doi:10.1016/j.jmb.2010.01.027 J. Mol. Biol. (2010) 397, 179–189

Available online at www.sciencedirect.com

Structure of theMembraneDomain of HumanErythrocyteAnionExchanger 1RevealedbyElectronCrystallography

Tomohiro Yamaguchi1, Yohei Ikeda1, Yoshito Abe2, Hiroyuki Kuma3,Dongchon Kang4, Naotaka Hamasaki3⁎ and Teruhisa Hirai1⁎

1Three-Dimensional MicroscopyResearch Team, RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo,Hyogo 679-5148, Japan2Department of Immunology,Graduate School ofPharmaceutical Sciences,Kyushu University, 3-1-1Maidashi, Higashi-ku, Fukuoka812-8582, Japan3Faculty of PharmaceuticalSciences, Nagasaki InternationalUniversity, 2825-7 Huis TenBosch-cho, Sasebo, Nagasaki859-3298, Japan4Department of ClinicalChemistry and LaboratoryMedicine, Graduate School ofMedical Sciences, KyushuUniversity, 3-1-1 Maidashi,Higashi-ku, Fukuoka 812-8582,Japan

Received 21 October 2009;received in revised form8 January 2010;accepted 13 January 2010Available online25 January 2010

*Corresponding authors. N. HamasaUniversity, 2825-7Huis Ten Bosch-chTeam, RIKEN SPring-8 Center, 1-1-1hamasaki-nao@niu.ac.jp; thirai@sprinPresent address: T. Yamaguchi, D

University, 46-29 Yoshida-ShimoadaAbbreviations used: 2D, two-dime

transmembrane.

0022-2836/$ - see front matter © 2010 E

The membrane domain of human erythrocyte anion exchanger 1 (AE1)works as a Cl−/HCO3

− antiporter. This exchange is a key step for CO2/O2circulation in the blood. In spite of their importance, structural informationabout AE1 and the AE (anion exchanger) family are still very limited. Weused electron microscopy to solve the three-dimensional structure of theAE1 membrane domain, fixed in an outward-open conformation by cross-linking, at 7.5-Å resolution. A dimer of AE1 membrane domains packed intwo-dimensional array showed a projection map similar to that of theprokaryotic homolog of the ClC chloride channel, a Cl−/H+ antiporter. In athree-dimensional map, there are V-shaped densities near the center of thedimer and slightly narrower V-shaped clusters at a greater distance fromthe center of the dimer. These appear to be inserted into the membrane fromopposite sides. The structural motifs, two homologous pairs of helices ininternal repeats of the ClC transporter (helices B+C and J+K), are wellfitted to those AE1 densities after simple domain movement.

© 2010 Elsevier Ltd. All rights reserved.

Keywords: AE1; band 3; ClC transporter; cryo-electron microscopy; two-dimensional crystal

Edited by W. Baumeister

ki is to be contacted at Faculty of Pharmaceutical Sciences, Nagasaki Internationalo, Sasebo,Nagasaki 859-3298, Japan. T.Hirai, Three-DimensionalMicroscopyResearchKouto, Sayo, Hyogo 679-5148, Japan. E-mail addresses:g8.or.jp.epartment of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyotochi, Sakyo-ku, Kyoto 606-8501, Japan.nsional; 3D, three-dimensional; AE1, anion exchanger 1; AE, anion exchanger; TM,

lsevier Ltd. All rights reserved.

