Transmembrane α-Helix Interactions are Required for the Functional Assembly of theEscherichia coliTol Complex

JMB—MS 314 Cust. Ref. No. YAN 31/94

[SGML] J. Mol. Biol. (1995) 246, 1–7

COMMUNICATION

Transmembrane a-Helix Interactions are Required for the Functional Assembly of the Escherichia coli Tol Complex Jean Claude Lazzaroni1, Anne Vianney1, Jean Luc Popot2 He´le`ne Be´ne´detti3, Fadel Samatey2, Claude Lazdunski3 Raymond Portalier1 and Vincent Ge´li3* 1

Laboratoire de Microbiologie et Ge´ne´tique Mole´culaire CNRS Universite´ Lyon I 69622 Villeurbanne cedex France 2

Institut de Biologie Physicochimique, 13, rue Pierre et Marie Curie, 75005 Paris, France 3

Laboratoire d’Inge´nierie et de Dynamique de Syste`mes Membranaires, GDR 1000 CNRS, 31, chemin Joseph Aiguier, 13402 Marseille cedex 20, France

*Corresponding author

TolQ, TolR and TolA are membrane proteins involved in maintaining the structure of Escherichia coli cell envelope. TolQ and TolR span the inner membrane with three and with one a-helical segments, respectively. The tolQ925 mutation (A177V), located in the third putative transmembrane helix of TolQ (TolQ-III), induces cell sensitivity to bile salts and tolerance towards colicin A but not colicin E1, unlike a null tolQ mutation, which induces tolerance to all group A colicins. Since TolQ is required for colicin A and E1 uptake, in contrast to TolR, which is necessary only for colicin A, we hypothesized that the tolQ925 mutation might affect an interaction between TolQ and TolR. We therefore searched for suppressor mutations in TolR that would restore cell envelope integrity and colicin A sensitivity to the tolQ925 mutant. Five different tolR alleles were isolated and characterized. Four of these suppressor mutations were found to be clustered in the single putative transmembrane helix of TolR (TolR-I) and one was located at the extreme C terminus of the protein. In addition, we isolated a spontaneous intragenic suppressor localized in the first transmembrane helix of TolQ (TolQ-I). These observations strongly suggest that TolR and TolQ interact via their transmembrane segments. Sequence analysis indicates that Ala177 lies on the a-helix face of TolQ-III that, according to its composition and evolutionary conservation, is the most likely to be involved in protein/protein interaction. Energy minimization of atomic models of the wild-type and mutated forms of TolQ-III and TolR-I suggests that the deleterious effect of the A177V substitution arises from a direct steric hindrance of this residue with neighboring transmembrane segments, and that suppressor mutations may alleviate this effect either directly or indirectly, e.g. by affecting the stability of conformational equilibrium of the transmembrane region of the complex. Keywords: membrane protein; intramembrane helix-helix interaction; colicin transport; Tol system

The envelope of Escherichia coli provides a selective barrier against deleterious agents in the environment and allows the passage of small hydrophilic molecules only. Larger molecules, like siderophores or vitamin B12 , use energy-coupled uptake systems consisting of a specific receptor and the TonB, ExbB and ExbD proteins (reviewed by Kadner, 1990; Postle, 1990; Braun et al., 1991). Some bacteriophages and colicins have parasitized these bacterial transport systems in order to enter into the target bacteria. Four other proteins (TolQ, TolR, TolA and TolB), involved in the maintenance of the cell 0022–2836/95/060001–07 $08.00/0

envelope structure (Lazzaroni et al., 1989) but with no known physiological transport function, have also been parasitized by another group of bacteriophages and colicins (reviewed by Webster, 1991). The TonB and Tol systems share common features. ExbB and ExbD are related to TolQ and TolR, respectively, since sequence comparisons reveal 25% identity and 75% similarity (Eick-Helmerich & Braun, 1989). In addition, both TolQ and TolR proteins can be exchanged with ExbB and ExbD, respectively, in order to restore the uptake of colicins (Braun, 1989; Braun & Herrmann, 1993). TolQ and ExbB span the 7 1995 Academic Press Limited

