412

The bacterial protein TolC assembles into an α-helical trans-periplasmic tunnel, which is embedded in the outer membraneby a contiguous β-barrel channel. TolC and its homologues thusprovide large exit ducts for a wide range of substrates, includingprotein toxins and antibacterial drugs, that are engaged byspecific recognition proteins in the cytosolic membrane.

AddressesCambridge University Department of Pathology, Tennis Court Road,Cambridge CB2 1QP, UK*Correspondence: Vassilis Koronakis*e-mail: vk103@mole.bio.cam.ac.uk

Current Opinion in Cell Biology 2001, 13:412–416

0955-0674/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsIM inner membrane OM outer membrane

IntroductionIn Gram-negative bacteria such as Escherichia coli,Pseudomonas aeruginosa and Salmonella spp, proteins des-tined for the cell surface or surrounding medium mustcross the cytoplasmic (inner) and outer membranes, andthe periplasm which lies between them. Several two-step mechanisms employ periplasmic intermediates(e.g., the type II general secretion pathway and theassembly of adhesion pili) or utilise a large number ofproteins in machineries spanning the envelope (e.g., thetype III assembly of flagella and invasomes) [1•]. Thetype I export mechanism, which exports large virulenceproteins like 110 kDa hemolysin of E. coli, contrasts withthese. It does not generate periplasmic intermediates,and it requires only an energised substrate-specific com-plex of two proteins in the inner membrane (IM) andTolC, or one of its homologues, in the outer membrane(OM) [2,3]. A closely related mechanism effects efflux ofsmall noxious compounds, including detergents, organicsolvents and antibacterial drugs such as nalidixic acid,and antibiotics like tetracycline, erythromycin and chloramphenicol [4]. As in the type I protein exportmachineries, these ‘efflux pumps’ comprise an OM TolChomologue cooperating with a substrate-specific IMcomplex [5,6].

The central mystery was how IM complexes could con-nect with a TolC homologue to bypass the periplasm,believed to measure at least 130 Å across, especially ifTolC only spanned the OM, like the widespread chan-nel-forming porins. This article reviews how elucidationof the TolC structure has resolved the underlying mech-anism and has revised our view of protein export anddrug efflux.

The TolC structure: a trans-periplasmicchannel-tunnelSince 1992 several OM proteins have been crystallised,including the OmpF and LamB porins [7,8], OmpA [9],phospholipase C [10] and the siderophore transporters FhuAand FepA [11,12]. The first indication that TolC might havea fundamentally different structure, and possibly contributeto a periplasmic bypass, was provided by electron microscopyof two-dimensional crystals, which indicated that it had a single pore and a domain outside of the membrane [13].Subsequent X-ray crystallography has revealed a remarkablestructure for TolC [14••]. Seen at 2.1 Å resolution, TolC is ahomotrimer that forms a hollow tapered cylinder 140 Å inlength. This comprises a 100 Å long α-helical barrel project-ing across the periplasmic space (the tunnel domain),anchored in the OM by a 40 Å long β-barrel (the channeldomain). A mixed α/β structure (the equatorial domain)forms a ‘strap’ around the mid section of the helical barrel.TolC thus assembles a water-filled conduit or exit duct witha diameter of 35 Å and a volume of 43,000 Å3. It is open tothe cell exterior, but the inner diameter decreases so that it isvirtually ‘closed’ at the periplasmic end (Figure 1).

