Component Structures Bring a Closer View of Tripartite Drug Efflux Pumps

7/28/2019 Component Structures Bring a Closer View of Tripartite Drug Efflux Pumps

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Threes company: component structures bring a closerview of tripartite drug efflux pumpsJeyanthy Eswaran1, Eva Koronakis1, Matthew K Higgins1,2,

Colin Hughes

1

and Vassilis Koronakis

1,3

Bacterial multidrug resistance is a serious clinical problem

and is commonly conferred by tripartite efflux pumps in the

prokaryotic cell envelope. Crystal structures of the three

components of a drug efflux 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.

Addresses1Cambridge University Department of Pathology, Tennis Court Road,

Cambridge CB2 1QP, UK2Current address: MRC Laboratory of Molecular Biology, Hills Road,

Cambridge CB2 2QH, UK3e-mail: [email protected]

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

0959-440X/$ see front matter

# 2004 Published by Elsevier Ltd.

DOI 10.1016/j.sbi.2004.10.003

Abbreviations

FAD flavin adenine dinucleotide

IM inner membrane

ITC isothermal calorimetry

MFS major facilitator superfamilyOM outer membrane

RND resistance nodulation division

TM transmembrane

IntroductionGram-negative pathogens such as Escherichia coli and

Pseudomonas aeruginosa employ membrane efflux 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 efflux pumps

comprise three components, each a member of an exten-

sive protein family [47]. An energy-providing integral

IM protein, either an ABC transporter or more often a

proton antiporter of the resistance nodulation division

(RND) or major facilitator superfamily (MFS) [7], coop-

erates 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 specificities; for example, P.

aeruginosa has at least four distinct major efflux (Mex)

systems [8], whereas the major efflux 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 efflux pump in the bacterial cell

envelope and how such pumps may operate to expel

drugs from the cell.

Structure of the tripartite pump componentsAcrB: an energy-providing, substrate-binding

component in the inner membrane

The architecture of the E. coli 1049 amino acid protonantiporter AcrB, a drug efflux transporter of the RND

family, has been solved at 3.5 A [11,12] (Figure 1).

AcrB is a trimer with a 50 A long and 100 A diameter TM

domain comprising 36 a helices (12 from each mono-

mer). There is minimal contact between monomers in

the TM domain, forming a chamber thought to be filled

with lipid. At the core of this domain, TM helices 4 and10 are suggested to form the proton translocation path-

way, 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 membrane-located substrates. In the large periplasmic domain,

the monomers, each 700 amino acids, are tightly inter-

locked and form a closed central pore. An internal cavity

open to the periplasm could provide access to periplas-

mic substrates, allowing RND transporters to act as

cytosolic membrane and periplasmic vacuum cleaners.

The top of the periplasmic domain forms a funnel-like

structure with an internal diameter of30 A, similar to

the diameter of the modelled open state of the TolC

entrance, and has been termed the TolC-docking

domain (Figure 1).

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TolC: an exit duct for polypeptide and drug substrates

At 2.1 A resolution, the TolC homotrimer (Figure 1) is

seen as a tapered cylinder 140 A in length. This comprisesa 40 A long OM b barrel, which anchors a contiguous a-

helical barrel projecting 100 A across the periplasmic

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

single central TolC pore is 19.8 A. Three TolC monomerseach contribute four b strands to form the twelve-

stranded b barrel, which is constitutively open to the cell

exterior. The periplasmic a barrel comprises twelve anti-

parallel 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

helices form six pairs of regular two-stranded coiled coils,

but one from each monomer folds inwards at the peri-

plasmic end. This constricts the entrance to establish aresting closed state with an effective diameter of approxi-

mately 3.9 A; this is reflected in the small conductance of

TolC in lipid bilayers [16,17].

MexA: a periplasmic adaptor linking the inner and

outer membrane componentsThe structure of approximately two-thirds of the 360-

residue 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

47 A (64-residue) a-helical hairpin comprising a straight

C-terminal helix and an N-terminal helix with a left-

handed 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

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 shownat 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.

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


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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 meansquare deviation (rmsd) of only 1.6 A [20]. Each lipoyl

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 five 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 tothe barrel. This structural element has been found in

diverse contexts, such as the FAD-binding domain of

flavodoxin reductase [21], odorant-binding domains [22],

isomerase FKBP [23] and the pleckstrin homology (PH)

domain [24].

Assembly of the efflux pumpsInteractions 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 acontiguous 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 efflux machinery appearsconstitutively assembled (i.e. independent of the drug

substrate) [26]. This apparent difference between the

export and efflux systems possibly reflects the require-

ments imposed by the different substrates. Whereas

polypeptide export systems translocate substrates of

1000 amino acids or more, it is estimated that approx-

imately 500 toxic ethidium molecules are expelled persecond by each P. aeruginosa MexABOprM pump [27].

