Transmembrane Gate Movements in the Type II ATP-binding

Transmembrane Gate Movements in the Type II ATP-bindingCassette (ABC) Importer BtuCD-F during Nucleotide Cycle*□S

Received for publication, June 14, 2011, and in revised form, September 8, 2011 Published, JBC Papers in Press, September 27, 2011, DOI 10.1074/jbc.M111.269472

Benesh Joseph‡, Gunnar Jeschke‡, Birke A. Goetz§, Kaspar P. Locher§, and Enrica Bordignon‡1

From the ‡Laboratory of Physical Chemistry, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland and the §Institute ofMolecular Biology and Biophysics, ETH Zurich, HPK D17, Schafmattstrasse 20, 8093 Zurich, Switzerland

ATP-binding cassette (ABC) transporters are ubiquitousintegral membrane proteins that translocate substrates acrosscell membranes. The alternating access of their transmembranedomains to opposite sides of themembrane powered by the clo-sure and reopening of the nucleotide binding domains is pro-posed to drive the translocation events. Despite clear structuralsimilarities, evidence for considerable mechanistic diversitystarts to accumulate within the importers subfamily. We pres-ent here a detailed study of the gating mechanism of a type IIABC importer, the BtuCD-F vitamin B12 importer from Esche-richia coli, elucidated by EPR spectroscopy. Distance changes atkey positions in the translocation gates in the nucleotide-free,ATP- and ADP-bound conformations of the transporter weremeasured in detergent micelles and liposomes. The transloca-tion gates of the BtuCD-F complex undergo conformationalchanges in line with a “two-state” alternating access model. Weprovide the first direct evidence that binding of ATP drives thegates to an inward-facing conformation, in contrast to type Iimporters specific for maltose, molybdate, or methionine. Fol-lowing ATP hydrolysis, the translocation gates restore to anapo-like conformation. In thepresenceofATP, an excess of vita-min B12 promotes the reopening of the gates toward theperiplasm and the dislodgment of BtuF from the transporter.The EPR data allow a productive translocation cycle of the vita-min B12 transporter to be modeled.

ATP-binding cassette (ABC)2 transporters couple the energyof ATP hydrolysis to the translocation of substrates across bio-logicalmembranes. They constitute the largest transmembraneprotein family present in all branches of life and mediate theactive transport of various substances such as sugars, aminoacids, peptides, vitamins, iron siderophores, opines, metals, etc.across the membrane (1–3). Biochemical evidence supports a

“two-state, alternating access”mechanisticmodel for bothABCexporters and importers (4). In this model, an ATP-bound con-formation of the transmembrane domains facing the extracel-lular side of the membrane is converted to an inward-facingconformation via ATP hydrolysis in the nucleotide-bindingdomains (NBDs). This conformational transition ensures netsubstrate uptake by the importers or net expulsion by theexporters. Among the canonical ABC importers, which arecharacterized by the presence of a soluble periplasmic substratebinding protein, two structurally different types exist, namelytype I (e.g. maltose, molybdate, and methionine systems) andtype II (e.g. vitamin B12, heme, and metal systems) (3). For type IABC importers the details of the alternating access mechanismhave been confirmed by a large body of experimental evidence,including the crystal structures showing the maltose trans-porter in different states during the nucleotide cycle (5–8). Thetransmembrane domains (TMDs) of type I importers, featuring10–14 helices, alternate from an ATP-bound outward-facingconformation where the substrate binding protein releases itscargo to the low affinity binding site in the TMDs, to an ADP-bound inward-facing conformation where the substrate isreleased in the cytoplasm. Type II importers comprise up to 20TM helices, which can translocate substrates up to 1 order ofmagnitude bigger than those imported by the type I (e.g. vita-min B12, 1355 Da; methionine, 141 Da). To date, only two typeII importers have been crystallized: the BtuCD vitamin B12importer from Escherichia coli in the presence and absence ofthe substrate binding protein BtuF (9, 10) and the BtuCDhomologous putative metal chelate importer HI1470/1 fromHaemophilus influenzae (11). The first BtuCD structureobtained with the translocation pathway open to periplasm in anucleotide-free state suggested that this transporter mightoperate by a mechanism opposite to that of type I transporters.However, the homologousHI1470/1 importerwas later crystal-lized in a nucleotide-free inward-facing conformation, similarto the apo-state of the type I importers. The structure ofBtuCD-F with an occluded asymmetric translocation pathwaysuggested further mechanistic diversity between type II andtype I importers. Overall, the three crystallized states confirmthe possibility of an alternating access mechanism for substratetransport. However, unlike the maltose transporter, theabsence of nucleotides in all the three structures makes itimpossible to delineate the sequence of the conformationalswitches during the transport cycle.Besides the structural diversity between the two types of

importers, remarkable differences exist in the interactionsbetween the substrate binding proteins and the TMDs. The

* This work was supported by the ETH Research Grant ETH-21 09-1 (to G. J.and E. B.) and by Grant Swiss National Science Foundation (SNF) 31003A-131075/1 (to K. P. L.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1, Figs. S1–S7, Equation S1, data, and additionalreferences.

1 To whom correspondence should be addressed: Laboratory of PhysicalChemistry, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzer-land. Tel.: 41-44-6337570; Fax: 41-44-633-1448; E-mail: [email protected].