JMB—MS 314 2 cytoplasmic membrane three times, with their N termini facing the periplasm (Kampfenkel & Braun, 1993a,b; Vianney et al., 1994), whereas TolR and ExbD are anchored to the cytoplasmic membrane by a single N-terminal transmembrane segment, their C termini facing the periplasm (Kampfenkel & Braun, 1992; Muller et al., 1993). TolA and TonB determine bacterial sensitivity to the different groups of phages and colicins (Braun & Herrmann, 1993). Both TolA and TonB are anchored to the cytoplasmic membrane by an N-terminal hydrophobic segment and feature a large periplasmic carboxy-terminal domain. The periplasmic domain of TonB has been shown to link the inner and outer membranes (Hannavy et al., 1990; Levengood et al., 1991; Roof et al., 1991; Levengood-Freyermuth et al., 1993). Exchange of the plasma membrane spanning regions of TonB and TolA indicates that these regions specify the dependence for ExbBD and TolQR, respectively (Karlsson et al., 1993a,b). These data, together with experiments showing that TonB and ExbD are stabilized by ExbB indicate that TonB probably forms a complex with ExbB and ExbD in the inner membrane and that the N-terminal transmembrane region of TonB participates in this interaction (Fischer et al., 1989; Skare & Postle, 1991). Furthermore, TonB can be chemically crosslinked in vivo into complexes that include ExbB and the outer membrane receptor FepA (Skare et al., 1993; Jaskula et al., 1994). Cofractionation of the Tol proteins with contact sites between the outer and inner membranes is consistent with the hypothesis that the Tol proteins form a similar complex (Bourdineaud et al., 1989; Karlsson et al., 1993a; Guihard et al., 1994). However, an interaction between the Tol proteins has not been demonstrated directly. In an earlier study, we isolated mutant tolQ925 (previously called exc-925 ), which carries a mutation (A177V) located in the third transmembrane segment of TolQ (TolQ-III; Lazzaroni et al., 1989; Vianney et al., 1994). A null tolQ mutant has a pleiotropic phenotype: it is tolerant to all group A colicins, resistant to filamentous phages, sensitive to bile salts, and it releases periplasmic proteins (Sun & Webster, 1987). In contrast, the tolQ925 mutant exhibits the last two phenotypes, but is tolerant only to colicin A (ColA) and not to colicin E1 (ColE1) or to filamentous phage f1 (Vianney et al., 1994). To further understand the ColA tolerance of the tolQ925 mutant, we have examined its sensitivity to hybrid colicins containing various regions of ColA and ColE1 (Be´ne´detti et al., 1991; Frenette et al., 1991). The results show that the cytotoxicity of the hybrids depends on their N-terminal domains (Table 1): colicins carrying the N-terminal domain of ColE1 are active against tolQ925, in contrast to colicins bearing the N terminus of ColA. Since N-terminal domains of colicins confer specificity in colicin uptake and TolQ is necessary for ColA and ColE1 to enter the cells, in contrast to TolR, which is required only for ColA uptake (Be´ne´detti et al., 1991), we hypothesized that the tolQ925 mutant

Communication

Table 1 Sensitivity of tolQ925 mutants to hybrid colicins Colicin(s) A E1 AE1E1 AE1A AAE1 E1AA E1AE1 E1E1A

Cytotoxic activity of hybrid × colcins† Indicator strain 1292 (wild-type)‡ JC5041 (1292tolQ925 ) 104 104 102 10 10 104 103 104

nk 104 nk nk nk 104 103 104

Colicins A, B and E1 were purified as described (Schwartz & Helinski, 1972; Cavard & Lazdunski, 1979; Pressler et al., 1986). Hybrid colicins between ColE1 and ColA were purified according to Be´ne´detti et al. (1991). For colicin activity tests, purified colicins (1 mg/ml) were diluted 1:10 serially from 1 to 10−4 in phosphate buffer (10 mM, pH 6.8, 0.2% (v/v) Triton X-100). Samples (1 ml) of each dilution were spotted onto a lawn of the indicator strain. † The colicin sensitivity was monitored by the highest dilution of colicin stock solution for which a clear zone of growth inhibition is observed on a lawn of the indicator strain; nk, the indicator strain is not killed by undiluted colicin. ‡ The genotype of strain 1292 is supE hsdS met gal lacY fhuA.