Frequent assembly and disassembly of the drug efflux

pumps might be energetically inappropriate.

The periplasmic contact between the IM and OM com-

ponents has been suggested to involve the TolC entrancecoils 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. How-

ever, although AcrB and TolC can be isolated as a com-plex after in vivo cross-linking, no interaction is seen

when the two purified proteins are studied by isothermal

calorimetry (ITC) [26]. By contrast, ITC confirms 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

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-terminaldomain to interact with cognate IM transporter compo-

nents [26,28].

Structural model of the assembled pumpThe direct efflux of drugs and other substrates across the

Gram-negative cell envelope requires assembly of a con-

tiguous 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 reflect the known

component structures, interactions and stoichiometries;

that in Figure 2 illustrates a 870 kDa transenvelope

Drug efflux pumps Eswaran et al. 743

Figure 2

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.



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complex over 270 A long. Although the solved adaptor

protein structure is that of P. aeruginosa MexA, this

protein is closely related to AcrA, the adaptor in the

AcrAAcrBTolC drug efflux pump ofE. coli. The MexA

structure is therefore included with the AcrB and TolC

structures to depict a possible model of the completepump 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 mem-

brane components. During assembly of the active efflux

pump, the hairpins of the adaptor could directly engage

the inner and/or outer coiled coils of the TolC a-helicalbarrel, compatible with the adaptor assembling into tri-

mers or hexamers. Cross-linking of in vivo complexes

using the short-arm chemical cross-linker DSG (disucci-

nimidyl glutarate) has identified adaptor trimers in both

the drug efflux 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 inter-

faces 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.

This would have a curvature similar to that observed in

the crystal packing (Figure 3), and would be sufficiently

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 flexible equa-torial 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 ahelices acting to draw together the IM and OM. However,

the MexA structure shows that residues 29 and 259 lie

within 5 A of each other in the b-barrel domain, position-

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 translocationDrug efflux substrates could enter the transenvelope

efflux conduit via AcrB, from either side of the cytoplas-

mic membrane or from the periplasm. Soaking AcrB

crystals with efflux substrates suggested that they bindat different positions in the TM domain [31], although this

view was not confirmed by similar experiments [32] and

analysis of hybrid transporters indicates that the antiporter

periplasmic domain plays a major role in substrate speci-

ficity [33,34]. In the protein export system, the largesubstrate is not only engaged by the IM traffic ATPase,

but also contacts the short cytoplasmic domain of the

adaptor [35]. A signal must be transduced to triggerrecruit-

mentand openingof TolC. In the constitutively assembled

drug efflux pumps, it is specifically the TolC opening step

that wouldneedto be substrate responsive. Howthismight

be coordinated with opening of the AcrB pore domain isone of manyopen 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 pre-

vented from escaping back into the periplasm.

Once past the AcrBTolC junction, substrates encounterthe 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 chan-nel, whereas later it might catalyse the release of basic

molecules.

Twist to open access to TolC byrealignment of entrance helicesAccess through the TolC periplasmic entrance is a key

event in the function of export and efflux machineries.

744 Proteins

Figure 3

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.



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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 ofthe inner coils

with respect to the outer coils (Figure 4) [13]. Experi-

mental disruption of three intermolecular and intramo-

lecular links that constrain the three inner coils in the

closed conformation allows enlargement of the aperture

diameter, as seen by increased conductivity of theseengineered TolC variants in planar lipid bilayers [36].

Furthermore, when movement of these helices is con-

strained by introducing disulfide bonds, translocation of

polypeptide substrate is abolished [37], further support-

ing a model in which transition to the TolC open stateis 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 polypeptidesubstrates.

Perspective efflux pumps as targets inmultidrug-resistant bacteria?The combined biochemical and structural data have

provided a closer vision of the assembly and operationof the large efflux 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 tripartitestructure. 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

of TolC. This is the sole constriction of the exit duct and

is lined by an electronegative ring of six aspartate resi-

dues, Asp371 and Asp374 from each monomer, which

determine a high-affinity metal-binding site [38,39].

TolC function in artificial lipid bilayers is severely inhib-

ited by divalent cations, and trivalent cations such as Cr3+,

Tb3+ and hexammine cobalt block the TM ion flux at

nanomolar concentrations. When the entrance aspartates

are substituted, high-affinity binding is abolished andblocking of the membrane pore is alleviated [38,39]. A

crystal structure of the TolCCo(NH3)63+ complex (Fig-

ure 4) confirms a ligand molecule bound at this site [39].

