folding of acrb subunit precedes trimerization

11
Folding of AcrB Subunit Precedes Trimerization Wei Lu, Meng Zhong and Yinan WeiDepartment of Chemistry, University of Kentucky, Lexington, KY 40506, USA Received 18 April 2011; received in revised form 20 May 2011; accepted 26 May 2011 Available online 2 June 2011 Edited by J. Bowie Keywords: membrane protein oligomerization; obligate oligomer; thiol trapping; protein tertiary and quaternary structures; folding and assembly AcrB and its homologues are major players in the efflux of anti-microbials out of Gram-negative bacteria. The structural and functional unit of AcrB is a homo-trimer. The assembly process of obligate membrane protein oligomers, including AcrB, remains elusive. It is not clear if an individual subunit folds into a monomeric form first followed by association (three- stage pathway) or if association occurs simultaneously with subunit folding (two-stage pathway). To answer this question, we investigated the feasibility of creating a folded monomeric AcrB mutant. The existence of well-folded monomers in the cell membrane would be an evidence of a three-stage pathway. A monomeric AcrB mutant, AcrB Δloop , was created through the truncation of a protruding loop that appeared to contribute to the stability of an AcrB trimer. AcrB Δloop expressed at a level similar to that of wild-type AcrB. The secondary structure content and tertiary conforma- tion of AcrB Δloop were very similar to those of wild-type AcrB. However, when expressed in an acrB-deficient strain, AcrB Δloop failed to complement its defect in drug efflux. Results from blue native polyacrylamide gel electrophoresis and chemical cross-linking experiments suggested that AcrB Δloop existed as a monomer. The expression of this monomeric mutant in a wild-type Escherichia coli strain did not have a significant dominant- negative effect, suggesting that the mutant could not effectively co-assemble with genomic AcrB. AcrB Δloop is the first monomeric mutant reported for the intrinsically trimeric AcrB. The structural characterization results of this mutant suggest that the oligomerization of AcrB occurs through a three- stage pathway involving folded monomers. © 2011 Elsevier Ltd. All rights reserved. Introduction More than one-third of all cellular proteins exist and operate as oligomers, the majority of which are homo-oligomers. 15 Many homo-oligomers are ob- ligate oligomersmeaning they exist and function exclusively as oligomers. The assembly of obligate oligomers is an interesting and yet poorly defined process. Studies with soluble protein oligomers indicate that oligomerization may occur via a two- stage or a three-stage pathway. 69 In the two-stage pathway, individual monomers remain largely unfolded prior to oligomerization, while in the three-stage pathway, individual monomers fold independently first into a structure, which may or may not be the same as the final structure in the oligomer. 10 It is not clear how proteins oligomerize in the plasma membrane of a cell. Approximately 2030% of proteins encoded in sequenced genomes are predicted to be membrane proteins, many of which function as oligomers. 11 Several studies have shown that the assemblies of hetero-oligomeric membrane proteins are sequential. For example, Green and Claudio had studied the sequential folding and assembly of the hetero-pentameric *Corresponding author. 305 ChemistryPhysics Building, University of Kentucky, Lexington, KY 40506-0055, USA. E-mail address: [email protected]. Abbreviations used: MIC, minimum inhibitory concentration; Flu-MAL, fluorescein-5-maleimide; IAM, iodoacetamide; BN-PAGE, blue native polyacrylamide gel electrophoresis; DSP, dithiobis(succinimidyl propionate). doi:10.1016/j.jmb.2011.05.042 J. Mol. Biol. (2011) 411, 264274 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.

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doi:10.1016/j.jmb.2011.05.042 J. Mol. Biol. (2011) 411, 264–274

Contents lists available at www.sciencedirect.com

Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

Folding of AcrB Subunit Precedes Trimerization

Wei Lu, Meng Zhong and Yinan Wei⁎Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA

Received 18 April 2011;received in revised form20 May 2011;accepted 26 May 2011Available online2 June 2011

Edited by J. Bowie

Keywords:membrane proteinoligomerization;obligate oligomer;thiol trapping;protein tertiary andquaternary structures;folding and assembly

*Corresponding author. 305 ChemisUniversity of Kentucky, Lexington,E-mail address: [email protected] used: MIC, minimu

concentration; Flu-MAL, fluoresceiniodoacetamide; BN-PAGE, blue natielectrophoresis; DSP, dithiobis(succi

