Threes company: component structures bring a closer view of tripartite drug efux pumps

Jeyanthy Eswaran1, Eva Koronakis1, Matthew K Higgins1,2, Colin Hughes1 and Vassilis Koronakis1,3
Bacterial multidrug resistance is a serious clinical problem and is commonly conferred by tripartite efux pumps in the prokaryotic cell envelope. Crystal structures of the three components of a drug efux pump have now been solved: the outer membrane TolC exit duct in the year 2000, the inner membrane AcrB antiporter in 2002 and the periplasmic adaptor MexA in 2004. These structures have enhanced our understanding of the principles underlying pump assembly and operation, and present pumps as new drug targets.
Addresses 1 Cambridge University Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK 2 Current address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK 3 e-mail: vk103@mole.bio.cam.ac.uk

Current Opinion in Structural Biology 2004, 14:741747 This review comes from a themed issue on Proteins Edited by Wim GJ Hol and Natalie C Strynadka

(RND) or major facilitator superfamily (MFS) [7], cooperates with a protein of the TolC exit duct family, which is anchored in the OM and projects across the periplasm. The third essential component of active pumps is an adaptor protein, which is largely periplasmic and anchored to the IM by a single transmembrane (TM) helix or an N-terminal lipid moiety. Pathogenic bacteria typically have several tripartite pumps with broad and often overlapping substrate specicities; for example, P. aeruginosa has at least four distinct major efux (Mex) systems [8], whereas the major efux pump of E. coli, AcrAArcBTolC, determines resistance to antibiotics, dyes, detergents, bile salts and organic solvents [9,10]. The crystal structures of the pump components MexA, AcrB and TolC have now been solved. In this review, we discuss how the components might assemble into an active tripartite drug efux pump in the bacterial cell envelope and how such pumps may operate to expel drugs from the cell.

Structure of the tripartite pump components

0959-440X/$ see front matter # 2004 Published by Elsevier Ltd. DOI 10.1016/j.sbi.2004.10.003

AcrB: an energy-providing, substrate-binding component in the inner membrane

Abbreviations FAD avin adenine dinucleotide IM inner membrane ITC isothermal calorimetry MFS major facilitator superfamily OM outer membrane RND resistance nodulation division TM transmembrane

Introduction
Gram-negative pathogens such as Escherichia coli and Pseudomonas aeruginosa employ membrane efux systems to export antibacterial drugs and other small noxious chemicals, as well as large protein toxins, from the cell [13]. This requires translocation across both the inner (cell) and outer membranes (IM and OM), and the intervening periplasmic space. Multidrug efux pumps comprise three components, each a member of an extensive protein family [47]. An energy-providing integral IM protein, either an ABC transporter or more often a proton antiporter of the resistance nodulation division
www.sciencedirect.com

The architecture of the E. coli 1049 amino acid proton antiporter AcrB, a drug efux transporter of the RND [11,12] (Figure 1). family, has been solved at 3.5 A diameter TM AcrB is a trimer with a 50 A long and 100 A domain comprising 36 a helices (12 from each monomer). There is minimal contact between monomers in the TM domain, forming a chamber thought to be lled with lipid. At the core of this domain, TM helices 4 and 10 are suggested to form the proton translocation pathway, in which residues Asp407, Asp408 and Lys940 are possibly central to gating. On the membrane-exposed surface of the domain is a vertical groove between helices TM7 and TM8, at the base of which is tilted helix TM9, perhaps providing access for membranelocated substrates. In the large periplasmic domain, the monomers, each 700 amino acids, are tightly interlocked and form a closed central pore. An internal cavity open to the periplasm could provide access to periplasmic substrates, allowing RND transporters to act as cytosolic membrane and periplasmic vacuum cleaners. The top of the periplasmic domain forms a funnel-like , similar to structure with an internal diameter of 30 A the diameter of the modelled open state of the TolC entrance, and has been termed the TolC-docking domain (Figure 1).
Current Opinion in Structural Biology 2004, 14:741747

742 Proteins

Figure 1

Structures of the three drug efflux pump components. The solved AcrB, TolC and MexA structures are shown as ribbon diagrams. Protomers of the trimeric AcrB and TolC proteins are coloured blue, red and green, whereas the MexA monomer is coloured by secondary structure: a helices red, b strands green and loops blue. The dashed lines in MexA indicate the unsolved structure (28 residues of the N terminus and 101 residues of the C terminus); the asterisk indicates the fatty acid modification. A surface representation of the MexA monomer is shown at far right. Small conserved residues in the hairpin domain are coloured light green, whereas the larger hydrogen-bonding residues at either end of the a-helical hairpin are coloured dark green.