180 EM Structure of AE1

Introduction

Anion exchanger 1 (AE1; also known as band 3 orSLC4A1) is the most abundant membrane protein(25% of membrane protein, 50% of integral mem-brane protein) in human erythrocytes.1 AE1 consistsof a 40-kDa N-terminal cytoplasmic domain (resi-dues 1–359) and a 55-kDa C-terminal membranedomain (residues 360–911). The N-terminal hydro-philic cytoplasmic domain of AE1 serves as ananchoring point for cytoskeletal proteins, such asankyrin,2 protein 4.1,3 and protein 4.2.4 The structureof this domain has been solved by X-ray crystallog-raphy at 2.6 Å.5 On the other hand, the membranedomain of AE1 works as a Cl−/HCO3

− antiporter.CO2 produced in the tissue is converted into HCO3

−

by carbonic anhydrase II, which is bound to theC-terminus of AE1 in the red blood cell.6 AE1exchanges HCO3

− with Cl−; the “chloride shift”accomplished by this transporter lowers pH andfacilitates the release of O2 from hemoglobin. Thecontrolled release of O2 by sensing CO2 ensures thattissues accept adequate levels of O2; otherwise,tissues can be damaged by either excess or lack ofO2.

7 AE1 belongs to the anion exchanger (AE) familyof electrochemical potential-driven transporters†.Members of the AE family are widespread inanimals, plants, and yeast. Substrates are not limitedto anions; the family includes animal bicarbonatetransporters, such as the Cl−/HCO3

− antiporter andNa+/HCO3

− cotransporter,8 and plant boron trans-porters.9 AE1 transports not only Cl− andHCO3

− butalso sulfate, phosphate, phosphoenolpyruvate, andpyridoxal 5-phosphate.10,11 AE1 also works as aflippase.12 The AE1 membrane domain behaves as adimer when solubilized by detergent,13–15 althoughnative AE1 is known to make tetramers whenankyrin is bound to the N-terminal domain.16

AE1 is expressed not only in erythrocytes but alsoin the cells of renal collecting duct. SpecificmutationsofAE1are related to suchdiseases as SoutheastAsianovalocytosis and distal renal tubular acidosis.17,18

The function of AE1 is inhibited by several stilbenederivatives, such as DIDS (4,4′-diisothiocyanatostil-bene-2,2′-disulfonic acid), SITS (4-acetamido-4′-iso-thiocyano-stilbene-2,2′-disulfonic acid), and DNDS(4,4′-dinitrostilbene-2,2′-disulfonic acid). H2DIDS(4,4′-diisothiocyanatodihydrostilbene-2,2′-disulfonicacid)19 can cross-linkAE1molecules almost 100%; thebinding sites for the inhibitor are located at residuesK539 and K851.20 H2DIDS binds to AE1 from theextracellular side, but the reaction sites of H2DIDSare hidden from the cell surface when the diethylpyrocarbonate modification takes place at His83421on the intracellular side of the membrane.22 Con-versely, the intracellular binding site of diethylpyrocarbonate is hidden when H2DIDS binds toAE1 from the extracellular side, suggesting theexistence of two conformations.22 Many biologicalexperiments have been performed, including mono-clonal antibody experiments,23,24 in situ proteolytic

†http://www.tcdb.org/tcdb/

digestion,25,26 scanning N-glycosylation muta-genesis,27–29 cysteine-scanning mutagenesis,30–32 re-assembly from fragments,33,34 assessment oftopogenic function,29,35,36 and chemicalmodificationof each type of residue (Met,37 His,21 Glu,38 Tyr,39

Arg,40 or Lys20,41), to confirm the important residuesand topology of AE1. From the results of thesebiological experiments, AE1 is expected to span themembrane up to 14 times with both the N- and C-termini on the cytoplasmic side.32,42 Transmembranesegments 1–5 (TM1–TM5) are expected to be clus-tered. While co-expressed fragments TM1–TM5+TM6–TM14 showed transport activity, any comple-mentary pair of fragments divided between TM1 andTM5 was not active.43 There was no tryptic cleavagesite between TM2 and TM5.26 However, the details ofthe protein's topology remain unclear. The 10thmembrane region is thought to be very flexiblebecause residues near K743 are observed on theexternal side in some experiments27,31 and on thecytoplasmic side in others.41,44 Although the proteinhas been studied for a long time, only low-resolutionstructures, derived from two-dimensional (2D) crys-tals by negative stain45 and tubular crystal,46 wereavailable for the AE1 membrane domain. Here, wereport 7.5-Å-resolution projection and a three-dimen-sional (3D) map solved by cryo-electron crystallogra-phy using a 2D crystal. This result enables us todiscuss the architecture of the AE1 transporter.We are also going to discuss the relationship

