colicin survival tests and thevarious bacteriocins (6, 15) were tested with cells grown inLB medium (0.1 ml of suspension at OD600 = 0.5 unit).Various dilutions (101 to 106) of colicins A or N were addedto the cells in 0.15 M NaCl/3 mM KCl/1 mM potassiumphosphate/10 mM sodium phosphate, pH 7, and incubatedfor 20 min at 37"C. The cell suspension was then diluted with15 vol of fresh LB medium. In the direct test, the percentageof surviving cells with or without bacteriocin treatment wasmonitored after 2 hr at 37"C by determining the ratio of theoptical densities at 600 nm. When the normal pathway wasbypassed (6, 16) by treatment at low ionic strength ("bypass"experiments), cells were washed twice in 10 mM sodiumphosphate (pH 6.8) and resuspended in the same buffer at theinitial density (ODWO = 0.5 unit). Portions (0.1 ml) of the cellsuspension were incubated with various dilutions of bacte-riocins (101 to 106) in this buffer and treated as in the directassay. The extent of cell survival was monitored by theprocedure described for the direct test.Measurement of K+ efflux resulting from the insertion of

colicin in the cytoplasmic membrane of sensitive bacteriaallows a quantitative determination of the colicin action.Variation of K+ concentrations was followed using a K+-selective electrode (17, 18). The buffer was 0.11 M sodiumphosphate/0.5 mM KCl, pH 7.2, and 109 cells per ml wereenergized by addition of 0.2% glucose. Initial rates werecalculated from the linear part of the kinetic of potassiumefflux after addition of colicin at various multiplicities (18).Planr Lipid Bilayers and Veside Swelling Assays. Double

quartz-distilled water and reagent grade chemicals wereused. Bilayers were formed across a 0.15-mm hole in Teflonsepta, pretreated with a solution of 2% n-hexadecane inn-hexane. Conductance measurements and the criteria forbilayer formation were as -described (19, 20). The transcompartment was held to virtual earth. The sign of themembrane potential referred to that on the cis side of themembrane, and the values quoted, therefore, refer to Vci, -Vtra8. Porins were always added to the subphase on the cisside of preformed bilayers, with the aqueous solution stirredby a magnetic bar. The membrane current was amplified witha current-voltage converter with an operational amplifier(Burr Brown, model 3528) and feedback resistors rangingfrom 106 to 109 fl. Recordings were filtered at 1 kHz with alow-pass filter [EG & G (Salem, MA) model 113] and re-corded on an FM tape recorder (Racal FM, Southampton,U.K.). All experiments were performed at room tempera-ture. Measurements of channel conductivities and voltagedependence were performed in 1 M NaCl/10 mM Hepes/5mM CaCl2, with a final pH 7.0. For the evaluation of ionselectivities, the reverse potential was generated by applying0.1 M NaCl in the trans side and 1 M NaCl in the cis side.Selectivities are expressed as the ratio of the permeability ofNa+ and Cl- ions, PNa/Pci (21). Swelling assays were per-formed as described (22).X-Ray Crystallography. Crystallization of the mutant

(G119D) was performed as described for wild-type protein(23). The crystals diffracted to 2.7 A but suffered fromradiation damage. A data set was, therefore, collected to3.0-A resolution (85% complete) from a single crystal on a

FAST area detector and processed by the program MADNES(24). The crystals of space group P321 have cell constants a= b = 117.9 A, c = 52.8 A, a = 13 = 90°, and fy = 1200. Theagreement between symmetry related reflections was R8y. =9.4% (34% in the highest resolution shell). Data reduction andmap calculations were performed by programs from theCCP4 package (25). The wild-type OmpF model includingordered water molecules and detergent fragments (10) servedas the starting model. The crystallographic R factor was23.9% (20-3.0 A). After remodeling of the site of mutationand conventional positional refinement using the programXPLOR (26), the final model had anR factor of 16.6% and goodstereochemistry (rms deviations of bond lengths and anglesfrom ideal values are 0.014 A and 1.8°, respectively). Tem-perature factors were taken from the wild-type structurewithout further refinement. The negative difference electrondensity at residue Asp-119 disappeared after assigning atemperature factor of 45 A2 to its atoms. The final differenceelectron density has a rms deviation of 0.034 electron perA3and extreme values of ±0.16 electron per Ak.