2 The abbreviations used are: ABC, ATP-binding cassette; NBD, nucleotide-binding domain; TMD, transmembrane domain; MTSSL, 1-oxyl-2,2,5,5,-te-tramethyl-d-3-pyrroline-3-methyl)methanethiosulfonate; AMPPNP, 5�-ad-enylyl-�,�-imidodiphosphate; LDAO, lauryldimethylamine N-oxide; DEER,double electron electron resonance; cw, continuous wave.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 47, pp. 41008 –41017, November 25, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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affinity of the substrate binding protein is orders of magnitudelarger in type II importers (Kd� 10�13 and 10�9 M, for BtuCD-FandHi1470/1–2, respectively) (12) than in type I (Kd � 10�4 M)(13). Additionally, no substrate binding site could be identifiedin the type II TMDs.Another striking difference is that the BtuFaffinity for BtuCD is decreased by 2 orders of magnitude in thepresence of ATP (and in the transition state intermediate) (12).The affinity is further reduced by high concentrations of sub-strate. By contrast, the highest affinity of the substrate bindingprotein to type I importers is in the transition state intermedi-ate (14), and no evidence for substrate-induced changes inaffinitywas found.All of these observations highlight themech-anistic differences between type I and type II importers.Probe techniques such as site-directed spin labeling (15)

allow for studying conformational transitions during the trans-port cycle in ABC transporters (16–21). Methanethiosulfonatespin labels (MTSSL) were previously attached in BtuCD to cys-teines strategically placed at the cytoplasmic end of TM5 (Ser-141) and in the short periplasmic loop between TM5 and helix5a (Thr-168) (10, 20), which are key residues in the substratetranslocation channel (see Fig. 1). It was shown by EPR that inthe absence of BtuF, the dynamics of the two gates were unaf-fected by the presence of nucleotides. In contrast, the presenceof BtuF induced changes in the dynamics of both gates, indicat-ing a downstream communication from the periplasmic to thecytoplasmic region of the transporter mediated by BtuF. Addi-tion of AMPPNP (a non-hydrolyzable analog of ATP) switchedthe cytoplasmic gate to a highly mobile conformation (20).Although an increase in mobility is not generally correlatedwith an increase in distance between the two spin labels, Goetzet al. (20) proposed that the higher mobility in the AMPPNP-bound BtuCD-F may reflect an inward-facing conformation.In this work, we used spin-labeled cysteines preceding and

following TM5 (residues Ser-141, Thr-142, and Thr-168,respectively) to quantitatively follow themovement of the cyto-plasmic and periplasmic gates during the nucleotide cycle usingpulse EPR techniques. In the present work, we delineate thesequence and molecular details of the opening and closing ofthe translocation pathway gates during the nucleotide cycle.The combined effects of substrate and nucleotides on the affin-ity of BtuF for the TMDs are also described. We could conclu-sively observe that the cytoplasmic gate of BtuCD-F opens uponATP binding, which could facilitate the release of vitamin B12into the cytoplasm. An EPR-basedmodel of a productive trans-location event is provided.

EXPERIMENTAL PROCEDURES

Expression and Purification of BtuF and BtuCD—BtuF waspurified as described previously (22). BtuF loaded with vitaminB12 was stored at 4 °C up to 3 months. Protein concentrationwas determined by absorption at 361 nm.Cysteine mutations in BtuCD at positions 141, 142, and 168

were introduced on a “Cys-less” plasmid as described before(10). BtuCDwas extracted and purified in lauryldimethylamineN-oxide (LDAO) as described previously (9) with some modi-fications. BtuCD mutants were overexpressed in E. coliBL21(DE3) Gold cells. The frozen cells were solubilized in 1%LDAO (Anatrace) in 50 mM Tris-HCl (pH 7.5) containing 25

mM imidazole-HCl (pH 8.0), 5mM �-mercaptoethanol, and 500mM NaCl. The cells were lysed by sonication using a Sonicsvibra-Cell sonicator. The lysate was centrifuged for 30 min at40,000 � g and then loaded onto a nickel-nitrilotriacetic acidcolumn, prewashed with 50 mM Tris-HCl (pH 7.5) containing0.1% LDAO, 25 mM imidazole-HCl (pH 8.0), 5 mM �-mercap-toethanol, and 500 mM NaCl. The column was washed with 50mM Tris-HCl (pH 7.5) containing 0.1% LDAO, 90 mM imidaz-ole-HCl (pH8.0), and 500mMNaCl. BtuCDwas elutedwith 200mM imidazole in 50 mM Tris-HCl (pH 7.5) containing 0.1%LDAO and 500 mM NaCl. The protein was immediatelydesalted with a HiPrep desalting column (GE Healthcare) into50 mM Tris-HCl (pH 7.5) containing 0.1% LDAO and 500 mM