0022-2836/$ - see front matter © 2011 E

AcrB and its homologues are major players in the efflux of anti-microbialsout of Gram-negative bacteria. The structural and functional unit of AcrB isa homo-trimer. The assembly process of obligate membrane proteinoligomers, including AcrB, remains elusive. It is not clear if an individualsubunit folds into a monomeric form first followed by association (three-stage pathway) or if association occurs simultaneously with subunit folding(two-stage pathway). To answer this question, we investigated thefeasibility of creating a folded monomeric AcrB mutant. The existence ofwell-folded monomers in the cell membrane would be an evidence of athree-stage pathway. A monomeric AcrB mutant, AcrBΔloop, was createdthrough the truncation of a protruding loop that appeared to contribute tothe stability of an AcrB trimer. AcrBΔloop expressed at a level similar to thatof wild-type AcrB. The secondary structure content and tertiary conforma-tion of AcrBΔloop were very similar to those of wild-type AcrB. However,when expressed in an acrB-deficient strain, AcrBΔloop failed to complementits defect in drug efflux. Results from blue native polyacrylamide gelelectrophoresis and chemical cross-linking experiments suggested thatAcrBΔloop existed as a monomer. The expression of this monomeric mutantin a wild-type Escherichia coli strain did not have a significant dominant-negative effect, suggesting that the mutant could not effectively co-assemblewith genomic AcrB. AcrBΔloop is the first monomeric mutant reported forthe intrinsically trimeric AcrB. The structural characterization results of thismutant suggest that the oligomerization of AcrB occurs through a three-stage pathway involving folded monomers.

© 2011 Elsevier Ltd. All rights reserved.

Introduction

More than one-third of all cellular proteins existand operate as oligomers, the majority of which arehomo-oligomers.1–5 Many homo-oligomers are ob-ligate oligomers—meaning they exist and functionexclusively as oligomers. The assembly of obligateoligomers is an interesting and yet poorly defined

try–Physics Building,KY 40506-0055, USA..m inhibitory-5-maleimide; IAM,ve polyacrylamide gelnimidyl propionate).

lsevier Ltd. All rights reserve

process. Studies with soluble protein oligomersindicate that oligomerization may occur via a two-stage or a three-stage pathway.6–9 In the two-stagepathway, individual monomers remain largelyunfolded prior to oligomerization, while in thethree-stage pathway, individual monomers foldindependently first into a structure, which may ormay not be the same as the final structure in theoligomer.10

It is not clear how proteins oligomerize in theplasma membrane of a cell. Approximately 20–30%of proteins encoded in sequenced genomes arepredicted to be membrane proteins, many ofwhich function as oligomers.11 Several studieshave shown that the assemblies of hetero-oligomericmembrane proteins are sequential. For example,Green and Claudio had studied the sequentialfolding and assembly of the hetero-pentameric

d.

265Folding of AcrB Subunit Precedes Trimerization

acetylcholine receptor.12 Taking advantage of thetemperature-sensitive assembly of torpedo acetyl-choline receptor subunits in the mouse cell line, theywere able to first express the subunits at 37 °C andthen trigger the assembly by lowering the temper-ature to 20 °C. Using this method, they found thatthe αβγ trimer was first assembled within a fewminutes, followed by the sequential addition of a δsubunit and, finally, a second α subunit. The authorsconclude that the subunit folding events contributeto subunit recognition site formation during assem-bly. One factor that was not addressed in this studyis how close the structures of each individualsubunit are to their final structure in the functionalhetero-oligomer.In the case of obligate oligomers, it is not always

clear how each subunit exists before their assemblyinto the final oligomeric state. In bacteria, themembrane insertion of inner-membrane proteinsoccurs co-translationally. It is not clear to whatextent each individual subunit folds before the onsetof the assembly. The anchoring of the subunits in amembrane restricts the movement of the subunits totwo dimensions, which may help to increase thelocal concentrations of the subunits and, therefore,the chance of encounters among them. Thus,oligomerization might occur more efficiently formembrane proteins. However, the mechanism of theoligomer assembly in the cell membrane of integralmembrane proteins remains elusive, mainly due tothe difficulty of characterizing, separately, proteintertiary and quaternary structures in the cellmembrane under the native condition.In this study, we investigated the relation between

subunit folding and oligomer assembly in a homo-trimeric membrane protein, AcrB. Our rationale is,since the major difference between the two-stagepathway and the three-stage pathway is theexistence of well-folded independent monomers,we will explore the possibility of creating andobserving a well-folded monomeric AcrB in the

Fig. 1. (a) Side view of an AcrB trimer. Different domains weach subunit was highlighted using an envelope to illustrate tThe part of the protruding loop that was removed in this studyProtein Data Bank file 2DHH.

cell membrane. It is important to have a method forprobing the structure of AcrB in the membraneunder the native condition to make this researchpossible, as detergent extraction and purificationmay affect the conformation and oligomeric state ofAcrB. For this purpose, we have previously createda folding reporter platform as a research tool thatcan reveal the tertiary structure of individual sub-units in the cell membrane.13 Taking advantage ofthis method, we created in this study a well-foldedmonomeric AcrB mutant, AcrBΔloop, in which partof a protruding loop was deleted (Fig. 1). Mono-meric AcrB has never been identified in the cellmembrane before, since AcrB is a structural andfunctional obligate trimer. We found that AcrBmonomers may exist in the cell membrane in afolded conformation that closely resembles thestructure in a trimer. In other words, the folding ofeach individual subunit does not need assistancefrom the neighboring subunits. Our result suggeststhat trimerization of AcrB occurs through a three-stage pathway.