between the AE1 transporter and the ClC transport-er based on their structural similarity observed inthis study. Members of the ClC chloride (Cl−)channel family are widespread among prokaryotesand eukaryotes. One of the well-known functions ofClC Cl− channels is to regulate electrical excitation inskeletal muscle.47 The X-ray structures of a bacterialhomolog of the ClC Cl− channel were reported byDutzler et al.48,49 Interestingly, the ClC Cl− channelhomolog is shown to be a Cl−/H+ antiporter.50 Fromthe structural pseudo-2-fold symmetry, the 14transmembrane segments of the ClC protein seemto originate from the duplication of 7 transmem-brane segments. Two repeated motifs have anti-parallel architecture, forming a Cl− binding poresurrounded by several N-terminal ends of heliceswith conserved Cl− binding motifs. This transporteris also inhibited by DIDS.51 The mechanism under-lying the conformational change is not known.

Results and Discussion

Structural analysis of AE1

For this study, we cross-linked AE1 with H2DIDSin order to increase protein stability and fix theconformation into the outward-facing openform.20,22 AE1 is crystallized two-dimensionally ina tube-like vesicle, which is about 2 μm wide andgreater than 4 μm long and which seems to beflattened when applied to an electron microscopygrid (Fig. 1a). In many cases, Fourier-transformed

Fig. 1. Two-dimensional crystals of AE1 and the quality of processed and merged data. (a) Electron microscopy imageof the negatively stained AE1 crystal. Scale bar represents 2 μm. (b) Phase deviation from either 0° or 180°. Phase deviationof averaged phases from symmetrical constraint (0° or 180°) is plotted as a function of resolution of the reflection.Averaged phase is calculated at each reflection from the four best untilted images. For this figure, IQ is defined based onthe output of the origtiltd program from the MRC package. (c) Example of a lattice line plot. Merged data, fitted curves,and error bars of phase and amplitude information are plotted for lattice lines (2, 3) and (6, 3).

181EM Structure of AE1

images gave two overlapping independent diffrac-tion patterns that could be processed separatelyafter selection by masking (Supplementary Fig. 1).The 2D plane symmetry was determined to bep22121. Based on the deviation of phase from thesymmetry requirements according to the resolution(Fig. 1b), a projection map was calculated, includingdata up to 7.5-Å resolution, by merging the four bestimages (Table 1). A 3D map of AE1 was calculatedalso at 7.5-Å resolution after merging 31 images

Table 1. Crystallographic data

Plane group p22121Lattice constants a=194.5 Å, b=182.0

Å, c=200 Å (assumed),α=β=γ=90°

Resolution limit for merging (Å) 7.5

2D (projection)No. of images 4Total no. of observations 2881 (IQ≤6)Completeness (%) 97.0Overall phase error (°) from

symmetrical requirement(0° or 180°)

22.5

3DNo. of images 31 (7 at 0°, 8 at 20°,

12 at 45°, 4 at 60°)Total no. of observations 9312 (IQ≤6)Completeness (%) 74.3 (0°–45°),

66.5 (0°–60°)Vertical resolution (Å)a 16.0Overall weighted R-factor (%) 19.4Overall weighted phase residual (°) 21.4

a Estimated from point spread function (Supplementary Fig. 2b).

tilted from 0° to 60° (7 at 0°, 8 at 20°, 12 at 45°, and 4at 60°). Examples of lattice lines are shown in Fig. 1cand Supplementary Fig. 2a. The overall weightedphase residual was 21.4° (Table 1).