NaCl. Protein aggregation was checked by size-exclusion chro-matography with a Superdex 200 10/300 column (GEHealthcare).Spin Labeling of BtuCD Mutants—After desalting, BtuCD

was concentrated to 15–20�Mwith 50-kDaAmiconUltraCon-centrators (Millipore). A 40 fold molar excess of MTSSL wasadded in 4 batches every 5 min at room temperature whileshaking. To prepare BtuCD-F, vitamin B12-bound BtuF wasadded at this stage at a molar ratio of 1:2 BtuCD:BtuF. ExcessMTSSL was removed using PD10 desalting columns (GEHealthcare). The functionality of protein preparationswas ana-lyzed with ATPase assay as described (22). For experiments inLDAO, the protein was concentrated to 100 �M using AmiconUltra Concentrators (Millipore). At this stage samples could besnap-frozen in liquid nitrogen in small aliquots and stored at�80 °C for further use up to three months.Reconstitution of BtuCD and BtuCD-F in Liposomes—For

reconstitution BtuCD and BtuCD-F were concentrated to 30�M and reconstitution was performed according to the proto-col described before by exchanging the LDAO to Triton X-100(22) or directly from LDAO supplementing the sample with0.14% Triton X-100 and incubating it with liposomes for 1 h atroom temperature. Liposomes were pre-incubated with 0.14%TritonX-100 for 1 h. A 1:500molar ratio of BtuCD to lipids wasused. Reconstitution was performed using BioBeads SM-2 asdescribed (22). ATPase activities were measured for all spin-labeled mutants according to Borths et al. (22).Sample Preparation for EPR Measurements—BtuCD and

BtuCD-F in LDAO or in liposomes were prepared in the apo-state (absence of nucleotides). The ATP-bound state was pre-pared incubating the sample for 5 min at 4 °C with 1 mM ATP,50�MEDTA. TheATP analogAMPPMPwas also used at 1mM

concentration. The post-hydrolytic state was induced by incu-bation of the sample for 10 min at 37 °C with 1 mM ATP and 2mMMgCl2 or directly by incubation at 4 °C for 5minwith 1mM

ADP and 2 mM MgCl2. For DEER measurements, 10% (v/v)deuterated glycerol was added to the samples before snap freez-ing them in liquid nitrogen.Continuous Wave and Pulse EPR—All continuous wave (cw)

X-band EPR experiments were performed with a Bruker Elex-sys E580 spectrometer equipped with a Bruker Elexsys SuperHigh Sensitive probehead at room temperature or at 160 Kusing a Bruker N2 flow cryostat. EPR spectra were detected atroom temperature in EPR glass capillaries (0.9-mm inner diam-eter; sample volume, 15 �l) with a 100 kHz field modulation, 2

Gate Movements in Type II ABC Importer

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milliwatts of microwave power, and a modulation amplitude of0.15 milliteslas. EPR spectra for interspin distance determina-tion were recorded at 160 K in EPR quartz capillaries (3-mmouter diameter; sample volume, 30 �l) with 100 kHz field mod-ulation, 0.08 milliwatts of microwave power, and a modulationamplitude of 0.25 milliteslas. Fitting of dipolar-broadened EPRpowder spectra was performed with the software DIPFIT (23).The line width parameters of the reference non-dipolar-broad-ened spectrum for each spin-labeled position in LDAO or inliposomes were obtained by fitting the spectrum in the nucleo-tide state, which showed distances �2 nm in DEER. The dipo-lar-broadened spectra were fitted by fixing all parameters,except Azz, distance and distance distribution (Gaussianmodel).DEER measurements were performed at X-band frequency

with a Bruker Elexsys E580 spectrometer equipped with aBruker Flexline split-ring resonator ER 4118X-MS3 using acontinuous flow He cryostat (ESR900; Oxford Instruments)controlled by an Oxford Instruments temperature controllerITC503S.DEERmeasurementswere also performed atQ-bandfrequency (34–35 GHz) on a home-made spectrometerequipped with a home-made rectangular resonator enablingthe insertion of X-band sample tubes with an outer diameter of3mm (24). Dipolar time evolution data were acquired using thefour-pulse DEER experiment. All DEER measurements wereperformed at 50 K. For X-band DEER, observer pulse lengthswere set to 32 ns for �/2 and � pulses, with the ELDOR � pulseset to 12 ns. The ELDOR frequency was set at the maximum ofthe echo-detected field swept spectrum, 65MHz lower than theobserver frequency. Deuterium nuclear modulations wereaveraged by increasing the first interpulse delay by 56 ns foreight steps. For Q-band DEER, all pulses were set to 12 ns, anddeuterium nuclear modulations were averaged by increasingthe first interpulse delay by 16 ns for eight steps. The ELDORfrequency was set at the maximum of the echo-detected fieldswept spectrum, 80 MHz higher than the observed frequency.Traces were accumulated for 2–6 h at Q band and for 24–48 hat X band, depending on spin concentration.The background of the DEER primary data (V(t)) was fitted,

and the resulting secondary data (F(t)) were converted by amodel-free Tikhonov regularization to distance distributionswith the software DeerAnalysis2010 (25). The simulation of thepossible spin label rotamers attached at a position was per-formed using the Matlab program package MMM based on arotamer library approach (26).