RESULTSHigh-Resolution Structure of G119D OmpF Porn. Trigonal

crystals (space group P321) of the mutant porin were readilyobtained using the established conditions for the wild-typeprotein (23). The structure was solved by the differenceFourier method at a 3.0-A resolution. The low R valueobtained with the wild-type model indicated that wild-typeand mutant crystals were virtually isomorphous. The differ-ence electron density map showed a distinctly altered courseof the protein backbone for segment at positions 119 and 120and a positive density for the aspartyl side chain at position119 (Fig. 1A). In the wild type, the segment at positions 119and 120 fit snugly to the inner barrel wall, whereas in themutant, it swung out toward the pore axis, with the carboxylgroup of Asp-119 near the cluster of basic residues at theopposite side of the pore (Fig. 1B). van der Waals distancesto residues Arg42, Arg-82, and Arg-132 were on the order of3 A. This effectively subdivides the channel into two sub-compartments, each with diameters of 3-4 A. Comparison ofthe mutant structure with that ofthe wild type shows no effecton the a-barrel. After superposition of all Ca-positions, therms deviation was 0.26 A, i.e., within the range of theestimated coordinate error.

Alterations of Channel Properties. In native porin, theconductance of OmpF porin channels is large (0.85 nS). Thechannels are slightly cation-selective and voltage-gated (21,27, 28). Channel activation usually occurs with high cooper-ativity of three channels, while inactivation and fluctuationsoccur independently as single steps. In vitro channel closingsare induced by characteristic threshold potentials (Vj), irre-spective of the polarity of the potential. The mutant pornshowed significant changes in its electrical properties com-pared to the wild type (Table 1). Conductance values weredecreased, the pores were more cation-selective, and thethreshold potential for channel closing was increased (Fig. 2).The permeation rates of sugars across the pores were altereddrastically. Also shown in Table 1 are the reductions of theflux rates of glucose and mannose by factors of 5- to 12-foldand that of arabinose by a factor of -2.

Sensitivity to Colicin N. Resistance of the mutant G119D tocolicin N, which requires the OmpF porin as the sole proteinto bind and to enter the bacterial cell, has been reported (7).In view of the structural results, it was of interest to deter-mine quantitatively the level of the sensitivity. Fig. 3 (A andB) shows that the sensitivity to colicin N ofthe G119D OmpFporin is decreased by a factor of =1000 relative to the wildtype. The effect on K+ efflux during colicin N action on E.coli cells is fully compatible with this result (Fig. 3C). Colicin

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E u 1 7

FiG. 1. (A) Stereoscopic view ofsegment containing positions 117-121 ofthe mutant G119D (the peptide backbone is indicated by solid bonds;the wild-type model is indicated by open bonds). Also shown is the electron density ofthe mutant porin, calculated with (2Fa,8-Fgjl) coefficientsand model phases (residues 118-121 were not used in the structure factor calculation). The contour level ofthe map is la,. (B) Model ofthe mutantOmpF porin structure in the channel constriction. The view is approximately along the pore axis. The slab shown is -25 A thick. Strands inthe a-barrel (periphery) are represented by broad arrows; the short helical segment is represented by a ribbon. Residues lining the poreconstriction are shown in full, and other loop segments are indicated by double lines. (C) For comparison, the model of the wild-type porin (10)is shown in the same view as in B.

binding, and its translocation across the outer membrane, iscontingent upon OmpF (6, 7) unless, at low ionic strength,colicin uses an alternative "bypass" pathway (6, 16). Under

Table 1. Comparison of the functional properties of thewild-type porin and G119D

Wild type Mutant RatioOmpF porin parameter (K-12) (G119D) mutant/wt

Electrical propertiesConductance, nS 0.85 0.55 0.65Selectivity, PNa/Pca 1.91 4.45 2.33Threshold voltage (Vj), mV 150 190 1.27

Sugar permeability*Glucose 0.354 0.060 0.17Mannose 0.388 0.030 0.08Arabinose 0.478 0.198 0.41

*Data are swelling rates (22) expressed as initial rates of decrease ofOD4. unit(s)/mm. All experiments were performed six times. SDvalues were 1-10% for OD values >0.3 unit and 5-20%6 for ODvalues <0.2 unit.

these conditions, the G119D mutation did not confer signif-icant resistance to colicin N (Fig. 3B).