Projection structure of AE1

A projection map is shown in Fig. 2a. In one unitcell (194.5 Å×182.0 Å), six dimers can be recognized.These dimers can be classified into two types: twostructural homodimers (A/A) and four structuralheterodimers (B/C). While the A/A dimer has a 2-fold axis in the center of dimer, molecules B and Care not crystallographically related. Half of them areupside down in the membrane, related by a screwaxis parallel with the membrane. Overall, moleculesA and B showed a similar density pattern, whilemolecule C showed a slightly dispersed densitypattern. Interestingly, the projection map of the AE1dimer (Fig. 2b) showed a pattern similar to that ofthe ClC transporter, which was reported by Mindellet al.52 (Fig. 2c). The dimension of the rhombus shapeof the dimer is ∼50 Å×100 Å in the case of the ClCtransporter, while it is slightly elongated,∼50 Å×120 Å, in the case of AE1. The densitypatterns for the AE1 and ClC transporters aresimilar, especially far from the center of the dimer,as shown in Fig. 2b and c; however, the area near thecenter of the dimer is less similar.

Three-dimensional structure of AE1

A 3D map of AE1 is shown in Fig. 3a–c andSupplementary Fig. 3. Horizontal sections of the

Fig. 2. Projection map of AE1 and comparison with ClC transporter. (a) Projection map of the AE1 whole unit cell(194.5 Å×182.0 Å). Each molecule (A, B, and C) is indicated by a dotted circle to illustrate the packing. Outlined charactersindicate that a molecule is upside down in the membrane (i.e., in inverted orientation compared with molecules annotatedwith plain characters). Two-fold axes are shown by their conventional graphic symbols. The one in the center is omittedfor clarity. Arrows indicate screw axes. Contour levels are incremented by one-tenth of the maximum value. (b) Projectionmap of AE1 A/A dimer. (c) Projection map of ClC transporter at 6.5-Å resolution, reported byMindell et al.52 [Adapted bypermission fromMacmillan Publishers Ltd.: Nature, copyright 2001.] Several features in (b) and (c) are colored or markedto make comparison of AE1 and ClC easier. Several very low-density and low-density areas are shown in purple and blue,respectively, in (b). White arrowheads are marked in both (b) and (c) to indicate corresponding high densities.

182 EM Structure of AE1

map are also shown in Fig. 4. Even though it was notpossible to count the 14 predicted transmembranesegments32,42 in molecule A, B, or C in the map,several long and highly tilted helices were easilyrecognized. The longest helices spanned the mem-brane, running more than 50 Å in the z-direction atdifferent heights, suggesting that those helices aresticking out from a membrane region (typically 40 Åwide) on different sides. In the portion of themolecule far from the center of the dimer (Fig. 3a),clustered densities are observed as an assembly oftwo possible helices at the V-shaped peripheraldensities, alongwith other extra densities between oradjacent to those helices (Fig. 3b, which is presentedupside down comparedwith Fig. 3a and c in order toallow easier comparison of the first and second V-shaped motifs). Separated by a cleft, another V-shaped density, which seems slightly rounder andwider, is recognized near the center of the dimer,withoutmuch extra density around it (Fig. 3c). ThoseV-shaped and wider V-shaped densities are insertedto the membrane from opposite sides (Fig. 3a).Taking the similarity in the projection map intoaccount, helices B+C and J+K (with connectingloop) of the ClC transporter, which had similarstructural features, could be fitted to the V-shapedand wider V-shaped densities, respectively. Toevaluate the possibility that the extra density aroundthe V-shaped helices (helices B+C) might corre-spond to helices E, F, and I, we included those helicesin the model used for fitting (Fig. 3b).