RESULTS

The crystal structures of BtuCD-F suggested that theperiplasmic (T168C) and the cytoplasmic (S141C, T142C) gateresidues offer key positions to follow conformational changesduring the transport cycle (9, 10). Fig. 1 shows the crystal struc-tures of the vitamin B12 importer, with the two gate positionsspin labeled in silico with MTSSL based on a rotamer libraryapproach implemented in the software MMM (26). The simu-lation provides the spin label rotamers, which can be populatedin the structures and predicts the distances between the nitrox-ides of the two labeled sites. Based on the simulations, interspin

distance measurements should reveal the effect of BtuF bind-ing, especially at the periplasmic gate.During the past few years, DEER (also known as pulsed elec-

tron electron double resonance) has been widely used as themost sensitive technique to extract distances in the 2–6 nmrange (27) on a variety of membrane proteins. For shorter dis-tances (1–2 nm), the analysis must be complemented with lineshape fittings of low temperature cw EPR spectra (23).In this work, DEER is used to measure interspin distances

between the spin-labeled R1 side chains at positions 168, 141,and 142 in BtuCD and BtuCD-F (in the following, R1 willdenote the unnatural side chain carrying the nitroxide radical).Prior to the EPR analysis, ATPase activities were measured forall spin-labeled mutants both in LDAO and in liposomes. InLDAO, the ATPase activities were found to be similar to thosemeasured in the wild type transporter (22), except for 142R1,which showed a 50% activity reduction. Nevertheless, the142R1mutant in LDAOwas proven to be able to bind ATP and

FIGURE 1. BtuCD and BtuCD-F, simulation of interspin distances. A, ribbonrepresentation of the x-ray structures of BtuCD and BtuCD-F (Protein DataBank codes 1L7V and 2QI9, respectively). The calculated spin label rotamersattached to the engineered cysteines S141C, T142C, and T168C with the soft-ware MMM are shown in red and blue ball and stick representation. Yellow,BtuC; green, BtuD; red, BtuF. The TM5 helices containing the strategicallyplaced spin-labeled residues at the cytoplasmic and periplasmic gates (posi-tions 141, 142, and 168, respectively) are highlighted in blue. B, interspin dis-tances between the gates in BtuCD and BtuCD-F calculated using MMM. Dot-ted gray line, BtuCD; dotted black line, BtuCD-F.




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ADP, undergoing conformational changes in line with thoseobserved in 141R1. In proteoliposomes, all spin-labeledmutants showed wild type-like ATPase activity, with 141R1being the most active (supplemental Table S1).Opening and Closing of Gates in Liposome-reconstituted

BtuCD-F during Nucleotide Cycle—The purified BtuCD-Fcomplexes were reconstituted in the presence of vitamin B12into liposomes. The final proteoliposomes were colorless, indi-cating the absence of vitamin B12, as observed previously (10).Specifically, the sample was measured in three different states:in the absence of nucleotides, in the presence ofATP andEDTA(to prevent hydrolysis), and in the post-hydrolytic stateobtained either by incubation with ATP andMgCl2 or by directaddition of ADP and MgCl2. The post-hydrolytic statesobtained with both methods revealed similar DEER distancedistributions in proteoliposomes, although incubation with

ATP and MgCl2 showed some residual distances of the ATP-bound state (supplemental Fig. S7).The dynamics of the R1 side chains in liposomes at positions

141 and 168 as revealed by room temperature cw EPR (supple-mental Fig. S1) were found to be in perfect agreement with thepreviously published spectra (20). The DEER analysis per-formed on BtuCD-F reconstituted in liposomes labeled at posi-tions 141, 142, and 168 is presented in Fig. 2.It has been shown that the reconstitution method used here

leads to 93% of the BtuCDmolecules exposing the NBDs to theoutside of the liposomes (22). In line with that, we were unableto see any interaction when BtuF was added to BtuCD recon-stituted in liposomes (data not shown). BtuCD-F was alsoproven by EPR to mainly expose the NBDs to the outside of theliposomes: for all positions investigated an almost completechange of the distance distributions (without residual apo-state

FIGURE 2. Nucleotide-dependent movement of periplasmic (168 –168) and cytoplasmic (141–141, 142–142) gates of BtuCD-F in proteoliposomes. Leftpanel, normalized experimental data V(t)/V(0) and exponentially decaying background arising from the distribution of remote spins (dashed lines), as fitted byDeerAnalysis2010. DEER traces were recorded at X band for positions 141 and 168 and at Q band for position 142. The background was fitted with anexponential function for position 142 (dimension 3 in DeerAnalysis2010) or stretched exponential function (dimension 3.5, for positions 141 and 168). Middlepanel, background-corrected normalized form factor F(t)/F(0) and fit by the Tikhonov regularization. Right panel, distance distribution P(r) obtained withDeerAnalysis2010. For positions 141 and 168, the excitation bandwidth correction was applied for all traces. The inset shows the spin-normalized cw EPRspectra detected at 160 K. The Gaussian distance distributions obtained by DIPFIT (red dotted line, position 168 (ATP-state); black dotted line, position 141(apo-state) (from supplemental Fig. S2)) are superimposed to the DEER-derived distributions in the P(r) panel. black, apo; red, ATP; green, ATP and MgCl2; seagreen, ADP and MgCl2.