Results

The expression levels of AcrBΔloop and wild-typeAcrB are similar

In an effort to probe if monomeric AcrB could foldindependently in the cell membrane, we designedand created an AcrB mutant, AcrBΔloop (Fig. 1). Toevaluate if the truncation of the protruding loopaffected the overall structure and stability of theprotein, we examined the expression level of themutant. To avoid contaminations from genomicAcrB, we transformed plasmids encoding AcrBΔloopand wild-type AcrB into an acrB-deficient strain,BW25114ΔacrB, for protein production. Membranevesicles were then extracted from the cells andsubjected to Western blot analysis (Fig. 2a). The

ere marked. (b) Top view of an AcrB trimer. The contour ofhe protruding loop. (c) Structure of a single AcrB subunit.was highlighted in green. The structure was created using

Fig. 2. Comparison of the expression levels of wild-typeAcrB and AcrBΔloop. (a) Western blot analysis of mem-brane vesicles extracted from BW25113ΔacrB expressingwild-type AcrB (WT) or AcrBΔloop (Δloop). Each samplewas diluted 1-, 10-, and 100-fold. (b) SDS-PAGE analysisof purified wild-type AcrB (WT) and AcrBΔloop (Δloop).Both proteins can be purified to near homogeneity. Theexpression levels and the yield of purification of the twoproteins were similar. The molecular masses (in kilo-daltons) of bands in the molecular mass marker weremarked on the left of the gel.

266 Folding of AcrB Subunit Precedes Trimerization

same samples were diluted 10- and 100-fold toreveal the expression levels of the two proteins in thecell. The densities of the blotting bands for the twoproteins were very similar, indicating that theexpression levels of the two proteins were veryclose.

AcrBΔloop is completely inactive

The activity of AcrBΔloop was examined througha drug susceptibility assay. Plasmids encodingAcrBΔloop or wild-type AcrB were transformed intostrain BW25112ΔacrB. In parallel, vector pQE70was also transformed into the same strain to serveas a negative control. The susceptibilities of thesestrains were measured for two established AcrBsubstrates, erythromycin and novobiocin (Table 1).The minimum inhibitory concentrations (MICs) ofthe strain containing the plasmid-encoded AcrB-Δloop were much lower than those of the samestrain containing the plasmid-encoded wild-typeAcrB and were similar to the MICs of the straincontaining the pQE70 vector. This result indicatedthat the mutant was completely nonfunctional.

Table 1. MICs of BW25113ΔacrB expressing plasmid-encoded acrB genes

Plasmid

MIC (μg/mL)

Erythromycin Novobiocin

pQE70 4 4pQE70AcrBΔloop 4 4pQE70AcrB 128 128

The secondary structure and the thermal stabilityof AcrBΔloop and wild-type AcrB are similar

To further compare the structures of AcrBΔloopand wild-type AcrB, we purified the proteins for amore detailed characterization. The mutant proteincould be purified with a comparable yield as wild-type AcrB (Fig. 2b). The far-UV circular dichroism(CD) spectra of the two proteins superimposed well,indicating that the mutant and the wild-type proteincontained a similar secondary structural componentwith a high percentage of α-helices (Fig. 3a).The thermal stabilities of the proteins were also

compared. Melting of the helical secondary struc-ture was monitored by recording the ellipticity at222 nm with an increase in temperature from 4 to98 °C (Fig. 3b). If the truncation of the loop doesaffect the folding and internal packing of the AcrBsubunit, we expect to see a change in the melting

Fig. 3. (a) Far-UV CD spectra of wild-type AcrB (opensquares) and AcrBΔloop (filled squares) superimposed wellonto each other, indicating that the two proteins hadsimilar secondary structure contents. (b) Temperaturedenaturation curves of wild-type AcrB (open diamonds)and AcrBΔloop (filled squares). The ellipticity valuesmonitored at 222 nm were normalized to the reading at4 °C. The thermal stabilities of the two proteins were verysimilar.

267Folding of AcrB Subunit Precedes Trimerization

temperature. Residual helical structure could beobserved even when the proteins were heated to98 °C, consistent with previous observations thattransmembrane helices in helical membrane pro-teins are very stable and that a portion of it existseven in the unfolded stage.14 The melting curves ofthe two proteins were very close to each other,indicating a lack of significant difference in thethermal stability between AcrBΔloop and wild-typeAcrB.

Tertiary structure of AcrBΔloop

The near-UV CD spectra (260–350 nm), whichreveal information about a protein's tertiary andquaternary structure, were collected for the twoproteins (Fig. 4a). Aromatic residues, includingtryptophan, tyrosine, and phenylalanine, are majorcontributors to the near-UV CD signal. A change inthe microenvironment of these aromatic residueswill affect their UV absorption and the CD spec-trum. As revealed in Fig. 4a, the spectra of wild-typeand mutant proteins were different, indicating adifference in the local structure of these residues.This change may be at the tertiary structure level orat the quaternary structure level, as both maypotentially change the microenvironment of theresidues.