Fitting structural motifs of the ClC transporter tothe AE1 map

After manual fitting, positions are refined bysimple domain movement, assuming two rigidbodies per molecule: one consisting of helices A+B,E, F, and I, and the other consisting of helices J+K.All models presented in Figs. 3a–c and 4 are theresults of this rigid-body refinement; they fit well tothe observed densities, especially considering theyare derived from the different transporter family. Forexample, as shown in Fig. 3c, helix J is very long, buthelix K is relatively short, and it ends withoutprotruding from the membrane. Those features ofthe ClC model matched quite well with the 3D mapof AE1. Helices B and C also fit well, though not aswell as helix J+K did. Still the sharp turn made byhelices B and C fits quite well to the 3D map. Thedensity to which helix J is assigned is very fat andmight include a contribution from another part of thestructure (see the “+10 Å” section in Fig. 4). Thedensity to which helix J is assigned is also connectedto the large density (the upper density in Fig. 3c andthe central density in the “+20 Å” section in Fig. 4)that resides on the extracellular side of membranenear the center of the dimer (2-fold axis for the A/Adimer and pseudo-2-fold axis for the B/C dimer).The longest loop, 7–8 (loop between TM7 and TM8),is on the extracellular side between the N-terminaland C-terminal halves of AE1, similar to loop I–J inthe ClC protein. This unexplained density might

Fig. 3. Three-dimensional mapof AE1 and the fitted motifs fromthe ClC transporter (stereo view).(a) Map of the B/C dimer withmolecule B on the right side. (b)Map around the first motif (mole-cule B). Helices B and C in the firstmotif are labeled. (c) Map aroundthe second motif (molecule C).Helices J and K in the secondmotif are labeled. In the case of (a)and (c), the extracellular side is up,but in the case of (b), the extracel-lular side is down to allow easiercomparison of the first and secondV-shaped motifs. The 3D map wascalculated and drawn at a contourlevel of 1.8σ using CCP453 andCCP4mg.54 All models presentedin (a)–(c) are the results of rigid-body refinement. The first andsecond motifs of molecule B areshown in magenta and coral, re-spectively. The first and secondmotifs of molecule C are shown incyan and yellow, respectively.

183EM Structure of AE1

include a contribution from this central loop and/oranother unmodeled part of AE1. Unexplained smalldensity is noticeable in the vicinity of loop B–C(lower right in Fig. 3b; Supplementary Fig. 3) besidesthe large central density. Intra-dimeric cross-linkingof single-cysteine mutants suggested that loops 1–2,5–6, and 7–8 are close to the (pseudo-)2-fold axis,55

although loop 1–2 is too far from the center of dimerat the current helix assignment of this conformation.

Sequence similarity between the AE family andthe ClC family

Because of their structural similarity, we lookedfor sequence similarity between AE1 and ClC

proteins. Specifically, the sequences of helices B+Cof the ClC proteins are well aligned with TM1+TM2(transmembrane helices 1+2) of the AE family,without many gaps or insertions (Fig. 5a; Supple-mentary Fig. 4). The sequences of helices J+K of theClC proteins are also well aligned with those ofTM8+TM9 of the AE family. To confirm the validityof the alignment between the ClC family and the AEfamily, we presented characteristics of the sites ofthe AE family on the ClC models (Fig. 5b). Thecharged residues clustered mainly outside of themembrane region, showing that the heights of thetransmembrane segments are at least roughlyaligned. The distribution of variable hydrophobicsites,56 which normally face the lipid bilayer, seems

Fig. 4. Horizontal sections fromthe 3D map of the AE1 B/C dimer.They are sectioned at intervals of10 Å from the center of the map,with thickness of 12 Å. Models arecolored and helices are labeled as inFig. 3.

184 EM Structure of AE1

Fig. 5. Sequence alignment be-tween the AE and ClC families. (a)Multiple sequence alignmentaround helices B+C of the ClCfamily and TM1+TM2 of the AEfamily and the following (putative)Cl− binding motif. Within the pre-sented region, the sequence identitybetween B3AT_HUMAN (AE1)and CLCA_ECOLI (Escherichia coliClC protein) is 20%. Sites aroundthe (putative) binding motif arehighlighted in an orange box.Charged sites, variable hydropho-bic sites, and small residue sites ofthe AE family (Materials and Meth-ods) are marked by small filledcircles shown in magenta, green,and blue, respectively. (b) Charac-teristics of aligned sequences of theAE family are shown on the ClCmodel as balls, colored as in (a)(stereo view). Possible Cl− bindingsite is shown in orange. Boundariesof the membrane region, estimatedbased on the distribution of char-acteristics of residues, are indicatedby horizontal bars.