protein carries a defect that perturbs TolR when the Tol complex is assembled. As a consequence, the putative TolAQ925R complex would be able to translocate ColE1 but not ColA. This led us to search for tolR mutants able to suppress the tolQ925 mutation. The tolR gene was cloned into pT7-5 (Tabor, 1990) under the control of the phage T7 promoter. After treatment with nitrosoguanidine (0.4 mg/ml), a NcoI-HindIII fragment containing the mutated tolR gene (tolR*) was exchanged with the wild-type tolR gene from plasmid pAV553 (pT7-5 carrying the orf1tolQ295RA genes). The resulting plasmids were used to transform a tolQ925 mutant (JC5041). Suppressors of the tolQ925, able to grow in the presence of cholic acid, were selected. Nine tolR mutants were isolated and further characterized. The first step was to obtain quantitative data concerning the effect of each tolR mutation on the suppression of the tolQ925 allele. Plasmids carrying the tolQ925R*A cluster were used to transform a tolQ925 mutant. The level of periplasmic secretion was quantified by assaying the release of alkaline phosphatase to the extracellular medium (Table 2). Most of the tolR suppressor mutations restored the wild-type basal level of secretion. We then analysed the sensitivity of the suppressor mutants to cholic acid. All of them exhibited the wild-type level of resistance to cholic acid (Table 2). Finally, each JC5041 strain carrying the tolQ925R*A mutations was tested for its sensitivity to ColA and ColE1, as well as to colicin B (ColB), whose translocation depends on the TonB system (Table 2). All tolQ925*A strains were fully sensitive to ColE1 and ColB. The strains were also sensitive to ColA, except that carrying the tolR207 allele (Table 2). We conclude that the pleiotropic effects of the tolQ925 mutation are efficiently suppressed by most of the tolR suppressor mutations, while the tolR207 mutation suppresses the permeability defect of the

JMB—MS 314 3

Communication

Table 2 Phenotype of the tolR suppressors Cytotoxic activity§ Plasmid-borne allele +

Amino acid change in TolR

+

tolQ tolR tolQ925 tolR+ tolQ925 tolR201 tolQ925 tolR203 tolQ925 tolR205 tolQ925 tolR207 tolQ925 tolR210

Pro to Leu at 20 Ser to Asn at 42 Thr to Met at 139 Pro to Leu at 37 Ala to Val at 36

Alkaline phosphatase release (%)† 5 46 5 6 4 4 11

Sensitivity to cholic acid‡ R S R R R R R

Colicin A 4

10 nk 104 103 103 nk 104

Colicin E1 4

10 104 104 104 104 104 104

Colicin B 104 104 104 104 104 104 104

Strain JC7703 (tolQ925 metB pstS lacI ) was transformed with each plasmid and grown overnight in BD medium (10 g/l Difco Bactotryptone, 5 g/l Difco yeast extract, 5 g/l NaCl, adjusted to pH 8.3) supplemented with ampicillin and isopropyl-b,D-thiogalactopyranoside. Cells were centrifuged (8000 g for 10 min) and the supernatant was taken as the extracellular medium. The pellet was resuspended in 10 mM Hepes (pH 7.4) and cells were broken by passage through an Aminco French pressure cell 1138,000 Pa at 4°C). The resulting fluid was taken to assay the intracellular activity. Enzymatic assays for alkaline phosphatase (Torriani, 1967) and b-galactosidase (Miller, 1972) activities have been described. † The percentage of alkaline phosphatase activity recovered in the growth medium. Less than 2% of total b-galactosidase activity (used as a cytoplasmic marker) was recovered in the extracellular medium under such conditions. ‡ Ability of the bacteria to grow (R) or not grow (S) on plates containing 58 mM cholic acid. § Maximal dilution of colicin for which a plaque of growth inhibition of the indicator strain is detected. nk, the indicator strain is not killed by undiluted colicin.