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distances) was induced upon ATP addition (Fig. 2). This selec-tive preferential orientation of the transporters in liposomesfacilitated data interpretation.The main distance in the apo-state between positions 168 in

the periplasmic gate is 2 nm (Fig. 2A). ATP binding decreasesthe interspin distance to �2 nm (Fig. 2A), toward the detectionlimit of DEER. Low temperature cw spectra (160 K) wereacquired to complement the analysis. Spin normalized cw spec-tra clearly show dipolar broadening upon ATP binding visibleas a decrease in the spectral intensity (Fig. 2A, inset in the thirdpanel). The mean distance revealed by line shape analysis (sup-plemental Fig. S2) is 1.6 nm (Gaussian dotted line in the P(r)panel of Fig. 2A), in line with the DEER data. ATP hydrolysisreopens the periplasmic gate, restoring an apo-like conforma-tion, as shown by DEER and cw EPR (supplemental Fig. S2).In the apo-state, the distance between the two labels at posi-

tion 141 in the cytoplasmic gate is centered at 1.7 nm (Fig. 2B).ATP binding opens the cytoplasmic gate by 0.7 nm, opposite tothe 0.4-nm decrease between the R1 side chains in the periplas-mic gate. ATP hydrolysis restores the cytoplasmic gate back toan apo-like conformation. Spin normalized low temperatureEPR spectra (inset in the third panel in Fig. 2B) and line shapeanalysis (supplemental Fig. S2) confirmed the movementsobserved by DEER.A similar distance increase was detected also between posi-

tions 142 in the cytoplasmic gate. It is worth noting that forpositions 142, the distance increase observed upon ATP bind-ing is accompanied by a decrease in mobility in the cw spectra(supplemental Fig. S1). The narrow distance distribution in theapo-state is indicative of spin labels strongly interacting withneighboring amino acids. The ADP-bound state is found to beindistinguishable from the apo-state. In summary, the gates ofBtuCD-F are shown to switch synchronously upon ATP bind-ing, with the cytoplasmic gate opening and the periplasmic gateclosing, suggesting an inward-facing conformation.Opening and Closing of Gates in Detergent (LDAO)—All data

presented above were obtained in proteoliposomes in the pres-ence of the substrate binding protein BtuF to mimic as closelyas possible the physiologically relevant state of the transporter.The question of whether the membrane is necessary for thenucleotide-driven conformational switch was investigated byanalyzing the complex solubilized in LDAOmicelles. It is worthnoting that the available crystal structures were also obtainedfrom detergent-solubilized samples, which could impose con-formational constraints during crystal formation. All deter-gent-solubilized samples were formed by addition of vitamin-boundBtuF in a 2-fold excess to BtuCD.The room temperaturecw spectra of the complex in LDAO showed an increased con-formational flexibility in both gates with respect to the proteo-liposomes, indicated by an increased mobility of the R1 sidechains (supplemental Fig. S1). In detergent, the dynamics of theR1 side chain in BtuCD-F as detected by cw EPR are not signif-icantly altered by the presence of nucleotides, probably due tothe high intrinsic flexibility of the complex. However, DEERanalysis showed that the gates open and close similarly to theliposome-embedded samples, with the periplasmic gates clos-ing and the cytoplasmic gates opening upon ATP binding (Fig.3). The restoration of the gates toward the apo-state following

ATP hydrolysis was complete, confirming that the detergent-solubilized transporter can undergo the nucleotide-inducedconformational changes.Unlike the proteoliposomes, the LDAO samples contained

vitamin B12 in a 1:1 molar ratio with respect to BtuF. In LDAO,vitamin B12 is released in the solution upon binding of BtuF toBtuCD. Nevertheless, to address a possible role of the vitaminto the observed gate movements, BtuCD-F complexes spinlabeled at position 168 were washed repetitively until theybecame colorless (no absorbance detected at 360 nm). DEERanalysis of the samples revealed that the gate response is inde-pendent on the presence of vitamin B12 (supplemental Fig. S6).To verify that the observed movements of the TMDs are

mediated through the nucleotide cycle catalyzed by NBDs, thenon-hydrolyzableATP analogAMPPNPwas added to the com-plex. AMPPNP locked the complex in an ATP-like state, whichwas not further modified by incubation with MgCl2 (supple-mental Fig. S3). The EPR data obtained in liposomes andmicelles suggest that ATP triggers an inward-facing conforma-tion of BtuCD-F, opposite to what is validated for the type Iimporters by a large amount of biochemical and crystallo-graphic data.Transmembrane Communication in Absence of Substrate

Binding Protein BtuF—Without BtuF, the characteristic move-ment of the gates upon ATP binding and hydrolysis was notobserved either in LDAO or in liposomes. The DEER datarecorded in LDAO micelles reveal a very disordered periplas-mic gate region, characterized by a broad distribution of dis-tances between the R1 side chains (Fig. 4A). ATP bindinginduced a conformation with a slightly higher propensitytoward shorter distances and ATP hydrolysis restored an apo-like conformation. The cytoplasmic gate (positions 141 and142) shows analogous broad interspin distance distributions(Fig. 4, C and E), which are only slightly affected by nucleotidebinding. In proteoliposomes, the periplasmic gate of BtuCDshows as well a broad distance distribution, which is shiftedtoward shorter distances by addition of ATP (Fig. 4B). Thecytoplasmic gate (position 141) shows a main distance at �3nm, which is only slightly affected by ATP binding (Fig. 4D).Overall, the data suggest that the gates are disordered in theabsence of BtuF, but some communication exists betweenNBDs and TMDs, in agreement with the futile ATP hydrolysiscycle detected in BtuCD.However, only in the presence of BtuFthe gates adopt a well defined conformation and fully accom-plish the switch from the open to the closed state.BtuF: Nucleotide- and Vitamin-dependent Affinity to BtuCD—