Although an embedded β-barrel is a feature common to theother characterised OM proteins, that of TolC is quite distinctin its architecture. The other known OM proteins form onebarrel per protein monomer, including the porins (Figure 1),which are trimeric structures with each monomer forming aβ-barrel of 16 (e.g. OmpF [7]) or 18 (e.g. LamB [8]) β-strands.In TolC, the three molecules each contribute four β-strands toform a single 12 strand β-barrel. Electrophysiological mea-surements show that TolC does form a channel ([15];C Andersen, C Hughes, V Koronakis, unpublished data), butthe TolC interior is also unique with its cross sectional area of960 Å2, which is 15-fold larger than that of the general diffu-sion pore OmpF. TolC lacks the common structural elementof channel-forming proteins — an inwardly folded loop thatconstricts the internal diameter of the β-barrel [16] — and itdoes not have the plug domain of the larger β-barrels of irontransporters [11,12]. Nevertheless, TolC conductance is low([15], C Andersen, C Hughes, V Koronakis, unpublisheddata), presumably due to the closed periplasmic entrance.Notwithstanding these singularities of the TolC β-barrel, themost striking difference from other known OM proteins is the100 Å long periplasmic tunnel (Figure 1), comprising a previ-ously unknown fold, a 12 strand α-helical barrel. Like theβ-barrel channel, the tunnel is assembled from four antiparal-lel strands per monomer (two continuous long helices and twopairs of shorter helices).

A large family of channel-tunnelsThe TolC family is widespread among Gram-negative bac-teria and is variously involved in the export of largeproteins like toxins and the efflux of cations and noxious

Protein export and drug efflux through bacterial channel-tunnelsChristian Andersen, Colin Hughes and Vassilis Koronakis*

Protein export and drug efflux through bacterial channel-tunnels Andersen et al. 413

agents such as antimicrobial drugs [17]. Figure 2 depictsfamily members with known or putative function, arrangedas a phylogenetic tree on the basis of their amino acidsequence relatedness. The sequences of the amino- andcarboxy-terminal halves of the family members are similarto each other [18•], indicating that the family has evolvedby a gene duplication. The strongest identity between thetwo halves (29.1%) is seen in Bordetella pertussis CyaE,which is involved in cyclolysin toxin export. CyaE is alsonearest to the root of the tree, suggesting that it is closestto the family progenitor.

Sequence similarity correlates with exporter substrate-speci-ficity; indeed the proteins can be grouped into threesubfamilies corresponding to roles in protein export, cationefflux and drug efflux [17]. Although TolC and CyaE of theprotein export subfamily combine with IM complexes ener-gised by traffic ATPases [19,20], homologues of the drug andcation efflux subfamilies typically cooperate with the RND(resistance nodulation division) family of IM transport com-plexes, which derive energy from proton antiport [6,21•,22•].TolC homologues and their IM partners are usually encodedwithin the same operons, for example, the B. pertussis

cyclolysin operon cyaCABDE and the P. aeruginosa MDR(multi drug resistant) operon mexABoprM [23,24]. E. coliTolC is an exception, as it is not encoded by an export/effluxoperon (it is suggested to be part of the stress-induced mar-sox regulon [25]), and it can act with multiple IM partners toeffect protein export and drug efflux [17,21•,26,27••].

Conservation of the channel-tunnel structureThe amino- and carboxy-terminal halves of the 471 residueE. coli TolC protein can be structurally superimposed [14••],and this structural duplication is evident throughout thefamily. Furthermore, sequences determining the α-helicesand β-strands of the channel-tunnel structure do not varysubstantially in length: the long α-helices are constant at 67residues and the shorter helices are 23 and 34 residues. Thissuggests that the basic fold of the structure is conserved, andstudies of P. aeruginosa OprM confirm that deletions or inser-tions in the barrel domains are poorly tolerated [28•,29•].Although reported TolC homologues vary in length from414 to 541 amino acids, this is due primarily to variableextensions at the periplasmic amino and carboxyl termini.Significant sequence gaps or insertions occur only in themixed α/β equatorial domain, which is not part of the α/βbarrel structure, and in the extracellular loops.

Only a small number of amino acids are common to all theTolC homologues, and these and others that are well

Figure 1

The structures of TolC and porin proteins. (a) View down the OMchannels. In the case of TolC, this extends to approximately two-thirds ofthe height of the OM (i.e. the top 100 Å). (b) Side view, at right angles tothe plane of the OM. (c) Cross-section of TolC near the tunnel entrance(i.e. the bottom 50 Å). Red, green and blue indicate individual monomers.