TolC: an exit duct for polypeptide and drug substrates

resolution, the TolC homotrimer (Figure 1) is At 2.1 A in length. This comprises seen as a tapered cylinder 140 A long OM b barrel, which anchors a contiguous aa 40 A across the periplasmic helical barrel projecting 100 A space [13,14]. A third domain, a mixed a/b structure, forms a strap around the mid-section of the a-helical barrel. The average accessible interior diameter of the . Three TolC monomers single central TolC pore is 19.8 A each contribute four b strands to form the twelvestranded b barrel, which is constitutively open to the cell exterior. The periplasmic a barrel comprises twelve antiparallel a helices (two continuous long helices and two pairs of shorter helices from each monomer) that pack laterally side-by-side and form two separate interfaces. The helices follow a left-handed superhelical twist that tends to be underwound in the upper half compared to helices in a conventional two-stranded coiled coil, enabling the helices to lie on the surface of a cylinder [15]. In the lower half of the a barrel, neighbouring
Current Opinion in Structural Biology 2004, 14:741747

helices form six pairs of regular two-stranded coiled coils, but one from each monomer folds inwards at the periplasmic end. This constricts the entrance to establish a resting closed state with an effective diameter of approxi ; this is reected in the small conductance of mately 3.9 A TolC in lipid bilayers [16,17].
MexA: a periplasmic adaptor linking the inner and outer membrane components

The structure of approximately two-thirds of the 360residue mature MexA protein from P. aeruginosa has been solved (the 28 N-terminal and 101 C-terminal residues were not ordered in the crystal) [18,19]. The monomer (Figure 1) has an elongated structure of three linearly arranged subdomains; a b barrel, a lipoyl domain, and a (64-residue) a-helical hairpin comprising a straight 47 A C-terminal helix and an N-terminal helix with a lefthanded superhelical twist. The exposed faces of the two helices, directly opposite the core of the hairpin, contain conserved residues, such as alanine in the f position of the
www.sciencedirect.com

Drug efflux pumps Eswaran et al. 743

helical heptad, and serine and glutamic acid in the c position (Figure 1). At either end of the a-helical hairpin lie large hydrophilic residues with the potential to engage in hydrogen bonding. Flanking the hairpin are elements structurally homologous to the lipoyl domains of pyruvate dehydrogenase; their carbonyl chains have a root mean [20]. Each lipoyl square deviation (rmsd) of only 1.6 A domain comprises two interlocking motifs of four b strands, but although these are conserved throughout the family of adaptor proteins, they are separated by variable lengths of intervening sequence, which forms the a-helical hairpin [5]. MexA has four heptad repeats in each of its helices; other adaptor proteins have ve or six heptads and will form longer hairpins. The third subdomain contains six antiparallel b strands, forming a b barrel with a single a helix situated at one entrance to the barrel. This structural element has been found in diverse contexts, such as the FAD-binding domain of avodoxin reductase [21], odorant-binding domains [22], isomerase FKBP [23] and the pleckstrin homology (PH) domain [24].

alone form a stable interaction, illustrating the central role of the adaptor protein in bridging the integral IM and OM components and stabilizing their assembly. The elongated modular structure of periplasmic adaptors would allow contact with the OM exit duct via their long periplasmic hairpin, while using a distinct C-terminal domain to interact with cognate IM transporter components [26,28].