It has been shown that BtuCD or the preformed complexBtuCD-F cannot bind vitamin B12 (12, 22). The available struc-tural and biochemical data suggest that BtuCD does not have abinding site for the substrate, unlike the type I importers. DEERand cw data in the presence and absence of BtuF clearly showthat the substrate binding protein binds to the TMDs in all thethree states of the transporter both in liposomes (no vitaminB12) and in LDAO (vitaminB12 to BtuF 1:1 ratio). Excess of ATPorADP-MgCl2 (up to 5mM) had no effect on the affinity of BtuFfor BtuCD at the micromolar protein concentrations used.Interestingly, a vitamin B12 excess was shown recently to pref-erentially impair BtuF binding in theATP-state of the transporter




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(12).We conducted experiments using cw and pulse EPR tomon-itor the influence of a molar excess of vitamin B12 to BtuF on thecomplex formation and the consequent effects on the periplasmicand cytoplasmic gates in detergent. We used as reporter moietiesthe spin labels atpositions168and142.Position141wasnot inves-tigated as the interspin distance distributions in theATP-state aresimilar for BtuCD and BtuCD-F in detergent.The room temperature cw EPR spectra of positions 168 in

BtuCD and BtuCD-F in LDAO (supplemental Fig. S1 and insetin Fig. 5A) show that binding of BtuF produces a characteristicpeak in the low field region due to the reduced mobility of theR1 side chains (asterisk in the inset in Fig. 5A). By increasing thevitamin to BtuF molar ratio, this characteristic peak graduallyvanishes in the presence of ATP. This suggests a gradual disso-ciation of BtuF from the periplasmic region of the transporter.DEER measurements confirmed that at high vitamin concentra-

tions the periplasmic and cytoplasmic gates adopt a conformationclose to the one observed in the ATP-bound state of BtuCD (Fig.5). In contrast, the same experiments performed in the ADP-statein both mutants showed a reduced dissociation of BtuF (supple-mental Fig. S4). Attempts to conduct similar experiments in pro-teoliposomes were not conclusive due to the inside-out orienta-tion of BtuCD-F and the difficulty to introduce excess of vitamininto the lumen of the liposomes.

DISCUSSION

The present study reveals how the transmembrane gates inthe vitamin B12 transporter BtuCD-F respond to the presenceof BtuF, substrate, andnucleotides. The vitaminB12 transporteris suggested to operate by an alternating access mechanism inwhich the ATP-bound state shows an inward-facing conforma-tion of the TM5 helices. The presence of BtuF is shown to be

FIGURE 3. Nucleotide-dependent movement of periplasmic (168 –168) and cytoplasmic (141–141, 142–142) gates of BtuCD-F in LDAO micelles. Leftpanel, normalized experimental data V(t)/V(0) and exponentially decaying background arising from the distribution of remote spins (dashed lines), as fitted byDeerAnalysis2010. The DEER traces for the apo- and ATP-states of the periplasmic gate were detected at X band. All of the other traces were detected at Q band.The background was fitted with an exponential function (dimension 3 in DeerAnalysis2010) or stretched exponential function (dimension 3.5, for position 168).Middle panel, background-corrected normalized form factor F(t)/F(0) and fit by the Tikhonov regularization (black dashed lines). Right panel, distance distribu-tion P(r) obtained with DeerAnalysis2010. For position 142, the inset shows the spin-normalized cw EPR spectra detected at 160 K. The Gaussian distancedistributions obtained by DIPFIT (see also supplemental Fig. S2) in the apo- and ADP-states are superimposed to the DEER-derived distributions in the P(r) panel(dotted lines). Black, apo; red, ATP; green, ATP and MgCl2; sea green, ADP and MgCl2.




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necessary for tight coupling between the NBDs and the trans-membrane gate movements, and the substrate is confirmed tomodulate the complex dissociation.BtuCD as shown by EPR adopts a dynamic conformation in

the apo-state, with the gates overall displaying broader distancedistributions than those expected by simulation of the interspindistances in the crystal structure. The comparison betweensimulations performed on the BtuCD crystal structure (ProteinData Bank code 1L7V) with MMM (26) and experimental dis-tances detected in LDAO in the apo-state is presented in sup-plemental Fig. S5. The broad experimental distance distribu-tion between positions 168 is in line with the simulation(supplemental Fig. S5). The experimental distances betweenpositions 141 and 142 are longer than the simulated ones. Toanalyze the effects induced by the R1 neighboring residues onthe simulated distances, an additional simulation was per-formed with the “any rotamers?” function available in MMM.All of the 210R1 rotamers of theMMMlibrarywere attached tothe mutated cysteines, a condition that represents the absenceof any steric constraints for the spin label rotamers in the struc-ture but maintains the original position of the backbone towhich the rotamers are attached. The latter simulation showedthat the measured interspin distances in the cytoplasmic gatecould not be completely reconciled with the available backboneconformation of the crystal. Among the possible explanations