DISCUSSIONIn the structure of wild-type OmpF porin, there is no spacefor a side chain in position 119. A change in the backboneconformation of the G119D mutation was, therefore, to beanticipated. X-ray structure analysis showed that the localstructural perturbation in the mutant protein is substantialand well defined (Fig. 1). Probably facilitated by the inher-ent conformational flexibility of the neighboring residueGly-120, the dipeptide (Asp119-Gly'20) adopts a new con-formation by protruding into the channel lumen. The con-formation of this segment may be largely stabilized byelectrostatic interactions between the carboxylate group ofAsp-119 and the cluster of basic residues on the oppositeside of the channel. The center-to-center distance to thenearest arginine residue (Arg-42) is 6.0 A and is, therefore,too large for a salt bridge, but the strong transversalelectrostatic field that exists across the pore (11) is further

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FIG. 2. Channel properties of the G119D OmpF in asolectin bilayers. Purified trimers from the G119 OmpF mutant were incorporated intoplanar bilayers by injecting detergent-solubilized protein into the bathing solution. The scanned trace shows the stepwise increases of themembrane current that followed protein injections. Each upward step corresponds to the conductance ofa trimer (1.6 nS). The closing downwardstep at the end of the recording corresponds to the closing of a single channel. (Inset) Current-voltage curve for G119D. The ionic current startsto decrease at applied potentials >200 mV. This negative resistance is due to channel closings. The critical threshold voltage (VY) ofthe wild-typeOmpF occurs >160 mV. The buffer used was 10 mM Hepes/1 M NaCl/5 mM CaCl2 at pH 7.0.

enhanced by the additional negative charge in the anioniccluster. Due to the changed backbone at positions 119 and120 and the protruding side chain of Asp-119, the cross-section of the mutant pore is drastically reduced. In the wildtype, the elliptical cross-section of the pore at the constric-tion site is 7 x 11 A (using van der Waals radii ofthe atoms),whereas in the mutant, the carboxylate group of Asp-119approaches Arg-42, Arg-82, and Arg-132 as close as 3 A,resulting in an effective division of the pore constrictioninto two subcompartments having diameters of 3 and 4 A,and a concomitant reduction of the overall cross-section byapproximately one-third.The reduced conductance of the G119D mutant and the

drastically reduced permeation rates of uncharged sugars(Table 1) may thus be attributed to the steric alterations andto the altered charge distribution. With ions, dehydrationmay play a vital role in the diffusion rates across the con-striction sites, since the hydrodynamic diameter of a hy-drated sodium ion is -5.5 A. The observed increase in cationselectivity is likely to be explained qualitatively by thepresence of the additional negatively charged carboxylategroup at the pore constriction, analogous to the anion selec-tivity that in PhoE porn is due to a single lysine residue(Lys-131). To assess this phenomenon quantitatively, mo-lecular dynamics calculations based on the x-ray structuresare required. The decreased sensitivity of the mutant porn

with respect to channel closing is significant, as these valuesare highly reproducible. It is conceivable that the energy levelof the closed state, the kinetic barrier for the transition, orboth are increased due to steric hindrance by the additionalside chain. In this context, it is interesting to compare amutation in OmpC porin (29) that conveys to cells the abilityto grow on substrates larger than the exclusion size of600 Da(30). Thus, maltodextrin is unable to diffuse across channelsof native OmpC porin but is able to diffuse into strains witha constriction-forming L3 loop that either carry mutationsencoding smaller side-chain residues or short deletions in thisloop. These changes increase the conductance of the porinand its sensitivity to voltage. These opposite effects in theOmpC mutants and that presented here indicate that the L3loop is indeed critical in the pore closing mechanism andsuggest that the voltage gating might correlate with theinherent flexibility of the L3 loop.A challenging problem persists: although the structural

alteration observed in the mutant porin explains the func-tional modifications rather well, its resistance to colicin N, onthe basis ofwhich it was selected, remains puzzling. The genesequence (7) and our present results show that the G119Dmutation has a strictly local effect in the constriction loop.The x-ray data reveal that the conformation ofthe barrel andof the six surface-exposed loops are unchanged. Since theseloops are involved in only a few weak crystal lattice inter-