Structural model of the assembled pump

The direct efux of drugs and other substrates across the Gram-negative cell envelope requires assembly of a contiguous proteinacious structure that allows passage from the cell cytosol or membrane to the outside medium, without leakage into the periplasmic space. A preliminary model of a complete pump must reect the known component structures, interactions and stoichiometries; that in Figure 2 illustrates a 870 kDa transenvelope
Figure 2

Assembly of the efux pumps

Interactions between the three components were initially established for the closely related type I protein export machinery [25]. In vivo chemical cross-linking showed that, when the IM transport ATPaseadaptor complex is engaged by substrate, it recruits TolC to establish a contiguous structure spanning the envelope. Assembly of this tripartite machinery is transient; once the large substrate is translocated, the components disengage and revert to the resting state [25]. By contrast, the AcrA adaptorAcrB antiporterTolC efux machinery appears constitutively assembled (i.e. independent of the drug substrate) [26]. This apparent difference between the export and efux systems possibly reects the requirements imposed by the different substrates. Whereas polypeptide export systems translocate substrates of 1000 amino acids or more, it is estimated that approximately 500 toxic ethidium molecules are expelled per second by each P. aeruginosa MexABOprM pump [27]. Frequent assembly and disassembly of the drug efux pumps might be energetically inappropriate. The periplasmic contact between the IM and OM components has been suggested to involve the TolC entrance coils and the apex of the AcrB antiporter (Figure 1), whether restricted to the six hairpins at the tip [11] or extending further down the antiporter structure. However, although AcrB and TolC can be isolated as a complex after in vivo cross-linking, no interaction is seen when the two puried proteins are studied by isothermal calorimetry (ITC) [26]. By contrast, ITC conrms that the AcrA adaptor establishes energetically favourable interactions with both AcrB and TolC. This is compatible with the view that, although the periplasmic domains of AcrB and TolC are in close proximity in vivo, they cannot
www.sciencedirect.com

Model of the assembled tripartite drug efflux pump. This possible model of an RND class drug efflux pump is based on the open-state model of TolC (red) forming a minimal contact interface with the six hairpins at the apex of AcrB (green). A ring of nine MexA molecules (blue) is modelled to form a sheath around AcrB and the a barrel of TolC (MexA is a close homologue of AcrA, the natural partner of AcrB/TolC). Variants of the model might include a lower order oligomer of MexA [19], and more extensive interaction between AcrB and TolC. Models of assembled pumps containing distinct IM transporters, such as traffic ATPases or MFS class antiporters, will presumably differ, especially as they have smaller periplasmic domains. Current Opinion in Structural Biology 2004, 14:741747

744 Proteins

long. Although the solved adaptor complex over 270 A protein structure is that of P. aeruginosa MexA, this protein is closely related to AcrA, the adaptor in the AcrAAcrBTolC drug efux pump of E. coli. The MexA structure is therefore included with the AcrB and TolC structures to depict a possible model of the complete pump assembly. TolC and AcrB are clearly trimeric proteins located in the OM and IM, respectively, but the oligomeric state of the adaptor in active pumps is not known. The adaptor is monomeric in solution, and oligomerisation may be induced by contact with one or both of the other membrane components. During assembly of the active efux pump, the hairpins of the adaptor could directly engage the inner and/or outer coiled coils of the TolC a-helical barrel, compatible with the adaptor assembling into trimers or hexamers. Cross-linking of in vivo complexes using the short-arm chemical cross-linker DSG (disuccinimidyl glutarate) has identied adaptor trimers in both the drug efux and protein export systems [25,29], whereas a hexamer has been suggested on the basis of the relative cellular abundance of the components [27]. On the other hand, in the MexA crystal, molecules pack side-by-side to form two twisted arcs of six and seven monomers (Figure 3) [18,19], with interaction interfaces formed by the stripes of conserved residues that lie on the exposed faces of the a-helical hairpins (Figure 1). Based on this propensity of MexA to pack side-by-side, a ring formed from nine MexA molecules can be modelled.
Figure 3

This would have a curvature similar to that observed in the crystal packing (Figure 3), and would be sufciently large to form a sheath around the open-state model of TolC [13] and so provide a seal against the periplasm. This ninefold symmetry might correlate with the nine short a helices that are located within the exible equatorial domain around the TolC a barrel. Notwithstanding these possibilities, the stoichiometry of the adaptor in the pumps and details of adaptor interaction with TolC remain uncertain. Indeed, conservation of the a-helical hairpin among adaptors has encouraged comparison with viral membrane fusion proteins [10,30]. The suggested mode of action would require that the adaptor spans the periplasm, with the N terminus anchored in the IM and the C terminus interacting with TolC or the OM. Hairpin formation would occur reversibly, with the two long a helices acting to draw together the IM and OM. However, the MexA structure shows that residues 29 and 259 lie of each other in the b-barrel domain, positionwithin 5 A ing the C terminus in close proximity to the N terminus, not near the OM, and reversible disruption of the three stable subdomains of the adaptor seems unlikely.