for the discrepancies are the following: (i) the crystallizationtrapped the BtuCD transporter in the energetically favoredconformation, (ii) both gates are dynamic, which would explainthe Hi1470/1 structure trapped in the opposite conformation,and (iii) the R1 side chain destabilizes the cytoplasmic gate inBtuCD. However, the last explanation appears unlikely becausefunctionality tests showed that spin labeling did not affectATPase activity (supplemental Table S1).The simulations performed with MMM on the BtuCD-F

structure (ProteinData Bank code 2QI9) (supplemental Fig. S5)showed a better agreement for both gates (positions 168 and141)with the experimental data obtained in proteoliposomes inthe apo-state (or ADP-state). For positions 142 the simulatedrotamers yielding the distance experimentally detected (blackrotamers in supplemental Fig. S5C) are under-represented inthe simulation.We suggest that the semi-occluded asymmetricBtuCD-F crystal structure is a good representative for the com-plex in the apo- or ADP-state.In this study, we also confirmed by EPR that the substrate

acts synergistically with ATP to decrease the affinity of BtuF forthe transporter. Moreover, in presence of ATP and high vita-min to BtuF molar ratios, both periplasmic and cytoplasmicgates are shown to adopt a conformation close to the ATP-bound state of BtuCD. How do the in vitro observations corre-late with the physiologically relevant conditions in vivo?Uptake of the scarce molecule vitamin B12 in the milieu by

E. coli involves the high affinity outer membrane receptor BtuB(28), the cytoplasmic membrane bound TonB (29), theperiplasmic substrate binding protein BtuF (30), and the trans-membrane associated BtuCD (31). The TonB-ExbB-ExbDcomplex is proposed to harness the energy of proton motiveforce to drive the release of vitamin B12 from BtuB. BtuF, whichseems to interact also with the TonB system (32), binds thereleased vitamin B12 in the periplasm and forms a stable com-plex with BtuCD. ATP hydrolysis in BtuD is thought to provideenergy for the release of vitamin B12 through the translocationchannel formed by BtuC with a mechanism that is still underinvestigation. BtuB is expressed at �200–500 copies per cell(33), and it is post-transcriptionally regulated through alternateRNA structures induced by the binding of adocobalamine tothe 5�-UTR region of the mRNA (34). Unlike genes of otherimport systems, btuCD and btuF are transcribed independ-ently. Both BtuCD (35) and BtuF (28, 30, 36) are expressed atvery low levels, and their expression is not regulated by vitaminB12 (35, 37). Although a quantitative analysis of the amount ofBtuCD and BtuF in the cell has not yet been performed, aboutthree vitamin B12 binding sites per cell were reported in theperiplasm of E. coli (28), which are potentially enough forthe observed rate of vitamin B12 import (38). Considering theextreme stability of the BtuCD-F complex (12), essentially all ofthe BtuF in the cell will be in complex with BtuCD. Hence, the

FIGURE 4. Nucleotide-dependent movement of periplasmic (168 –168) and cytoplasmic (141–141 and 142–142) gates of BtuCD in LDAO micelles andliposomes. Left panel, normalized experimental data V(t)/V(0) and exponentially decaying background arising from the distribution of remote spins (dashedlines), as fitted by DeerAnalysis2010. The DEER traces were detected at Q band (except for position 168 in liposomes). Middle panel, background-correctednormalized form factor F(t)/F(0) and fit by the Tikhonov regularization with a regularization parameter � � 100 or 1000 (black dashed lines). Right panel, distancedistribution P(r) obtained with DeerAnalysis2010. Colors are as described in the legends to Figs. 2 and 3.

FIGURE 5. Effect of vitamin B12 and ATP on the periplasmic gate inBtuCD-F in LDAO micelles. A, inset shows room temperature cw EPR spectraof the periplasmic gate (168 –168) in the presence of ATP without BtuF (gray),with 1:1 (black) and 1:50 (violet) molar ratio of BtuF to vitamin B12. The char-acteristic immobile component in BtuCD-F (indicated by an asterisk) showsthe interaction between BtuCD and BtuF. The arrow indicates the decrease ofthe immobile component at high vitamin concentrations. Left panel, normal-ized form factors F(t)/F(0) with the fit by Tikhonov regularization with a regu-larization parameter � � 100 (dashed lines). Right panel, corresponding dis-tance distribution P(r) obtained with DeerAnalysis2010. B, same experimentsas described in A performed in the cytoplasmic gate (142–142). The arrowsindicate the reappearance of the BtuCD-like distances in the ATP-state at highvitamin concentration. All DEER traces were recorded at Q band.