outer membrane without restoring the sensitivity to ColA. tolR suppressor mutations were then tested for their own phenotypes. Plasmids containing the tolR mutations and the wild-type orf1, tolQ and tolA genes were used to transform strain JC8031 (JC1292DtolRA, which carries a deletion including the entire tolR gene and the beginning of tolA ). The transformants were tested for the phenotypes associated with a null tolR mutation. All the cells behaved like the wild-type strain, indicating that none of the tolR mutations caused an alteration leading to the loss of TolR activity. The allele specificity of the tolR suppressors was established by transforming the tolQ856, tolQ925, tolQ522 and tolQ528 mutant strains (Vianney et al., 1994) with plasmids containing each of the tolR suppressor mutations. Only the tolQ925 mutation originally used for isolating the tolR suppressors was suppressed (not shown). This allele specificity strongly suggests that tolQ and tolR interact directly. TolR is anchored in the inner membrane by a single transmembrane segment, TolR-I, corresponding to residues 16 to 43 (Mu¨ller et al., 1993). Given that the tolQ925 A177V mutation is located in transmembrane region of TolQ, we expected most of the tolR suppressors to affect TolR-I. Sequence analyses indeed revealed that eight out of the nine suppressor mutations are localized in this region (Table 2 and Figure 1). Five mutations correspond to the same P20L transition. Three other mutations, A36V, P37L and S42N, map close one to another in the same region of TolR-I. The mechanism(s) by which these four mutations might suppress the effect of tolQ925 will be discussed below. The last suppressor mutation, T139M, is located at the extreme C terminus of TolR, in a region whose residue distribution suggests that it may form an amphiphilic a-helix. The helix overall is rather hydrophilic and likely to be external to the

membrane. The effect of the T139M mutation, however, suggests that it may interact with the transmembrane region. In order to examine its sensitivity to externally added proteases, the Tol proteins were labelled with [35S]methionine and submitted to carboxypeptidase and proteinase K degradation after the cells had been converted to spheroplasts. The wild-type TolR protein was degraded by proteinase K, as were TolA, TolQ and the periplasmic TolB protein (Figure 2). As expected, the TolQ protein was only partially degraded, mainly

Figure 1. Position of the altered residues in the suppressor strains. The A177V transition corresponding to the tolQ925 mutation is boxed. The positions of the altered residues in the suppressor mutations are represented by asterisks(*).

JMB—MS 314 4

Communication

Figure 2. Susceptibility of the TolQ, TolR and TolA proteins to carboxypeptidase and proteinase K. 35Slabelled Tol proteins were expressed from pT7-1QRA derivatives as described (Vianney et al., 1994). Proteins were analysed after separation by SDS-PAGE (8% acrylamide) and autoradiography. Spheroplasts (O) were treated with 0.1 mg/ml carboxypeptidase A and B (C) or with 0.1 mg/ml proteinase K (K). The periplasmic TolB, Bla' (truncated polypeptide, 20 kDa), and the N-terminal part of the outer membrane protein Pal (Pal', 7 kDa) were used as controls. Note that untreated TolR207 migrated more rapidly than wild-type TolR, at the same level as the 17 kDa TolQ degradation product.

giving 17 and 5.5 kDa degradation products (Vianney et al., 1994). The tolQ925 protein was less degraded by proteinase K in the presence of the tolR201, tolR205 and tolR207 mutants than in the presence of wild-type TolR, suggesting that interaction between the two proteins affects the accessibility to TolQ. The C terminus of TolQ, which is located in the cytoplasm (Vianney et al., 1994), was not proteolysed by carboxypeptidase. Although the C termini of TolR and TolA are both located in the periplasm, they were protected from the carboxypeptidase treatment, in contrast to that of TolB (Figure 2). These results suggest that the C terminus of TolR is poorly accessible, as might be the case if it was involved in protein/protein interaction. Despite the presence in the NcoI-HindIII mutagenized DNA fragment of a region encoding the N-terminal transmembrane region of TolA, none of the isolated suppressors mapped within this region. However, the mutagenesis was not carried out under saturating conditions. On the other hand, as we characterized the tolR suppressor mutants, we identified a suppressor plasmid devoid of any mutational change within the tolR region, even though no spontaneous reversion of the tolQ925 mutation had occurred. The EcoRI-AvaI region of the plasmid (containing the 5' part of tolQ ) was found to be responsible for the suppression of the tolQ925 mutation. Sequencing of the entire fragment revealed a single base change resulting in an L19P substitution. Leu19 is localized at the periplasmic end of the first transmembrane segment of TolQ, TolQ-I (Vianney et al., 1994). This intragenic suppressor mutant was named tolQ6 and subcloned independently of the tolQ925 allele to characterize its phenotype. A pT7-1QRA derivative carrying the tolQ6 allele was able to complement all the tolQ