Substrate binding and translocation

Drug efux substrates could enter the transenvelope efux conduit via AcrB, from either side of the cytoplasmic membrane or from the periplasm. Soaking AcrB crystals with efux substrates suggested that they bind at different positions in the TM domain [31], although this view was not conrmed by similar experiments [32] and analysis of hybrid transporters indicates that the antiporter periplasmic domain plays a major role in substrate specicity [33,34]. In the protein export system, the large substrate is not only engaged by the IM trafc ATPase, but also contacts the short cytoplasmic domain of the adaptor [35]. A signal must be transduced to trigger recruitment and opening of TolC. In the constitutively assembled drug efux pumps, it is specically the TolC opening step that would need to be substrate responsive. How this might be coordinated with opening of the AcrB pore domain is one of many open questions. As AcrB-type antiporters have a cavity that appears to communicate with the periplasm via the vestibules [12], it is unclear how drugs are prevented from escaping back into the periplasm. Once past the AcrBTolC junction, substrates encounter the electronegative interior surface of TolC [13,14], which may have implications for transport. A pulse of cations (protons) early in transport might favour the entry of acidic and hydrophobic substrates into the channel, whereas later it might catalyse the release of basic molecules.

Comparison of oligomeric MexA and trimeric TolC. The oligomeric crystal packing of MexA, with seven monomers forming a twisted spiral-arc (top), is compared to the diameter of trimeric TolC (bottom), both shown in top view. Current Opinion in Structural Biology 2004, 14:741747

Twist to open access to TolC by realignment of entrance helices

Access through the TolC periplasmic entrance is a key event in the function of export and efux machineries.
www.sciencedirect.com

Drug efflux pumps Eswaran et al. 745

Figure 4

States of the TolC periplasmic entrance (all viewed from the periplasm). Space-filled depictions of the closed and modelled open states of TolC, and a ribbon representation of the Co(NH3)63+-blocked a-barrel entrance, with the bound ligand in the centre coloured red. Pictured far right is the ligand coordinated by Asp374 sidechains.

A proposed allosteric mechanism for entrance opening is based on the small differences in superhelical twist between the inner and outer coiled coils, suggesting that opening occurs by inducing realignment of the inner coils with respect to the outer coils (Figure 4) [13]. Experimental disruption of three intermolecular and intramolecular links that constrain the three inner coils in the closed conformation allows enlargement of the aperture diameter, as seen by increased conductivity of these engineered TolC variants in planar lipid bilayers [36]. Furthermore, when movement of these helices is constrained by introducing disulde bonds, translocation of polypeptide substrate is abolished [37], further supporting a model in which transition to the TolC open state is achieved by an iris-like realignment of the entrance helices. Electrophysiology shows that the open state may be unstable [36]; if so, it might be stabilized by repacking the disrupted open-state TolC helices to those of the adaptor or by interaction with the antiporter. It remains possible that opening to a large aperture is required only for the transport of high molecular weight polypeptide substrates.

of TolC. This is the sole constriction of the exit duct and is lined by an electronegative ring of six aspartate residues, Asp371 and Asp374 from each monomer, which determine a high-afnity metal-binding site [38,39]. TolC function in articial lipid bilayers is severely inhibited by divalent cations, and trivalent cations such as Cr3+, Tb3+ and hexammine cobalt block the TM ion ux at nanomolar concentrations. When the entrance aspartates are substituted, high-afnity binding is abolished and blocking of the membrane pore is alleviated [38,39]. A crystal structure of the TolCCo(NH3)63+ complex (Figure 4) conrms a ligand molecule bound at this site [39]. This rst biochemical and structural characterisation of an in vitro inhibitor of TolC may suggest a strategy to develop bioactive molecules, especially as the electronegative entrance is widely conserved throughout pumps central to virulence and drug resistance.