Limited protease digestion is another well-established method that has been used extensivelyto probe structures of proteins.15–19 A well-foldedprotein is more resistant to protease digestion than apoorly folded one. The pattern of the resultantpeptide fragment may also reveal the disorderedregion of a protein. We treated AcrBΔloop and wild-type AcrB with trypsin and compared the digestionresults. As shown in Fig. 4b, wild-type AcrB wasmore resistant to trypsin digestion. No digestionwas observed after 15 min of incubation. Under thesame experimental condition, AcrBΔloop wasdigested much faster. A clear decrease in the amountof full-length protein was obvious within 5 min intodigestion, and at 15 min, a large portion of theprotein was digested into smaller fragments. As anegative control, we also digested a sample of wild-type AcrB, which had been subjected to five cycles offreezing and thawing. We found that such atreatment has a similar effect on purified wild-typeAcrB samples as extended storage at 4 °C, whichcaused AcrB trimers to dissociate into monomers.This monomeric form of AcrB was very sensitive totrypsin digestion (Fig. 4b, control). Approximately90% of the protein was digested within 5 min ofincubation. To further reveal the difference indigestion rates, we plotted the intensity of theremaining full-length protein bands in the three

Fig. 4. (a) Near-UV CD spectra ofwild-type AcrB (gray diamonds)and AcrBΔloop (black squares). (b)Limited trypsin digestion of puri-fied wild-type AcrB and AcrBΔloop.Under the current experimentalcondition, wild-type AcrB washighly resistant to the digestion.No apparent degradation could beobserved. AcrBΔloop was more sen-sitive to digestion. Degradation ofthe full-length protein was appar-ent after 5 min of incubation. In thenegative control, unfolded AcrBwas very sensitive to trypsin diges-tion. The molecular masses (in kilo-daltons) of bands in the molecularmass marker were marked on theleft of the gel. (c) The percentage ofthe remaining full-length proteinsat each time point during thedigestion of wild-type AcrB (filledsquares), AcrBΔloop (open squares),and control (filled diamonds) sam-ples was plotted against the incu-bation time.

268 Folding of AcrB Subunit Precedes Trimerization

gels against digestion time (Fig. 4c). The bandintensity before digestion in each gel was designatedas 100% and used to normalize the intensitiesat other time points. There was a clear differencein digestion rates between wild-type AcrB andAcrBΔloop and between AcrBΔloop and the negativecontrol monomeric AcrB.This difference in digestion rate between wild-

type AcrB and AcrBΔloop, again, can be explained bychanges occurring at the tertiary-structure and/orquaternary-structure level, as both will affect theaccessibility of potential cutting sites to trypsin andtherefore affect the rate of digestion. Two phenom-ena may result in the observed difference in trypsindigestion rate: First, AcrBΔloop is a monomer. Theinter-subunit interface in the trimeric wild-typeAcrB provides potential trypsin recognition sitesbetter protection from digestion. Second, the tertiarystructure of AcrBΔloop is different from that of wild-type AcrB. Since AcrBΔloop was digested faster, itstertiary packing might be “looser” than the tertiarystructure packing in wild-type AcrB.To determine which one is the case, we developed

a method based on disulfide bond trapping that canbe used to probe the protein tertiary structure in thecell membrane under the native condition.13 Mostcurrent protein structure characterization methods,such as CD spectroscopy and the limited proteasedigestion analysis discussed above, require theextraction of the target membrane protein from thecell membrane and purification. The sample prep-aration process may affect the structure of theprotein and cause protein oligomers to dissociate.Our method takes advantage of the fact that thereplacement of the two intrinsic Cys in AcrB withAla has no impact on AcrB activity.20,21 Using theCys-less AcrB (CLAcrB) as background, we intro-duced five reporter Cys pairs in the periplasmicdomain of AcrB.13 According to the crystal structureof AcrB, these five pairs of Cys should form disulfidebonds. The extent of disulfide bond formation in theprotein under the native condition (i.e., before thecell membrane was disrupted) was probed using ablocking–reducing–labeling scheme, as described inMaterials and Methods. Although the protein waseventually purified and analyzed using SDS-PAGE,the blocking step helped capture the state of theprotein before it suffered from the stress of detergentextraction and protein purification.The extent of disulfide bond formation reflects

protein structural change. If the distance betweenthe two Cys in a reporter pair changed for severalangstroms, the extent of disulfide bond formationwill change significantly and can be identified by thefluorescence labeling method.13 The extents ofdisulfide bond formation in five reporter pairswere used as a panel to reflect a potential structuralchange in AcrBΔloop. We have shown that all fivepairs formed a disulfide bond on the CLAcrB