This first biochemical and structural characterisation of

an in vitro inhibitor of TolC may suggest a strategy todevelop bioactive molecules, especially as the electro-

negative entrance is widely conserved throughout pumps

central to virulence and drug resistance.

AcknowledgementsOur work is supported by the Medical Research Council andWellcome Trust.

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

of special interest of outstanding interest

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2. Koronakis V, Eswaran J, Hughes C: Structure and functionof TolC: the bacterial exit duct for proteins and drugs.Annu Rev Biochem 2004, 73:467-489.

3. Thanassi DG, Hultgren SJ: Multiple pathways allow proteinsecretion across the bacterial outer membrane.Curr Opin Cell Biol 2000, 12:420-430.

4. Dinh T, Paulsen T, Saier MH Jr: A family of extra-cytoplasmicproteins that allow transport of large molecules across


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.



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the outer membranes of Gram-negative bacteria.J Bacteriol 1994, 176:3825-3831.

5. Johnson JM, Church GM: Alignment and structure predictionof divergent protein families: periplasmic and outermembrane proteins of bacterial efflux pumps.J Mol Biol 1999, 287:695-715.

6. Andersen C, Hughes C, Koronakis V: Chunnel vision: export andefflux through bacterial channel-tunnels. EMBO Rep 2000,1:313-318.

7. Saier MH, Paulsen IT, Sliwinski MK, Pao SS, Skurray RA,Nikaido H: Evolutionary origins of multidrug and drug-specificefflux pumps in bacteria. FASEB J 1998, 12:265-274.

8. Hancock RE, Brinkman FS: Function of Pseudomonas porins inuptake and efflux. Annu Rev Microbiol 2002, 56:17-38.

9. Sulavik MC, Houseweart C, Cramer C, Jiwani N, Murgolo N,Greene J, DiDomenico B, Shaw KJ, Miller GH, Hare R,Shimer G: Antibiotic susceptibility profiles ofEscherichia colistrains lacking multidrug efflux pump genes.Antimicrob Agents Chemother 2001, 45:1126-1136.

10. Zgurskaya HI, Nikaido H: Multidrug resistance mechanisms:drug efflux across two membranes. Mol Microbiol 2000,37:219-225.

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Murakami S, Nakashima R, Yamashita E, Yamaguchi A:Crystal structure of bacterial multidrug efflux transporterAcrB. 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 efflux system,indicates a possible proton translocation pathway through the membraneand allows speculation on how substrates gain access to the effluxmachinery. The trimeric AcrB structure is used in this review to presenta preliminary model of the assembled drug pump.

12. Murakami S, Yamaguchi A: Multidrug-exportingsecondary transporters. Curr Opin Struct Biol 2003,13:443-452.

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Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C:Crystal structure of the bacterial membrane protein TolCcentral to multidrug efflux and protein export. Nature 2000,405:914-919.

The structure of the OM protein TolC revised perceptions of the structureand function of efflux pumps, and is the core of the preliminary modelpresented in this review. In the novel TolC fold, a change in superhelicaltwist of the 100 A long a helices determines the tapering and closure ofthe periplasmic cylinder. This finding established the basis for subse-quent studies of the opening mechanism and the electrophysiologicalproperties 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 Biol2001,305:603-618.

16. Benz R, Maier E, Gentschev I: TolC ofEscherichia colifunctionsas an outer membrane channel. Zentralbl Bakteriol 1993,278:187-196.

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Higgins MK, Bokma E, Koronakis E, Hughes C, Koronakis V:Structure of the periplasmic component of a bacterial drugefflux pump. Proc Natl Acad Sci USA 2004, 101:9994-9999.

The structure of the third essential component of tripartite drug effluxpumps, the adaptor protein, postulated to bridge the IM and OM pumpsubunits. A possible oligomeric structure is presented, derived fromthe observed packing in the crystal. The adaptor protein MexA fromP. aeruginosa is closely related to AcrA of E. coli and its structure istherefore included in this review to depict a possible model of thecomplete pump assembly.

19.

Akama H, Matsuura T, Kashiwagi S, Yoneyama H, Narita S,Tsukihara T, Nakagawa A, Nakae T: Crystal structure ofthe membrane fusion protein, MexA, of the multidrugtransporter in Pseudomonas aeruginosa. J Biol Chem 2004,279:25939-25942.

This determination of the MexA crystal structure con firms the unusualcrystal packing of 13 monomers in a spiral-shaped double cylinder [18].Two alternative options for assembly in the complete drug pump arepresented: onea wrap-around version of theadaptor oligomer,as seen inthe crystal spiral-arc, the other suggesting that three dimers of MexAinteract with theperiplasmichelices of theOprM exit duct, thestructure ofwhich is modelled from that of TolC.