Acknowledgements
Our work is supported by the Medical Research Council and Wellcome Trust.

References and recommended reading Perspective efux pumps as targets in multidrug-resistant bacteria?
The combined biochemical and structural data have provided a closer vision of the assembly and operation of the large efux machines spanning the Gram-negative cell envelope. Further studies will pursue details of the underlying dynamics (e.g. key coiled-coil interactions and channel gating) and visualization of the entire tripartite structure. Such knowledge will also facilitate the design of potential antibacterial agents for the treatment of multidrug-resistant infections. Pump function could be inhibited at several points, including drug-binding sites in the IM transporter AcrB, the component interactions underlying assembly and the energy cycle of the IM transporter. An obvious target is the periplasmic entrance
www.sciencedirect.com Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Paulsen IT, Park JH, Choi PS, Saier MH Jr: A family of Gram-negative bacterial outer membrane factors that function in the export of proteins, carbohydrates, drugs and heavy metals. FEMS Microbiol Lett 1997, 156:1-8. Koronakis V, Eswaran J, Hughes C: Structure and function of TolC: the bacterial exit duct for proteins and drugs. Annu Rev Biochem 2004, 73:467-489. Thanassi DG, Hultgren SJ: Multiple pathways allow protein secretion across the bacterial outer membrane. Curr Opin Cell Biol 2000, 12:420-430. Dinh T, Paulsen T, Saier MH Jr: A family of extra-cytoplasmic proteins that allow transport of large molecules across Current Opinion in Structural Biology 2004, 14:741747

2.

3.

4.

746 Proteins

the outer membranes of Gram-negative bacteria. J Bacteriol 1994, 176:3825-3831. 5. Johnson JM, Church GM: Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efux pumps. J Mol Biol 1999, 287:695-715. Andersen C, Hughes C, Koronakis V: Chunnel vision: export and efux through bacterial channel-tunnels. EMBO Rep 2000, 1:313-318. Saier MH, Paulsen IT, Sliwinski MK, Pao SS, Skurray RA, Nikaido H: Evolutionary origins of multidrug and drug-specic efux pumps in bacteria. FASEB J 1998, 12:265-274. Hancock RE, Brinkman FS: Function of Pseudomonas porins in uptake and efux. Annu Rev Microbiol 2002, 56:17-38. Sulavik MC, Houseweart C, Cramer C, Jiwani N, Murgolo N, Greene J, DiDomenico B, Shaw KJ, Miller GH, Hare R, Shimer G: Antibiotic susceptibility proles of Escherichia coli strains lacking multidrug efux pump genes. Antimicrob Agents Chemother 2001, 45:1126-1136.