(a)

(b)

(c)

Porins TolC

Current Opinion in Cell Biology

Figure 2

The TolC family. The 36 TolC homologues for which the function isknown, or strongly implicated by the location of their genes in theexport or efflux operons, sorted by TREEVIEW [37] on the basis of asequence alignment using MULTIALIN [38]. SilC1 may be an exceptionas it is reported to be involved in silver efflux. Scale ‘0.1’ indicates 0.1nucleotide substitutions. Note: the database contains an increasingnumber of homologues with no ascribed function to date. Ea, Erwiniaamylovora; Ec, Escherichia coli; Ech, Erwinia chrysanthemi;Se, Salmonella enteritidis; Sm, Serratia marcescens;Pa, Pseudomonas aeruginosa; Pf, P. fluorescens.

Current Opinion in Cell Biology0.1

CnrCNccC

HelC

CzcC

CzrC

CyaE

RsaFSapF

HasF(Sm)

TolC(Ec)TolC(Se)

ZapDLipD

PrtF (Ea)AprF (Pa)

PrtF(Ech)

OmpX

TliFAprF(Pf) EprF

Orf140

FusA OprAOpcM

OprNNodT3NodT2NodT1

OprJ.SmeCSrpCTtgCOprM

MtrESilC1

Drug efflux

Proteinexport

Cation efflux

HasF(Pf)

414 Membrane permeability

conserved correlate with features that determine the corestructure [14••,17]. At or near the tunnel entrance, glycinesfacilitate a tight turn between the helices forming theentrance, and small residues such as alanine and serine atthe interface of tunnel-forming helices allow a very densepacking, which determines the tapering and closure.Aspartic acid residues maintain the electronegative innersurface at the entrance, which may influence substratemovement. Towards the other end of the structure, thetransition from the left handed α-helices of the periplasmicdomain to the right handed β-strands of the OM domain isaccommodated by proline-containing linkers (although forone of the four strands there is divergence in the efflux sub-families, with glycines mixed in with or replacing prolines).At the bottom of the β-strands, aromatic residues face out-wards to form a ring around the channel domain, delimitingthe inner edge of the lipid bilayer. This is seen in all knownOM protein structures and possibly has an anchor function.These comparisons show that the defining structural ele-ments of TolC are conserved.

A common mechanism of export and efflux viachannel tunnelsTolC is an integral part of the type I protein exportmachinery; without it there is no ‘half-way’ export to theperiplasm or from spheroplasts [13]. In vivo crosslinkinghas shown that export is effected by transient recruitment

of TolC by the IM complex in response to engagement ofthe substrate [27••]. The TolC structure has revealedvividly how the periplasmic space is bypassed. One canreadily visualise how substrate-laden IM complexesrecruit and open cognate channel-tunnels, providinggated exit of diverse molecules from the cell interiorthrough trans-periplasmic ducts to the external environ-ment (Figure 3). From the OM exit down to the equatorialdomain, TolC is a uniform cylinder with an inner diame-ter of 35 Å, through which even large proteins couldreadily pass. In contrast, the periplasmic entrance of TolC must undergo conformational change to allow pas-sage of substrate.

An allosteric mechanism has been proposed [14••], wherebya realignment of the inner pair of helices of each monomerrelative to the outer pair could open the entrance, like aniris, to a diameter of 30 Å. It is envisaged that this openingwould be triggered and stabilised by the recruiting IM com-plex. The principal contact is made by the IM accessoryprotein, which, like TolC, is trimeric [27••]. It can be regard-ed as a dynamic adaptor between the channel-tunnel andthe energy-providing, substrate-binding IM protein. Itmight contact TolC at the tunnel entrance, and its predictedcoiled-coil structures [18•] could repack against the coiled-coils of the tunnel α-barrel and/or reach to the equatorialdomain to effect opening of the entrance (Figure 3). Such a

Figure 3

Current Opinion in Cell Biology

ATP ∆H+

Protein export Drug efflux

OM

IM

Protein

Drugs

(f)(e)

(d) (c)

(b)

(a)

?

? ?