Dilution Dilution Multiplicity10 20 30 40 50

WT

FIG. 3. Sensitivity to colicin, as revealed by bacterial survival or by K+ efflux. (A and B) The mutant strain (G119D) is significantly lesssensitive to colicin in the presence ofporin, except under "bypass" conditions (low ionic strength), but is considerably more sensitive than strainslacking porin. (C) Colicin N causes rapid K+ efflux from the wild type (WT) but very little from the G119D mutant. Survival was evaluatedfrom culture turbidity (OD600) after the addition of colicin N (circles) to the final dilution indicated. Colicin A (squares) is shown for comparison.Open symbols and dotted lines show the porin-dependent pathway, and "bypass" conditions are indicated by solid symbols. The strains usedwere the "porin-deficient" strain, the wild-type strain (WT), and the G119D mutant. For efflux measurements, 2 x 109 cells per ml,corresponding to 1 mg (dry weight), were incubated for 15 min at 37TC in 10mM Hepes/0.15 M NaCl, pH 7.2, supplemented with 0.2%6 glucoseand 0.6 mM KCl. Initial rates were calculated from the linear part of the kinetics of K+ efflux after colicin addition and expressed in nmol permg per min for colicin N at the multiplicities indicated in the top of the panel.

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actions, their structures are unlikely affected by forces sta-bilizing crystal contacts. Moreover, the same loop structuresare also seen in a different (tetragonal) crystal form ofwild-type OmpF porin (S. W. Cowan, R. M. Garavito, J. N.Jansonius, J. Jenkins, R. M. Karlsson, N. Konig, E. Pai,R. A. Pauptit, P. J. Rizkallah, J.P.R., G.R., and T.S., un-published data). Finally, the antigenic profile of the mutantporin is unchanged relative to the wild-type protein (7). Theexternal surface thus appears very similar, suggesting thefollowing explanations. (i) The receptor function of themutant porin is impeded. The constriction site may bedirectly involved in the binding of colicin N or, alternatively,the dynamics of the external loops may be more severelyaffected by long-range conformational changes than appearsfrom the data. (ii) Translocation of colicin N occurs throughthe porin channel. In the mutant, this process could beprevented due to steric hindrance at the pore constriction.This latter possibility is intriguing, as unfolding has beensuggested for the passage of colicin A (31, 32). The differ-ences observed between the two colicins appear to supportthis hypothesis, as the size of the receptor-translocatordomain of colicin A is nearly twice as large as that of colicinN (15, 33). This difference could indeed affect the unfoldingprocess significantly. Approaching this question by studyingthe molecular dynamics of porin appears interesting alsobecause it may give, at the same time, clues to the equilibriumof open and closed states of the native porin in vivo.

We thank Drs. D. Cavard and B. I. Holland for the generous giftsof colicins and plasmids and Dr. M. Luckey for expert advice withthe vesicle swelling assays. We gratefully acknowledge J.-M. Bolla,D. Cavard, R. El Kouhen, J. H. Lakey, and M. Mallea for helpfuldiscussions. We thank S. Scianimanico and N. Bleimling for excel-lent technical assistance. This work was supported by the CentreNational de la Recherche Scientifique and the Institut National de laSante et de la Recherche Mddicale (CRE 930610) to J.-M.P. and bygrants of the Swiss National Science Foundation to T.S. and J.P.R.

1. Nikaido, H. & Vaara, M. (1985) Microbiol. Rev. 49, 1-32.2. Benz, R. & Bauer, K. (1988) Eur. J. Biochem. 176, 1-19.3. Buehler, L. K., Kusumoto, S., Zhang, H. & Rosenbusch, J. P.