20. Berg A, Vervoort J, de Kok A: Three-dimensional structure insolution of the N-terminal lipoyl domain of the pyruvatedehydrogenase complex from Azotobacter vinelandii.Eur J Biochem 1997, 244:352-360.

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. Cell 1995,83:1037-1046.

25. Thanabalu T, Koronakis E, Hughes C, Koronakis V:Substrate-induced assembly of a contiguous channel forprotein export from E. coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore.EMBO J 1998, 17:6487-6496.

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Touze T, Eswaran J, Bokma E, Koronakis E, Hughes C,Koronakis V: Interactions underlying assembly of theEscherichia coli AcrAB-TolC multidrug efflux system.Mol Microbiol 2004, 53:697-706.

The first demonstration, byin vivo cross-linking, that the E. colidrug effluxproteins 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 rever-sible. Underlying bilateral contacts of component proteins were analysedby in vivo cross-linking and their interaction characteristics analysed byITC. 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 efflux-pump expression level with substrate extrusion rate in theMexAB-OprM efflux pump of Pseudomonas aeruginosa.Biochem Biophys Res Commun 2003, 308:922-926.

28. ElkinsCA, Nikaido H: Chimeric analysisof AcrAfunctionrevealsthe importance of its C-terminal domain in its interactionwith the AcrB multidrug efflux pump. J Bacteriol 2003,185:5349-5356.

29. Zgurskaya HI, Nikaido H: Cross-linked complex betweenoligomeric periplasmic lipoprotein AcrA and theinner-membrane-associated multidrug efflux pumpAcrB from Escherichia coli. J Bacteriol 2000,182:4264-4267.

30. Ip H, Stratton K, Zgurskaya H, Liu J: pH-induced conformationalchanges of AcrA, the membrane fusion protein of

Escherichia coli multidrug efflux 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 theAcrB multidrug efflux pump. Science 2003, 300:976-980.

32. Pos KM, Schiefner A, Seeger MA, Diederichs K: Crystallographicanalysis of AcrB. FEBS Lett 2004, 564:333-339.

33. Middlemiss JK, Poole K: Differential impact of MexB mutationson substrate selectivity of the MexAB-OprM multidrugefflux 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 substratespecificity by resistance nodulation division (RND)-type

746 Proteins



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multidrug resistance pumps: the large periplasmic loops ofMexD from Pseudomonas aeruginosa are involved insubstrate recognition. Mol Microbiol 2002, 46:889-901.

35. Balakrishnan L, Hughes C, Koronakis V: Substrate-triggeredrecruitment of the TolC channel-tunnel during type I export ofhemolysin 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 TolCperiplasmic tunnel entrance. Proc Natl Acad Sci USA 2002,99:11103-11108.

37. Eswaran J, Hughes C, Koronakis V: Locking TolC entrancehelices to prevent protein translocation by the bacterial type Iexport apparatus. J Mol Biol 2003, 327:309-315.

38.

Andersen C, Koronakis E, Hughes C, Koronakis V: An aspartatering at the TolC tunnel entrance determines ion selectivity andpresents a target for blocking by large cations. Mol Microbiol2002, 44:1131-1139.

This study used protein reconstituted in black lipid bilayers to identify ahigh-affinity metal-binding site located at the TolC entrance constriction.The site is determined by six aspartate residues that form a highlyelectronegative gate. A series of divalent and trivalent cations were

shown to bind with nanomolar affinities and, in one case, cause irrever-sible blocking of the TM pore.

39. Higgins M, Eswaran J, Edwards P, Schertler G, Hughes C,Koronakis V: Structure of the ligand-blocked periplasmicentrance of the bacterial multidrug efflux protein TolC.J Mol Biol 2004, 342:697-702.


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Elsevier recently announced that six million articles are now available on its premier electronic

platform, ScienceDirect. This milestone in electronic scientific, technical and medical publishingmeans that researchers around the globe will be able to access an unsurpassed volume ofinformation from the convenience of their desktop.

ScienceDirects extensive and unique full-text collection covers over 1900 journals, including titlessuch 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 prestigiouspublications as well as ongoing addition to the Backfiles heritage collections in a number ofdisciplines. 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 onScienceDirect. Also available online for the first time are six Cell titles long-awaited Backfiles,

containing more than 12,000 articles highlighting important historic developments in the field oflife sciences.

The six-millionth article loaded onto ScienceDirect entitled Gene Switching and the Stability ofOdorant Receptor Gene Choice was authored by Benjamin M. Shykind and colleagues from theDept. 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 ofElseviers leading journal Cell, Volume 117, Issue 6, pages 801-815.

www.sciencedirect.com


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