background, and none of them drastically affectedthe drug efflux activity of AcrB.13 In this study,we introduced these reporter Cys pairs into theAcrBΔloop background. In practice, we truncated theloop in each of the five reporter constructs through aone-step QuikChange mutagenesis, as described inMaterials and Methods. The extents of disulfidebond formation in these reporter pairs with theAcrBΔloop mutation are shown in Fig. 5. The firstlanes in each gel contained samples that werereduced using DTT before they were labeled usingfluorescein-5-maleimide (Flu-MAL). The secondlanes contained samples that were purified in thepresence of iodoacetamide (IAM) to block free thiolsand then labeled using Flu-MAL. The third lanescontained samples that were purified in the presenceof IAM and then reduced using DTT before labeling.When treated with DTT alone (lane 1), all reporterswere fluorescently labeled, indicating the presenceof Cys in the sequence. In contrast, when treatedwith IAM alone (lane 2), none of the proteins waslabeled, indicating that the IAM treatment waseffective in blocking free thiols. Finally, whentreated first with IAM to block free thiols and thentreated with DTT to reduce the disulfide bond (lane3), only proteins containing a disulfide bond werelabeled (Fig. 5a). In a previous study, we haveshown that all reporter pairs formed a disulfidebond when introduced into CLAcrB.13 As shown inFig. 5b, the truncation of the protruding loop had nosignificant effect on the extents of labeling for allreporter pairs, suggesting that the tertiary structureof AcrB in the cell membrane was not disrupted bythis modification.All mutations and labeling experiments described

above were conducted using CLAcrB as the proteinbackground. To further demonstrate that the looptruncation has no impact on the overall conforma-tion of the protein, we created a loop truncationmutant on the wild-type AcrB background. Wild-type AcrB contains two intrinsic Cys (C493 andC887), which do not form a disulfide bond accord-ing to the crystal structure of AcrB. Our labelingexperiment further confirmed that these two Cysexisted as free thiols (Fig. 5c, WT). Furthermore, wefound that truncation of the loop (Fig. 5c,WTΔloop)had no effect on labeling, suggesting a lack of globalstructural change in the mutant.

AcrBΔloop exists as a well-folded monomer

Finally, to investigate how the deletion of the loopeliminated transport activity, we investigated theoligomeric state of AcrB. First, we analyzed purifiedAcrBΔloop using blue native polyacrylamide gelelectrophoresis (BN-PAGE). BN-PAGE is a methodthat is widely used in the determination of mem-brane protein oligomeric states22,23 and has beenused to confirm that wild-type AcrB is a trimer.24

Fig. 6. (a) After detergent purification, freshly preparedwild-type AcrB samples migrated as a trimer in BN-PAGE.Under the same condition, purified AcrBΔloop migrated as amonomer. A 4–20% gradient gel was used in thisexperiment. Proteins that made up the molecular markerinclude apoferritin band 1 (720 kDa), apoferritin band 2(480 kDa), β-phycoerythrin (242 kDa), lactate dehydroge-nase (146 kDa), and bovine serum albumin (66 kDa). (b)Western blot analysis of membrane vesicles extracted fromBW25113ΔacrB expressing the wild-type AcrB or AcrBΔloopafter chemical cross-linking. The molecular masses (inkilodaltons) of bands in the molecular mass marker weremarked on the left of the gels.

Fig. 5. (a) Schematic illustration of the blocking–reducing–labeling procedure. (b) Tertiary structure as revealed by thedisulfide trapping method. The extents of disulfide bond formation for each reporter were very similar in AcrBΔloop ascompared to those in wild-type AcrB. Therefore, the overall conformation, or tertiary structure, of AcrBΔloop was verysimilar to that of wild-type AcrB. (c) Control experiments showing that no disulfide bond was formed in the wild-typeAcrB or when the truncation mutation was introduced into the wild-type background (WT Δloop).

269Folding of AcrB Subunit Precedes Trimerization

The results of BN-PAGE showed that AcrBΔloopmigrated as a monomer. We have also confirmedthat, under the same experimental conditions, wild-type AcrB migrated as a trimer, consistent with theresult in the literature (Fig. 6a).The observation that AcrBΔloop migrated as a

monomer in BN-PAGE after purification does notcompletely rule out the possibility that the proteinmay associate weakly as a dimer or as a trimer in thecell membrane but may dissociate during theprocess of purification and electrophoresis analysis.To further investigate the impact of the mutation onthe quaternary structure of the protein, we con-ducted a cross-linking experiment using membranevesicles and dithiobis(succinimidyl propionate)(DSP) as the cross-linker. DSP cross-links two –NH2 groups. Cross-linking was performed usingmembrane vesicles extracted from BW25113ΔacrBcells expressing wild-type AcrB or AcrBΔloop.Immunoblotting with an anti-AcrB antibody wasused to detect the protein bands after SDS-PAGEanalysis. As shown in Fig. 6b, the wild-type AcrBtrimer was clearly visible, while AcrBΔloop onlymigrated as a monomer. Some wild-type AcrBmonomers were also visible, presumably due tothe incompleteness of the cross-linking reaction. Inthe absence of cross-linking or upon the addition ofDTT, wild-type AcrB only migrated as a monomerunder the same experimental conditions (data not

shown). This result further confirmed that AcrBΔloopexisted as a monomer in the inner membrane ofEscherichia coli.