This determination of the MexA crystal structure conrms the unusual crystal packing of 13 monomers in a spiral-shaped double cylinder [18]. Two alternative options for assembly in the complete drug pump are presented: one a wrap-around version of the adaptor oligomer, as seen in the crystal spiral-arc, the other suggesting that three dimers of MexA interact with the periplasmic helices of the OprM exit duct, the structure of which is modelled from that of TolC. 20. Berg A, Vervoort J, de Kok A: Three-dimensional structure in solution of the N-terminal lipoyl domain of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Eur J Biochem 1997, 244:352-360. 21. Ingelman M, Bianchi V, Eklund H: The three-dimensional structure of avodoxin reductase from Escherichia coli at resolution. J Mol Biol 1997, 268:147-157. 1.7 A 22. Vincent F, Spinelli S, Ramoni R, Grolli S, Pelosi P, Cambillau C, Tegoni M: Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes. J Mol Biol 2000, 300:127-139. 23. Van Duyne GD, Standaert RF, Karplus PA, Schreiber SL, Clardy J: Atomic structure of FKBP-FK506, an immunophilinimmunosuppressant complex. Science 1991, 252:839-842. 24. Ferguson KM, Lemmon MA, Schlessinger J, Sigler PB: Structure of the high afnity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell 1995, 83:1037-1046. 25. Thanabalu T, Koronakis E, Hughes C, Koronakis V: Substrate-induced assembly of a contiguous channel for protein export from E. coli: reversible bridging of an innermembrane translocase to an outer membrane exit pore. EMBO J 1998, 17:6487-6496. 26. Touze T, Eswaran J, Bokma E, Koronakis E, Hughes C,  Koronakis V: Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efux system. Mol Microbiol 2004, 53:697-706. The rst demonstration, by in vivo cross-linking, that the E. coli drug efux proteins AcrA, AcrB and TolC assemble into a tripartite complex in vivo, similar to the previously reported TolCHlyBHlyD protein export system [25]. AcrAAcrBTolC assembly appeared constitutive rather than reversible. Underlying bilateral contacts of component proteins were analysed by in vivo cross-linking and their interaction characteristics analysed by ITC. This revealed limitations of cross-linking analyses and indicated complexity of the component interaction patterns. 27. Narita S, Eda S, Yoshihara E, Nakae T: Linkage of the efuxpump expression level with substrate extrusion rate in the MexAB-OprM efux pump of Pseudomonas aeruginosa. Biochem Biophys Res Commun 2003, 308:922-926. 28. Elkins CA, Nikaido H: Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB multidrug efux pump. J Bacteriol 2003, 185:5349-5356. 29. Zgurskaya HI, Nikaido H: Cross-linked complex between oligomeric periplasmic lipoprotein AcrA and the inner-membrane-associated multidrug efux pump AcrB from Escherichia coli. J Bacteriol 2000, 182:4264-4267. 30. Ip H, Stratton K, Zgurskaya H, Liu J: pH-induced conformational changes of AcrA, the membrane fusion protein of Escherichia coli multidrug efux system. J Biol Chem 2003, 278:50474-50482. 31. Yu EW, McDermott G, Zgurskaya H, Nikaido H, Koshland DE Jr: Structural basis of multiple drug-binding capacity of the AcrB multidrug efux pump. Science 2003, 300:976-980. 32. Pos KM, Schiefner A, Seeger MA, Diederichs K: Crystallographic analysis of AcrB. FEBS Lett 2004, 564:333-339. 33. Middlemiss JK, Poole K: Differential impact of MexB mutations on substrate selectivity of the MexAB-OprM multidrug efux pump of Pseudomonas aeruginosa. J Bacteriol 2004, 186:1258-1269. 34. Mao W, Warren MS, Black DS, Satou T, Murata T, Nishino T, Gotoh N, Lomovskaya O: On the mechanism of substrate specicity by resistance nodulation division (RND)-type www.sciencedirect.com

6.

7.

8. 9.

10. Zgurskaya HI, Nikaido H: Multidrug resistance mechanisms: drug efux across two membranes. Mol Microbiol 2000, 37:219-225. 11. Murakami S, Nakashima R, Yamashita E, Yamaguchi A:  Crystal structure of bacterial multidrug efux transporter AcrB. Nature 2002, 419:587-593. The three-dimensional structure of the RND class proton antiporter AcrB, the integral IM component of the major E. coli drug efux system, indicates a possible proton translocation pathway through the membrane and allows speculation on how substrates gain access to the efux machinery. The trimeric AcrB structure is used in this review to present a preliminary model of the assembled drug pump. 12. Murakami S, Yamaguchi A: Multidrug-exporting secondary transporters. Curr Opin Struct Biol 2003, 13:443-452. 13. Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C:  Crystal structure of the bacterial membrane protein TolC central to multidrug efux and protein export. Nature 2000, 405:914-919. The structure of the OM protein TolC revised perceptions of the structure and function of efux pumps, and is the core of the preliminary model presented in this review. In the novel TolC fold, a change in superhelical long a helices determines the tapering and closure of twist of the 100 A the periplasmic cylinder. This nding established the basis for subsequent studies of the opening mechanism and the electrophysiological properties of the membrane channel. 14. Koronakis V, Andersen C, Hughes C: Channel-tunnels. Curr Opin Struct Biol 2001, 11:403-407. 15. Calladine CR, Sharff A, Luisi B: How to untwist an alpha-helix: structural principles of an alpha-helical barrel. J Mol Biol 2001, 305:603-618. 16. Benz R, Maier E, Gentschev I: TolC of Escherichia coli functions as an outer membrane channel. Zentralbl Bakteriol 1993, 278:187-196. 17. Andersen C, Hughes C, Koronakis V: Electrophysiological behaviour of the TolC channel-tunnel. J Membr Biol 2002, 185:83-92. 18. Higgins MK, Bokma E, Koronakis E, Hughes C, Koronakis V:  Structure of the periplasmic component of a bacterial drug efux pump. Proc Natl Acad Sci USA 2004, 101:9994-9999. The structure of the third essential component of tripartite drug efux pumps, the adaptor protein, postulated to bridge the IM and OM pump subunits. A possible oligomeric structure is presented, derived from the observed packing in the crystal. The adaptor protein MexA from P. aeruginosa is closely related to AcrA of E. coli and its structure is therefore included in this review to depict a possible model of the complete pump assembly. 19. Akama H, Matsuura T, Kashiwagi S, Yoneyama H, Narita S,  Tsukihara T, Nakagawa A, Nakae T: Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa. J Biol Chem 2004, 279:25939-25942. Current Opinion in Structural Biology 2004, 14:741747