Figure 3. Wheel representation of the 3rd putative transmembrane helix of TolQ (TolQ-III) and of the single putative transmembrane helix of TolR (TolR-I). Helices were built using ideal a-helix parameters (3.6 residues/ turn), without taking into account distortions introduced by the proline residues. The most hydrophobic 17-residue stretch in each putative transmembrane segment was determined using the GES scale (Engelman et al., 1986). The differential conservation of helix faces was examined using the sequence alignment of E. coli TolQ (resp. TolR) with ExbB (resp. ExbD) of E. coli and of Pseudomonas putida (Kampfenkel & Braun, 1993a,b). Shaded boxes indicate positions that are not strictly conserved. Arcs indicate the face of each helix that may face the inside of the transmembrane region, according to hydrophobicity estimates (Engelman et al., 1986) and to the lipid-facing propensity of the residues (Samatey et al., unpublished results).

mutations, indicating that tolQ6 expresses a functional TolQ protein. In order to understand the mechanism by which suppressor mutations in putative transmembrane helices compensate for the tolQ925 mutation, we built and analysed a-helical models of wild-type TolQ-I, TolQ-III, TolR-I and of each of their variants. The tolQ925 mutation, A177V, is located close to the N-terminal periplasmic extremity of TolQ-III (Figure 1), away from the most variable face of this helix (Figure 3). In integral proteins whose three-dimensional structure has been established crystallographically, conserved helix faces generally correspond to protein/protein rather than protein/ lipid interfaces, an observation that has been corroborated by sequence analysis of other protein families (see Rees et al., 1989). Analysis of the amino acid composition of each face of TolQ-III, using either

JMB—MS 314 Communication

a hydrophobicity scale (Engelman et al., 1986) or a ‘‘propensity scale’’ defining the tendency of each type of residue to face either the lipids or the protein interior (Samatey et al., unpublished results), is also consistent with Ala177 lying in a region of protein/protein contact (arc in Figure 3). The A177V mutation is a rather discrete substitution of two methyl groups for two hydrogen atoms. It does not introduce any polar group in the putative transmembrane helix. It might act either by direct steric hindrance of protein packing in the transmembrane region or, indirectly, by preventing TolQ-III from achieving a correct conformation. In order to distinguish between these two hypotheses, we built energy-minimized models of TolQ-III and its A177V variant (see the legend to Figure 4). Due to the presence of a proline residue at position 187, both sequence segments minimize to quite distorted helices. Inspection by molecular graphics indicates that the wild-type and TolQ925 structures superimpose exactly (Figure 4(a) and (b); root-mean-square deviation of backbone atom positions over 33 ˚ ). Since the A177V mutation can be residues 0.08 A accommodated within the TolQ-III helix without structural rearrangements, the effect of the mutation probably results from a direct steric clash between Val177 and a neighbouring transmembrane segment. Such an effect of the quite conservative A/V substitution at a helix/helix interface has been observed in bacteriophage T4 lysozyme (Alber, 1989), and probably causes poor packing of transmembrane helices in the major proteolipid of central nervous system myelin in the jimpy msd mouse mutants (Popot et al., 1991). However, the sole effect of the A177V mutation might not be to weaken the association of TolQ-III with the rest of the transmembrane region. It may, for instance, if several conformation states are accessible, upset the equilibrium between them in favour of the state(s) where no steric clash occurs (see below). Two of the suppressor mutations within or close to TolR-I involve Pro to Leu substitutions (P20L and P37L). The presence of the proline residues in the wild-type helix causes strong distortions (Figure 4(c)). As expected, suppressors P20L (not shown) and P37L (Figure 4(d)) each remove one of the kinks in the helix, yielding large root-meansquare values when superimposition to the wild-type helix is attempted. Pro20, located at the N-terminal, cytosolic end of the helix, may not be part of it at all. Pro37 is more likely to be part of the putative helix, towards its periplasmic end. Interestingly, the P37L mutation restores membrane integrity, but not sensitivity to colicin A. The other two suppressor mutations in this region map close to the C-terminal (periplasmic) extremity of TolR-I (Figure 4(c)). Examination of the variability of residue positions and of the distribution of residue classes in this helix does not lead to a clear-cut prediction as to which helix face, if any, is turned towards the lipids, since there is no good agreement between the two criteria (Figure 3). However, the most conservative substitution, A36V, lies at the edge