270 Folding of AcrB Subunit Precedes Trimerization

AcrBΔloop does not have a significantdominant-negative effect when expressedin a wild-type E. coli strain

To examine if the expression of AcrBΔloop in-terferes with the function of the wild-type AcrB, wetransformed plasmid-encoded AcrBΔloop into awild-type AcrB strain, BW25113, and examined thedrug susceptibility level of the resultant strain. In aprevious study, Tikhonova and Zgurskaya showedthat when another functionless AcrB mutant,AcrBD408A, was overexpressed in a wild-type E.coli strain, the drug susceptibility level of the strainwas drastically reduced.25 D407 and D408 are twohighly conserved acidic residues in the transmem-brane domain of AcrB that are critical for the protonrelay pathway, which provides the energy needed todrive the protein conformational change critical forthe function of AcrB.26 The crystal structure ofAcrBD407A, which forms a trimeric structure highlyresembling that of the wild-type AcrB, has beendetermined.27 The exact reason behind the observeddominant-negative effect when AcrBD408A wasexpressed in the wild-type E. coli strain remainselusive.25 AcrBD408A could potentially interrupt thenormal function of genomic AcrB in the wild-typestrain by competing for binding with AcrA and TolCor by co-assembling with the genomic AcrB.Takatsuka and Nikaido have shown that all threesubunits in an AcrB trimer need to be active for theprotein to function properly.24 Therefore, if an AcrBtrimer contains one or more AcrBD408A subunits, itwill not be functional.The MICs of the wild-type strain BW25113

transformed with plasmids encoding wild-typeAcrB or AcrBΔloop are shown in Table 2. BW25113transformed with the empty vector pQE70 was usedas a control. Other than established AcrB substrates,we also included a non-AcrB substrate, kanamycin,as a control for nonspecific effect. Expression ofmembrane proteins may cause stress in the cellmembrane and may lead to a nonspecific reductionof MIC. The MIC for kanamycin was monitored as acontrol for this non-AcrB-specific effect. The expres-sion of AcrBΔloop caused a small change in MIC (2-fold or less) for all drugs tested, including kanamy-cin. These results suggest that the expression ofAcrBΔloop does not specifically disrupt the functionof the genomic AcrB.

Table 2. MICs of BW25113 expressing plasmid-encodedacrB genes

Plasmid ormutant

MIC (μg/mL)

Erythromycin Novobiocin Kanamycin

pQE70 128 128 10pQE70AcrBΔloop 64 64 5pQE70AcrB 128 128 5

Discussion

The goal of this study is to examine if a monomericAcrB mutant could exist in the cell membrane and, ifso, how the structure of this mutant compares withthat of wild-type AcrB subunit in a trimer. Theanswer to this question will shed light on theoligomerization pathway of AcrB. The capabilityof AcrB monomer to fold, independent of theneighboring subunits in the cell membrane, willsuggest a three-stage oligomerization pathway,which involves a well-folded monomeric interme-diate state. AcrB is an obligate trimer. No mono-meric or dimeric AcrB has been previously observedin cell membranes. After extraction from themembrane using a detergent, AcrB predominantlyexists and crystallizes as a trimer.28–31 Change in thebuffer condition or extended storage may causeAcrB trimers to dissociate into monomers, but noevidence on whether these monomers could reas-semble into a functional trimer has been reported.32

To create monomeric AcrB in the cell membrane,we need to disrupt the inter-subunit interactionsand, at the same time, minimize the disturbance tointra-subunit interactions. Through examination ofthe structure of AcrB, we speculated that mutationsin the protruding loop, as illustrated in Fig. 1, mightgenerate monomeric AcrB mutants. Since the loop isdistant from the bulk of the protein structure, weexpected it not to be important for the packing of theprotein tertiary structure although, at the same time,it is critical for the inter-subunit interactions. Wedeleted 17 residues in the loop, corresponding to halfof the overall length of the loop. Next, we character-ized the mutant AcrBΔloop in several aspects.If a mutation drastically reduced the overall stability

of a protein, it could cause themutant protein to fail thequality control mechanism of the cell and result in adrastic decrease in the protein expression level.We firstcompared the expression levels of AcrBΔloop and wild-type AcrB and found that the two proteins wereexpressed at similar levels. The subsequent activityassay indicated that AcrBΔloop was completely inac-tive. Based on the current knowledge of the workingmechanism of AcrB, the drug efflux activity of AcrBcan be disrupted in several aspects, including thedisruption of the substrate binding or proton relaypathway, the folding of each subunit, the assembly of afunctional AcrB trimer, or the interaction betweenAcrB and its functional partners AcrA and TolC.33–41