185EM Structure of AE1

to validate the rotational positioning of transmem-brane segments. Small residues often appear atpositions of helix–helix contact, kinks, or turns.Their distribution seems reasonable. One of the Cl−

binding sites appears soon after the end of helix C inthe case of the ClC proteins. This conserved motif isrepresented as GSGIP, including a serine (S107) thatdirectly contacts the Cl− ion.48 In the AE family, theconserved motif GXSGP, which also includes aserine (S465), is aligned at the same position;however, the density around the possible Cl−

binding site is not clear in the 3D map of AE1.

Conformational difference between the AE1transporter and the ClC transporter

Structures of AE1 A/A and B/C dimers repre-sented by fitted motifs are aligned and shownsuperposed in Fig. 6a. The second motifs (TM8+TM9) are well aligned to each other, suggestingthat the dimer interface is the same between theA/A and B/C dimers. The positions of loop 1–2(between TM1 and TM2) of molecules B and C areshifted toward the center of the dimer by 5 and10 Å, respectively, compared with molecule A. TheAE1 membrane domain is cross-linked between theN-terminal and C-terminal halves, but some struc-tural flexibility probably still remains. Furthermore,

the AE1 A/A and ClC dimers are aligned andshown superposed in Fig. 6b (top view) and c (sideview). The AE1 dimer showed an elongated shapecompared with the ClC dimer when it was viewedfrom the top (Fig. 6b). The distance between thetips of loop 1–2 of the AE1 dimer (L1AE1) is∼110 Å, while the corresponding distance in theClC dimer (L1ClC) is ∼90 Å. On the other hand,the width around the center of the AE1 dimer issmaller. The distance between the tips of loop 8–9in the AE1 dimer (L2AE1) is ∼50 Å, while thecorresponding distance in the ClC dimer (L2ClC) is∼75 Å. Because of the narrower space between C-terminal halves of the AE1 dimer, the same foldthat appears in the ClC molecule is not possible forthe C-terminal half of AE1 (after the second motif)in the current structure. The displacement of heightbetween the tips of loop 1–2 and loop 8–9 is ∼60 Åfor AE1 (H1AE1), while the corresponding distanceis ∼40 Å for ClC proteins (H1ClC), resulting in alarger protrusion from the membrane regions ofAE1.

Conclusions

Based on the structural and sequence homologybetween AE1 and ClC proteins in the regions of the

Fig. 6. Comparison between the AE1 A/A dimer, AE1B/C dimer, and ClC dimer. (a) Superposition of the AE1A/A and B/C dimers. Molecules A, B, and C are shown inpurple, magenta, and cyan, respectively. (b and c)Comparison of the AE1 A/A and ClC dimers (b, topview; c, side view). The AE1 and ClC dimers are shown inpurple and coral, respectively. For this figure, only the firstand second motifs are shown. The A/A dimer in (a) isrotated 30° along the z-axis, compared with the A/Adimer in (c).

186 EM Structure of AE1

two-helix repeated motifs (TM1+TM2 and TM8+TM9 of AE1 and helices B+C and J+K of ClCproteins), we presume that the two protein familiesshare the same antiparallel structural framework,although it remains unknown as to what extent theyshare a transport mechanism. Because both AE1 andClC proteins share the same asymmetry between thefirst and second motifs, it is most likely that theydiverged after that asymmetry appeared in thecommon ancestor. However, the present 3D mapdoes not give detailed information, especially in theregion following the second motif, and sequencesimilarity is also very weak in that region. Asdiscussed, the C-terminal half of AE1 in the currentconformation will have a different fold comparedwith the reported structure of the ClC transporter