5

Figure 4. Atomic models of (a) wild-type and (b) A177V TolQ-III and (c) wild-type and (d) P37L TolR-I. Models are seen from the membrane plane (top) and in projection on its cytosolic face (bottom). Peptides 33 residues long comprising the most hydrophobic 17-residue segment flanked by 8 residues on either end were built into a-helices using standard parameters (IUPAC-IUB, 1970). Proline residues were set in 1 or 3 possible conformations (Ponder & Richards, 1987), yielding 9 starting configurations for each of the 2 wild-type helices. Side-chain conformations chosen from a rotamer library were optimized using the SMD procedure (Tuffery et al., 1991). Global optimization was performed using the FLEX force-field (Lavery et al., 1986), with a fixed dielectric constant of 3. For both TolQ-III and TolR-I wild-type sequences, two initial choices of proline conformers converged to low-energy structures that were essentially indistinguishable on the basis of either energy or conformation. In all cases, except for the tolR210 (A36V) mutant, mutated helices converged to either 1 or 2 very similar structures. The A36V TolR helix yielded 2 optimal structures with comparable energies, one of them very similar to wild-type conformations, while the other presented an increased kink at the level of Pro37. Atomic models of the central 25-residue stretch of each model helix were visualized on a Silicon Graphics Indigo workstation using the software PROEXPLORE (Oxford Molecular, Strasbourg, France).

of the most conserved region and close to the face whose composition suggests a protein/protein interface. When the whole Ile16-Pro48 region is modelled as an a-helix, the S42N mutation leads to a pertubation of the helix backbone at its C-terminal (periplasmic) end (not shown). This results from a hydrogen bond that established between the carboxylic group of Asp46 and the amino group of Asn42. Whether this may occur in situ is uncertain, however, particularly since Asn42 and Asp46 may well lie in the polar head region of the bilayer and are not necessarily part of the transmembrane helix. The A36V mutation is likely to be located in the transmembrane region, in the periplasmic half of the

JMB—MS 314 6 helix. The predicted effect of this substitution is complex (see the legend to Figure 4) and it is difficult to conclude whether it acts by direct steric hindrance, as the comparable A177V mutation in TolQ-III seems likely to do. Whatever its exact effect, the localization of this mutation, together with that of P37L and S42N, suggests that the periplasmic extremity of TolR-I is important for its association with the rest of the Tol transmembrane region. The effect of the intragenic suppressor mutation tolQ6 (L19P) was examined using the same procedures. Leu19 is located at the N-terminal (periplasmic) extremity of TolQ-I. As expected, introduction of a proline residue prevents this segment from forming a regular a-helix (not shown). All tolR suppressor mutations, as well as the intragenic tolQ6 suppressor, end up with similar phenotypes; i.e. restoration of the membrane integrity and, in most cases, of the sensitivity to colicin A. The diversity of these mutations and of their localizations suggests that they might reverse the effect of the tolQ925 mutation by an indirect mechanism. One possibility would be that association of wild-type TolR and A177V TolQ distorts the structure of TolR in such a way that TolR (or the whole Tol complex) is not recognized by ColA anymore. The suppressors might weaken the TolR/TolQ association in such a way that TolR recovers a wild-type-like conformation. Alternatively, the suppressors may restore an equilibrium between conformational states of the Tol complex that is upset by the A177V tolQ925 mutation. In conclusion, our data provide the first direct evidence of an interaction between the TolQ and TolR proteins. This interaction involves the transmembrane region of both proteins. Our work is now aimed at confirming this genetic evidence by a biochemical approach, either by using crosslinking agents or by performing co-immunoprecipitation experiments. We also plan to combine these genetic and biochemical approaches to elucidate the way the other components of the Tol complex interact.

Acknowledgements We thank Dr R Lloubes for helpful discussion, Dr D. Cavard for the gift of colicin E1, Dr C. Etchebest for her help with computer and programs and Drs R. Lavery and P. Tuffery for allowing us to use programs developed by them. Work in the group of J.-L.P. was supported by the research program CM2AO (‘‘Conception Macromole´culaire Assiste´ e par Ordinateur’’) of Organibio. A.V. was supported by an MESR fellowship. Work in the group of C.L. was supported by the C.N.R.S. and the Fondation pour la Recherche Medicale.