Since the site of mutation is distant from the sites ofsubstrate binding, proton relay, and AcrA/TolCdocking, the lack of function is more likely to be aresult of disrupting subunit folding or trimerization.Far-UV CD analysis showed that the secondary

structure ofAcrBΔloopwas very close to the structure ofwild-type AcrB. The thermal stabilities of the twoproteins were also comparable. Near-UV CD andlimited trypsin digestion results indicated that there

271Folding of AcrB Subunit Precedes Trimerization

were differences at either the tertiary structure level orthe quaternary structure level between the two pro-teins. To investigate which one is the case, we used amethod based on disulfide bond trapping to track thedistance between five reporter pairs located in theperiplasmic domain of the proteins. The distancesbetween each pair of Cys were largely unchanged inthe mutant, indicating that the overall tertiary foldingof the mutant was very close to that of the wild-typeAcrB. The quaternary structure of AcrBΔloop wasinvestigated using two methods, BN-PAGE and DSPcross-linking followed by SDS-PAGE. Results fromboth experiments suggested that AcrBΔloop existed asa monomer. Finally, we expressed AcrBΔloop in awild-type E. coli strain to examine if AcrBΔloopdisrupted the normal activities of genomic AcrB,which has been reported for another functionlessAcrB mutant, AcrBD408A. We observed a 2-folddecrease in MIC for both AcrB substrates and non-AcrB substrate, indicating that AcrBΔloop did not havea significant effect on the function of genomic AcrB.In summary, in this study, we reported a detailed

characterization of the first AcrB mutant that existedas a monomer in the cell membrane. The tertiarystructure of the mutant in the cell membrane wasvery close to the structure of individual subunits inthe final AcrB trimer. The observation of anindependent and well-folded AcrB monomer in-dicates that the folding of individual subunits doesnot need assistance from neighboring subunits.These results suggest that oligomerization of AcrBtrimer occurs through a three-stage pathway involv-ing a well-folded monomeric intermediate state.Folding of membrane proteins is an intriguing and

yet poorly understood biological process.42 In terms ofα-helical membrane proteins, the research communityhas acquired a decent understanding of the identifica-tion of transmembrane helices and the prediction oftheir topology in the membrane. However, howmembrane proteins achieve their tertiary and quater-nary structures remains largely elusive. Here, wedemonstrated, using AcrB as an example, that forproteins in which a soluble domain contributessignificantly to inter-subunit interactions, a three-stage oligomerization pathway is more likely to bethe case, as recognition between folded domains isrequired prior to oligomerization. For proteins contain-ing only transmembrane domains, folding and oligo-merization may happen co-translationally withmembrane insertion following a two-stage model.

Materials and Methods

Cloning, expression, and purification of AcrB andits mutants

Plasmid pQE70AcrB was used as template in theconstruction of plasmid pQE70AcrBΔloop.13 The QuikChange

mutagenesis kit (Agilent Technologies) was used to createthis deletion mutation, in which N211-G227 was deleted(Fig. 1). Complementary primers used for the construction ofAcrBΔloop are as follows: 5′-CATTACCGCCATCAAAGCG-CAGCAACAGCTTAACGCCTCTATTATTGC and 5′-GCAAT AATAGAGGCGTTAAGCTGTTGCTGCGCTTT-GATGGCGGTAATG. With the same method, loop deletionmutation was introduced into the AcrB reporter constructsCLAcrBV32C/I390C, CLAcrBT44C/T91C, CLAcrBM184C/V771C,CLAcrBT199C/T749C, and CLAcrBQ726C/G812C, which werecreated in a previous study.13 Protein coding sequences inall plasmids used in this study were confirmed by DNAsequencing. AcrB and its mutants were expressed in an E. colistrain deficient in the acrB gene (BW25113ΔacrB) andpurifiedas described previously.13,43

Drug susceptibility assay of AcrBΔloop mutant

AcrB activity could be measured using a drug suscep-tibility assay. The MICs of different strains were measuredas described previously.13,24 Briefly, E. coli strainBW25113ΔacrB was used as the host cell. BW25113ΔacrBstrains transformed with the plasmid-encoded wild-typeAcrB (pQE70AcrB) or the pQE70 vector were used aspositive and negative controls, respectively. Plasmidsencoding different AcrB mutants were used to transformBW25113ΔacrB as well. Freshly transformed cells wereplated on LB agarose plates containing 100 μg/mLampicillin and 50 μg/mL kanamycin. The same ampicillinand kanamycin concentrations were used throughout thestudy, when noted. A single colony was used to inoculateLB media supplemented with ampicillin and kanamycin.The exponential-phase cultures of different strains werediluted to an OD600 (optical density at 600 nm) unit of 0.1using LB broth. Five microliters of this culture was used toinoculate 2 mL of LB media containing the indicatedconcentration of erythromycin or novobiocin. The cultureswere incubated at 37 °C overnight, with shaking . The nextmorning, the OD600 of each sample was measured. Theactivity assay was conducted under the basal expressioncondition. Each experiment was repeated at least threetimes. Each experiment was repeated at least three times.