Drug efflux pumps Eswaran et al. 747

multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition. Mol Microbiol 2002, 46:889-901. 35. Balakrishnan L, Hughes C, Koronakis V: Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli. J Mol Biol 2001, 313:501-510. 36. Andersen C, Koronakis E, Bokma E, Eswaran J, Humphreys D, Hughes C, Koronakis V: Transition to the open state of the TolC periplasmic tunnel entrance. Proc Natl Acad Sci USA 2002, 99:11103-11108. 37. Eswaran J, Hughes C, Koronakis V: Locking TolC entrance helices to prevent protein translocation by the bacterial type I export apparatus. J Mol Biol 2003, 327:309-315.

38. Andersen C, Koronakis E, Hughes C, Koronakis V: An aspartate  ring at the TolC tunnel entrance determines ion selectivity and presents a target for blocking by large cations. Mol Microbiol 2002, 44:1131-1139. This study used protein reconstituted in black lipid bilayers to identify a high-afnity metal-binding site located at the TolC entrance constriction. The site is determined by six aspartate residues that form a highly electronegative gate. A series of divalent and trivalent cations were shown to bind with nanomolar afnities and, in one case, cause irreversible blocking of the TM pore. 39. Higgins M, Eswaran J, Edwards P, Schertler G, Hughes C, Koronakis V: Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efux protein TolC. J Mol Biol 2004, 342:697-702.

ScienceDirect collection reaches six million full-text articles

Elsevier recently announced that six million articles are now available on its premier electronic platform, ScienceDirect. This milestone in electronic scientic, technical and medical publishing means that researchers around the globe will be able to access an unsurpassed volume of information from the convenience of their desktop. ScienceDirects extensive and unique full-text collection covers over 1900 journals, including titles such as The Lancet, Cell, Tetrahedron and the full suite of Trends and Current Opinion journals. With ScienceDirect, the research process is enhanced with unsurpassed searching and linking functionality, all on a single, intuitive interface. The rapid growth of the ScienceDirect collection is due to the integration of several prestigious publications as well as ongoing addition to the Backles heritage collections in a number of disciplines. The latest step in this ambitious project to digitize all of Elseviers journals back to volume one, issue one, is the addition of the highly cited Cell Press journal collection on ScienceDirect. Also available online for the rst time are six Cell titles long-awaited Backles, containing more than 12,000 articles highlighting important historic developments in the eld of life sciences. The six-millionth article loaded onto ScienceDirect entitled Gene Switching and the Stability of Odorant Receptor Gene Choice was authored by Benjamin M. Shykind and colleagues from the Dept. of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, College of Physicians and Surgeons at Columbia University. The article appears in the 11 June issue of Elseviers leading journal Cell, Volume 117, Issue 6, pages 801-815.

www.sciencedirect.com
www.sciencedirect.com Current Opinion in Structural Biology 2004, 14:741747