References Alber, T. (1989). Mutational effects on protein stability. Annu. Rev. Biochem. 58, 765–798. Be´ne´detti, H., Frenette, M., Baty, D., Knibiehler, M., Pattus, F. & Lazdunski, C. (1991). Individual domains of

Communication

colicins confer specificity in colicin uptake, in pore-properties and in immunity requirement. J. Mol. Biol. 217, 429–439. Bourdineaud, J.-P., Howard, S. P. & Ladzunski, C. (1989). Localisation and assembly into the Escherichia coli envelope of a protein required for entry of colicin A. J. Bacteriol. 171, 2458–2465. Braun, V. (1989). The structurally related exbB and tolQ genes are interchangeable in conferring tonB-dependent colicin, bacteriophage, and albomycin sensitivity. J. Bacteriol. 171, 6387–6390. Braun, V. & Herrmann, C. (1993). Evolutionary relationship of uptake systems for biopolymers in Escherichia coli: Cross-complementation between the TonB-ExbBExbD and the TolA-TolQ-TolR proteins. Mol. Microbiol. 8, 261–268. Braun, V., Gu¨nter, K. & Hantke, K. (1991). Transport of iron across the outer membrane. Biol. Metals, 4, 14–22. Cavard, D. & Lazdunski, C. (1979). Purification and molecular properties of a new colicin. Eur. J. Biochem. 96, 517–524. Eick-Helmerich, K. & Braun, V. (1989). Import of biopolymers into Escherichia coli: nucleotide sequences of the exbB and exbD genes are homologous to those of the tolQ and tolR genes, respectively. J. Bacteriol. 171, 5117–5126. Engelman, D. M., Steitz, T. A. & Goldman, A. (1986). Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu. Rev. Biophys. Biophys. Chem. 15, 321–353. Fischer, E., Gu¨nter, K. & Braun V. (1989). Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB. J. Bacteriol. 171, 5127–5134. Frenette, M., Be´ne´detti, H., Bernadac, A., Baty, D. & Lazdunski, C. (1991). Construction and release of hybrid colicins. J. Mol. Biol. 217, 421–428. Guihard, G., Boulanger, P., Benedetti, H. Lloube´s, R., Besnard, M. & Letellier, L. (1994). Colicin A and the Tol proteins involved in its translocation are preferentially located in the contact sites between the inner and outer membranes of Escherichia coli cells. J. Biol. Chem. 269, 5874–5880. Hannavy, K., Barr, G. C., Dorman, C. J., Adamson, J., Mazengera, L. R., Gallagher, M. P., Evans, J. S., Levine, B. A., Trayer, I. P. & Higgins, C. F. (1990). TonB protein of Salmonella typhimurium. A model for signal transduction between membranes. J. Mol. Biol. 216, 897–910. IUPAC-IUB (1970). Comission on biochemical nomenclature. Abbreviations and symbols for the description of the conformation of polypeptide chains. Tentative rules (1969). Biochemistry 9, 3471–3479. Jaskula, J. C., Letain, T. E., Roof, S. K., Skare, J. T. & Postle, K. (1994). Role of the TonB amino terminus in energy transduction between membranes. J. Bacteriol. 176, 2326–2338. Kadner, R. J. (1990). Vitamin B12 transport in Escherichia coli: energy coupling between membranes. Mol. Microbiol. 4, 2027–2033. Kampfenkel, K. & Braun, V. (1992). Membrane topology of the Escherichia coli ExbD protein. J. Bacteriol. 174 (16), 5485–5487. Kampfenkel, K. & Braun, V. (1993a). Membrane topologies of the TolQ and TolR proteins of Escherichia coli: inactivation of TolQ by a missense mutation in the