Channel-tunnels acting in export and efflux. A possible model indicatingreversible interaction of trimeric TolC homologues (green) with substrate-specific IM complexes containing an adaptor protein (red/blue) and anenergy-providing protein of either the traffic ATPase (protein export, yellow)or proton antiporter families (small molecule efflux, purple). E. coli TolC canact in both pathways, whereas in other bacteria multiple TolC homologuesact separately in distinct pathways. The pre-assembled IM complex (a) is

engaged by substrate (b) and the trimeric adaptor protein contacts theperiplasmic tunnel (c), possibly via the predicted coiled-coil structures (d),prompting the conformational change that opens the entrance andpresents the exit duct (e). Following transport, the components revert tothe resting state (f). The steps have not been demonstrated in the effluxsystems, so there may be some differences. An animated model of proteinexport is available at http://archive.bmn.com/supp/ceb/ani1.html

Protein export and drug efflux through bacterial channel-tunnels Andersen et al. 415

view of protein export envisages signal transduction acrossthe IM, most likely via the transmembrane and amino-ter-minal cytosolic domains of the adaptor. Although somedifferences may emerge in the mechanism underlying effluxof small molecules (for example, some adaptor proteinhomologues involved in these systems have only periplas-mic domains anchored to the IM), the conservation of keyfeatures among TolC homologues establishes a commoncore mechanism for the export and efflux systems.

ConclusionsStructural and biochemical data have come together to pro-vide a clear view of how a wide range of molecules utilisethe TolC family to bypass the periplasm and exit the bac-terial cell. The mechanistic details of how large and smallsubstrates enter channel-tunnels are now accessible. Howthe tunnel entrance operates is clearly a core question; thedense packing of the helices at the periplasmic entrance ofthe channel-tunnel suggests a very stable structure, and ourin vitro experiments show that opening cannot be inducedby high voltage, low pH or urea (C Andersen, C Hughes,V Koronakis, unpublished data). An understanding of theopening mechanism will require assessment of the residuescentral to stability of the entrance iris and also to tunnelinteraction with the adaptor during substrate-triggeredrecruitment (the resting state might already involve a looseinteraction, and there may be unique features of substrateaccess in the efflux systems).

Other questions that remain to be answered include how is thetrimeric structure assembled and how is the peptidoglycanlayer negotiated (perhaps cellular enzymes that degrade thepeptidoglycan might have a function in TolC assemblyanalagous to the dedicated muraminidase needed for assem-bly of flagella on the cell surface [30]). Future structures of theIM transport components may eventually assimilate such datainto a comprehensive picture of export/efflux machinery oper-ation (traffic ATPase cytosolic domains have now beencrystallised and their structures determined [31, 32]). Whetherunderstanding TolC is relevant to other examples of bacterialmembrane translocation [1•], or indeed to mitochondrialimport [33•,34•], is not yet known. From an applied perspec-tive, the TolC-dependent type I export system has beeninvestigated as a means to deliver heterologous antigens fromattenuated bacteria used as live vaccines [35,36], and the con-served structure of the TolC family among Gram-negativebacteria could strengthen the case for its use. Perhaps ofgreater importance, the multifaceted contribution of channel-tunnels to bacterial survival during infection suggests theymay present a possible chemotherapeutic target. It is possible,for example, that drugs could be designed to bind the con-served negatively charged residues at the periplasmic entranceto effect irreversible closure of the tunnel, thus reducing viru-lence and survival against chemotherapeutic drugs.

AcknowledgementsWe thank Eva Koronakis for continued support and helpful criticism of themanuscript. Our work is supported by a Medical Research CouncilProgramme grant (VK & CH). CA is an EMBO fellow.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

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multidrug efflux and protein export. Nature 2000, 405:914-919.Recombinant TolC protein was detergent-extracted from E. coli membranes.Crystallisation occurred in a detergent mixture, and the TolC structure wassolved by multiple wavelength anomolous dispersion (MAD) using oxidizedselemnomethionine derivatives.

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416 Membrane permeability

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