Expression, purification, and labeling of AcrBand its mutants

Labeling of AcrB reporters was performed as describedpreviously.13 Briefly, AcrB reporter mutants freshlyexpressed in BW25113ΔacrB, treated with IAM whennoted, were purified with immobilized metal-affinitychromatography. IAM blocked free thiols in the proteinwithout affecting disulfide bonds. After purification,protein samples were treated with DTT to reduce disulfidebonds and to generate free thiols. These freshly generatedthiols were immediately labeled with Flu-MAL andanalyzed using SDS-PAGE.

Analysis of the expression levels of wild-type AcrBand AcrBΔloop by immunoblotting

Freshly transformed colonies of BW25113ΔacrB con-taining plasmid-encoded wild-type AcrB or AcrBΔloop

272 Folding of AcrB Subunit Precedes Trimerization

were used to inoculate LB medium containing ampicil-lin and kanamycin. After being cultured at 37 °Covernight, cells were harvested, resuspended in 10 mMK-Hepes buffer (pH 7.5) to an OD600 of 15, andruptured using a French press in the presence ofPMSF. Cell debris was removed via low-speed centri-fugation, and membrane vesicles were collected usingultracentrifugation at 150,000g for 1 h at 4 °C. Mem-brane vesicles were resuspended in a 10 mM K-Hepes(pH 7.5) buffer containing 2% (wt/vol) SDS, sonicatedon ice for 1 min with 10-s on/off intervals, and thenincubated at 37 °C for 2 h. SDS loading buffer was thenadded into the samples. The samples were resolvedwith SDS-PAGE on an 8% gel and transferred to anitrocellulose membrane for Western blot analysis usinga polyclonal rabbit anti-AcrB primary antibody and analkaline-phosphatase-conjugated anti-rabbit secondaryantibody. Protein–antibody conjugates were visualizedusing the substrates nitroblue tetrazolium chloride and5-bromo-4-chloro-3′-indolyl phosphate p-toluidine.

CD spectroscopy and heat stability analysis

CD was performed using a JASCO J-810 spectrometerwith a 1-nm bandwidth. Protein samples were dialyzedovernight into a low-salt buffer (10 mM sodium phos-phate, 50 mMNaCl, 10% glycerol, and 0.05% n-dodecyl-β-maltoside, pH 7.5) before the CD measurement. Blankscans were collected using the exterior dialysis buffer. Forfar-UV CD spectra, samples in a 1-mm path-lengthcuvette were scanned in the wavelength range of 250–190 nm. For the near-UV CD analysis, spectra werecollected in the wavelength range of 350–250 nm using a1-cm path-length cuvette. All spectra were corrected forblanks. Protein concentrations were determined using theBradford assay.

BN-PAGE analysis

BN-PAGE was performed as described previously.22,23

Briefly, purified protein samples were mixed with bluenative loading buffer to reach a final concentration of0.1 M 6-aminoocaproic acid, 10 mM 2-[bis(2-hydro-xyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol–HCl,6% sucrose, and 1% Coomassie brilliant blue G-250(pH 7.0). The protein samples were loaded onto a 4–20%gradient polyacrylamide gel containing 0.5 M 6-aminoo-caproic acid and 50 mM 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol–HCl (pH 7.0). The elec-trophoresis was performed using running buffer (25 mMTris–HCl, 192 mM glycine, and 0.01% Coomassie brilliantblue G-250, pH 8.3) at 60 V, in a 4 °C refrigerator for 12 h.The protein bands were visualized after Coomassie bluestaining. Band intensity was quantified using the softwareImageJ.44

Limited trypsin digestion

Trypsin was added into purified AcrB samples at amolar ratio of 1:200 in a reaction buffer containing 20 mMNa-phosphate buffer (pH 7.5), 0.1 M NaCl, 0.05% n-dodecyl-β-maltoside, and 10% glycerol. The samples were

incubated at room temperature for the indicated timeperiod before PMSF (final concentration, 2 mM) and SDSloading buffer were added to stop the digestion reaction.The samples were then heated at 95 °C for 2 min andresolved using SDS-PAGE.

Chemical cross-linking

Membrane vesicles were extracted from BW25112ΔacrBcells expressing the protein of interest as describedabove. Then, the membrane vesicles were suspended ina buffer containing 50 mM phosphate, 0.15 M NaCl, and0.5 mM DSP (pH 7.5). The suspension was incubated at37 °C for 40 min. Tris–Cl solution (pH 8.0) was added toa final concentration of 50 mM to quench the cross-linking reaction. Then, Triton X-100 was added to a finalconcentration of 1%, and AcrB was purified as describedabove. Purified AcrB and AcrBΔloop were subjected toSDS-PAGE analysis under the nonreducing condition.Western blot analysis was performed as describedabove.

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