JMB—MS 314 Communication

proposed first transmembrane segment. J. Bacteriol. 175, 4485–4491. Kampfenkel, K. & Braun, V. (1993b). Membrane topology of the ExbB protein in the cytoplasmic membrane of Escherichia coli. J. Biol. Chem. 268, 6050– 6057. Karlsson, M., Hannavy, K. & Higgins, C. F. (1993a). A sequence-specific function for the N-terminal signallike sequence of the TonB protein. Mol. Microbiol. 8, 379–388. Karlsson, M., Hannavy, K. & Higgins, C. F. (1993b). ExbB acts as a chaperone-like protein to stabilize TonB in the cytoplasm. Mol. Microbiol. 8, 389–396. Lavery, R., Parker, I. & Kendrick, J. (1986). A general approach to the optimization of the conformation of ring molecules with an application to valinomycin. J. Biomol. Struct. Dynam. 4, 443–462. Lazzaroni, J. C., Fognini-Lefebvre, N. & Portalier, R. (1989). Cloning of the excC and excD genes involved in the release of periplasmic proteins by Escherichia coli K-12. Mol. Gen. Genet. 218, 460–464. Levengood, S. K., Beyer, W. F. & Webster, R. E. (1991). TolA: a membrane protein involved in colicin uptake contains an extended helical region. Prac. Nat. Acad. Sci., U.S.A. 88, 5939–5943. Levengood-Freyermuth, S. K., Click, E. M. & Webster, R. E. (1993). Role of the carboxy-terminal domain of TolA in protein import and integrity of the outer membrane. J. Bacteriol. 175, 222–228. Miller, J. H. (1972). Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Mu¨ller, M. M., Vianney, A., Lazzaroni, J. C., Portalier, R. E. & Webster, R. E. (1993). Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity. J. Bacteriol. 175, 6059–6061. Ponder, J. W. & Richards, F. M. (1987). Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes. J. Mol. Biol. 193, 775–791. Popot, J. L., Pham-Dinh, D. & Dautigny, A. (1991). Major myelin proteolipid: the 4-a-helix topology. J. Membr. Biol. 120, 233–246. Postle, K. (1990). TonB and the Gram-negative dilemma. Mol. Microbiol. 4, 2019–2025. Pressler, U., Braun, V., Wittmann-Liebold, B. & Benz, R.

7 (1986). Structural and functional properties of colicin B. J. Biol. Chem. 261, 2654–2659. Rees, D. C., DeAntonio, L. & Eisenberg, D. (1989). Hydrophobic organization of membrane proteins. Science, 245, 510–513. Roof, S. K., Allard, J. D., Bertrand, K. P. & Postle, K. (1991). Analysis of the Escherichia coli TonB membrane topology by use of PhoA fusions. J. Bacteriol. 173, 5554–5557. Schwartz, S. A. & Helinski, D. R. (1972). Purification and characterization of colicin E1. J. Biol. Chem. 264, 6318–6327. Skare, J. T. & Postle, K. (1991). Evidence for a TonB-dependent energy transduction complex in Escherichia coli. Mol. Microbiol. 4, 2883–2890. Skare, J. T., Ahmer, B. M. M., Seachord, C. L., Darveau, R. P. & Postle, K. (1993). Energy transduction between membranes. TonB, a cytoplasmic membrane protein, can be chemically crosslinked to the outer membrane receptor FepA. J. Biol. Chem. 268, 16302–16308. Sun, T. P. & Webster, R. E. (1987). Nucleotide sequence of a gene cluster involved in entry of E colicins and single strand DNA of infecting filamentous bacteriophages into Escherichia coli. J. Bacteriol. 169, 2667–2674. Tabor, S. (1990). Expression using the T7 RNA polymerase/promoter system. In Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds), pp. 16.2.1–16.2.11, Wiley Interscience, New York. Torriani, A. M. (1967). Alikaline phosphatase from Escherichia coli. In Procedures in Nucleic Acid Research (Cantoni G. L. & Davies, D. L, eds), pp. 224–235, Harper and Row Publishers, Inc., NY. Tuffery, P., Etchebest, C., Hazout, S. & Lavery, R. (1991). A new approach to the rapid determination of protein side chain conformations. J. Biomol. Struct. Dynam. 8, 1267–1289. Vianney, A., Lewin, T. M., Bayer, W. E., Lazzaroni, J. C., Portalier, R. & Webster, R. E. (1994). Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity. J. Bacteriol. 176, 822–829. Webster, R. E. (1991). The tol gene products and the import of macromolecules into Escherichia coli. Mol. Microbiol. 5, 1005–1011.

Edited by M. Yaniv (Received 19 July 1994; accepted 27 October 1994)