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few copies of vitamin B12 binding protein detected byWhite etal. (28)may reflect the fraction of the free BtuF in the periplasm.The scarce vitamin B12 encountered by the bacteria in the

milieu may be concentrated in the periplasm by the action ofthe high affinity BtuB and TonB system (39). According to thein vitro observations, the synergistic action of intracellular ATPand increasing vitamin concentration in the periplasm canaccelerate the dissociation of BtuF from the complex (Fig. 5 andsupplemental Equation S1). It is worth mentioning that trans-membrane potential or pH gradient in the cell could affect thevitamin to BtuF ratio required to dissociate the complex in vivo.Concomitantly to the BtuF release, ATP is hydrolyzed, and thegates adopt the more flexible conformation as observed inBtuCD (Fig. 4). The released BtuF can bind the available sub-strate preferentially reforming the complex with BtuCD in theapo- or ADP-state (Fig. 5 and Lewinson et al. (12)), to initiate anew productive cycle. We speculate that at this stage (absenceof ATP), the vitamin is transiently released to the translocationpathway in the TMDs. Interestingly, radioactive traces of vita-min were found in membrane-embedded BtuCD-F only in theabsence ofATP (22). Althoughwe did not follow themovementof substrate during the transport, our results strongly suggestthat vitamin B12 is released to the cytoplasm uponATP bindingdue to the opening of the cytoplasmic gate with concomitantclosure of the periplasmic gate. The absolute requirement ofATP for vitamin B12 transport across the membrane, both invivo and in vitro (10) further supports this model. Consideringthe mM concentration of ATP in the cytoplasm, BtuCD will beprevalently either in theATP- or ADP-bound state. However, itis conceivable that the apo-state is also transiently populatedduring the exchange of ADP to ATP at the NBDs. A kineticanalysis of the complex formation using surface plasmon reso-nance in the presence of Mg2�-ATP suggested that on averageBtuCDmolecules reside longer in the ADP-bound state than inthe ATP-bound or the transition-state-like (Mg2�-ATP/vana-date) intermediates (12). It has been an intriguing question formost ABC transporters, whether it is one or two molecules ofATP hydrolyzed by NBDs in a transport event. In liposomesBtuCD has a high basal ATPase activity which is further stim-ulated by addition of BtuF (supplemental Table S1). Consider-ing this high basal ATPase rate and lack of any asymmetry at theNBDs upon BtuF binding (10), it is conceivable that both ATPsare hydrolyzed simultaneously. Based on the above consider-ations and the EPRdata, which are schematically represented inFig. 6A, we detail amodel for a productive vitaminB12 transportcycle mediated by BtuCD-F in the cell (Fig. 6B).The model proposed here points to a mechanistic difference

in coupling the energy of ATP hydrolysis to substrate transportbetween type I and type II importers. The physiological impli-cation of the model described here is that the BtuF affinity toBtuCD; thus, the transport rate is tuned to the availability ofvitamin in the periplasm: the higher the availability, the fasterBtuF is released to the periplasm to bind the new substrate anddeliver it to BtuCD preferentially in the apo- or ADP-states. InButCD-F, the ATP-induced dimerization of the NBDs opensthe translocation pathway toward the cytoplasm. A mechanis-tic implication of these observations is that the TMDs inButCD-F and in the maltose transporter are driven in opposite

directions along with the movements of NBDs. In both trans-porters the movement of TMDs along with NBDs is transmit-ted by the coupling helices. In BtuCD, the NBDs are tilted withrespect to the long axis of the BtuC dimer, positioning the cou-pling helices diametrically opposed (9). Based on the BtuCDstructure, it was speculated that the closure of NBDs uponATPbinding would pull the coupling helices apart opening theTMDs toward the cytoplasm. The EPR data indeed suggest thatbinding of ATP opens the translocation pathway of BtuCD-Ftoward the cytoplasm. How the movement of the gates corre-late to the overall TMD rearrangement and whether the mech-anistic model proposed is valid for all members of the type IIimporters will be a subject of further investigation.

Acknowledgments—We thank Roland Riek for providing facilities forprotein expression and purification andChristos Tzitzilonis for intro-duction to protein purification techniques.

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FIGURE 6. EPR distances and model of a productive vitamin transportcycle. A, schematic description of the EPR-derived distances during the nucle-otide cycle (apo-, ATP-, and ADP-states) for BtuCD (left panel) and BtuCD-F(right panel). The corresponding states of NBDs are indicated in black, red, andgreen, respectively. The TM5 helices are highlighted in black. Spin-labeledpositions are represented by yellow circles. Ovals were used for BtuCD to rep-resent the large distance distributions detected. B, model of a productivevitamin B12 transport in the cell. The vitamin B12-bound BtuF interacts withthe apo-state of BtuCD (1L7V), forming the BtuCD-F complex (model super-imposed to Protein Data Bank code 2QI9). Vitamin B12 is transiently releasedto the translocation channel. ATP binding to the NBDs inwardly opens thetranslocation channel, and the vitamin escapes into the cytoplasm. Simulta-neously, the presence of excess vitamin in the periplasm helps to promote thedissociation of BtuF, restoring BtuCD to an outward facing conformation,ready to interact with another vitamin-bound BtuF.




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BordignonBenesh Joseph, Gunnar Jeschke, Birke A. Goetz, Kaspar P. Locher and Enrica

Importer BtuCD-F during Nucleotide CycleTransmembrane Gate Movements in the Type II ATP-binding Cassette (ABC)

doi: 10.1074/jbc.M111.269472 originally published online September 27, 20112011, 286:41008-41017.J. Biol. Chem.

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Transmembrane Gate Movements in the Type II ATP-binding