(1991) J. Biol. Chem. 266, 24446-24450.4. Nikaido, H. (1994) J. Biol. Chem. 269, 3905-3908.5. Pugsley, A. P. (1984) Microbiol. Sci. 1, 168-176.6. Fourel, D., Hikita, S., Bola, J.-M., Mizushima, S. & Pages,

J.-M. (1990) J. Bacteriol. 172, 3675-3680.7. Fourel, D., Mizushima, S., Bernadac, A. & Pages, J.-M. (1993)

J. Bacteriol. 175, 2754-2757.

8. Nikaido, H. (1989) Antimicrob. Agents Chemother. 33, 1831-1836.

9. Benson, S. A., Occi, J. L. L. & Sampson, B. A. (1988) J. Mol.Biol. 203, 961-970.

10. Cowan, S. W., Schirmer, T., Rummel, G., Steiert, M., Ghosh,R., Pauptit, R. A., Jansonius, J. N. & Rosenbusch, J. P. (1992)Nature (London) 358, 727-733.

11. Karshikoff, A., Cowan, S. W., Spassov, V., Ladenstein, R. &Schirmer, T. (1994) J. Mol. Biol. 240, 372-384.

12. Jeanteur, D., Lakey, J. H. & Pattus, F. (1991) Mol. Microbiol.5, 2153-2164.

13. Jackson, M. E., Pratt, J. M., Stoker, N. G. & Holland, I. B.(1985) EMBO J. 4, 2377-2383.

14. Garavito, R. M. & Rosenbusch, J. P. (1986) Methods Enzymol.125, 309-328.

15. El Kouhen, R., Fierobe, H.-P., Scianimanico, S., Steiert, M.,Pattus, F. & Pages, J.-M. (1993) Eur. J. Biochem. 214, 635-639.

16. Cavard, D. & Lazdunski, C. (1981) FEMS Microbiol. Lett. 12,311-316.

17. Boulanger, P. & Letellier, L. (1988) J. Biol. Chem. 263,9767-9775.

18. Bourdineaud, J.-P., Boulanger, P., Lazdunski, C. & Letellier,L. (1990) Proc. Natl. Acad. Sci. USA 87, 1037-1041.

19. Schindler, H. (1980) FEBS Lett. 122, 77-79.20. Wilmsen, H. U., Pugsley, A. P. & Pattus, F. (1990) Eur.

Biophys. J. 18, 149-158.21. Benz, R., Janko, K., Boos, W. & Lauger, P. (1978) Biochim.

Biophys. Acta 511, 305-319.22. Nikaido, H. & Rosenberg, E. Y. (1983) J. Bacteriol. 153,

241-252.23. Pauptit, R. A., Zhang, H., Rummel, G., Schirmer, T., Janso-

nius, J. N. & Rosenbusch, J. P. (1991) J. Mol. Biol. 218,505-507.

24. Messerschmidt, A. & Pflugrath, J. W. (1987) J. Appl. Crystal-logr. 20, 306-315.

25. CCP4 (1979) Science and Engineering Research Council Col-laborative Computing Project No. 4 (Daresbury Laboratory,Warrington, U.K.).

26. Brunger, A. T. (1990) X-PLOR Manual (Yale Univ., New Haven,CT).

27. Schindler, H. & Rosenbusch, J. P. (1978) Proc. Natl. Acad.Sci. USA 75, 3751-3755.

28. Schindler, H. & Rosenbusch, J. P. (1981) Proc. Natl. Acad.Sci. USA 78, 2302-2306.

29. Lakey, J. H., Lea, E. J. A. & Pattus, F. (1991) FEBS Lett. 278,31-34.

30. Misra, R. & Benson, S. A. (1988) J. Bacteriol. 170, 3611-3617.31. Bdn6detti, H., Lloubes, R., Lazdunski, C. & Letellier, L.

(1992) EMBO J. 11, 441-447.32. Webster, R. E. (1991) Mol. Microbiol. 5, 1005-1011.33. Baty, D., Frenette, M., Lloubes, R., Geli, V., Howard, S. P.,

Pattus, F. & Lazdunski, C. (1988) Mol. Microbiol. 2, 807-811.

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