after the second motif. The region around TM10 isknown to be very flexible based on the accessibilityof K743 from both sides of the membrane. TM10 alsohas a very hydrophilic character, and it can be easilyreleased by in situ proteolytic digestion after alkalitreatment (10 mM NaOH, pH 12). (The regionsreleased only after alkali treatment are called“category 2”.25) An analysis of topogenic functionsshows that TM10 itself does not have any topogenicfunction.35 This kind of flexibility could be one of thereasons why the map does not show a clear densityaround that region.Although the AE1 and ClC transporters have

structural similarity, their conformations are signif-icantly different. It is not yet known whether thisreflects a family-wide difference or rather representsa conformational change in the chloride transporter.Further investigation is necessary to understand thetransport mechanism of AE1 and to determine thesignificance of the similarity between AE1 and ClCtransporters.

Materials and Methods

Purification of AE1

Human blood stored at 4 °C in acid/citrate/dextrosesolution was obtained from the Fukuoka Red Cross BloodCenter. Erythrocyte membranes (white ghosts) wereprepared as described previously.57 White ghosts(∼400 mg) were incubated with H2DIDS (2 mg, MolecularProbes) in 10 mM borate buffer (pH 9.5) at 37 °C for90 min. To remove the N-terminal cytoplasmic domain ofAE1, we treated the membranes with TPCK (L-1-tosyla-mido-2-phenylethyl choloromethyl ketone)-trypsin(0.4 mg, Roche Diagnostics) in 5 mM Na2CO3 for 30 minon ice. Peripheral membrane proteins were stripped by10 mM NaOH. Membrane proteins were solubilized with0.1% (v/v) C12E8 (Wako) and deglycosylated with N-glycosidase F (100 U, Roche Diagnostics) simultaneouslyfor 24 h at room temperature. Solubilized AE1 was thenpurified using POROS HQ/20 anion exchange column(Applied Biosystems) by HPLC (BioCAD 700E perfusionchromatography). Purified AE1 was concentrated by thebatch method with DEAE Sepharose resin (GE Health-care). In this step, the detergent C12E8 was exchanged withC13E8.

Two-dimensional crystallization

After mixing solubilized protein and lipid, we per-formed 2D crystallization of AE1 by removing detergentusing button dialysis. The protein was mixed with DOPS(1,2-dioleoyl-sn-glycero-3-phospho-L-serine, SIGMA) at2.5 mg protein/ml at a lipid-to-protein-ratio of 0.14−0.16(w/w) in 0.09% (v/v) C13E8 (Anatrace) and 0.01% (v/v)C12E8, and dialysis was performed in a dialysis button,initially against 10 mM Tris, pH 8.0, 100 mM NaCl, and0.02% NaN3 at 26 °C for 8 days and then against 10 mMTris, pH 8.0, 20 mM NaCl, 10 mM Na4P2O7, and 0.02%NaN3 at 26 °C for 5 days. For screening of 2Dcrystallization conditions, samples were negativelystained with 2% (w/v) uranyl acetate on glow-dischargedcarbon-coated Cu grids and then observed using a JEOL

187EM Structure of AE1

JEM-2010 transmission electron microscope at an acceler-ation voltage of 100 kV.

Cryo-electron microscopy

The 2D crystals were embedded in 14% trehalose58 onMo grids using the carbon sandwich method.59 Excessbuffer was removed by pipetting and blotting with filterpaper, and then the grid was manually plunged into liquidnitrogen. These operations were carried out at 4 °C.Electronmicrographs were recorded on a JEOL JEF3000SFFwith a liquid helium stage cooled to a temperature of 4 K60

at an acceleration voltage of 300 kV. Images were recordedon KODAK SO-163 films at a nominal magnification of40,000× with an exposure time of 2 s. Defocus values usedwere 10,000–25,000 Å. The quality of micrographs waschecked by optical diffraction, and good images werescanned by a ZEISS SCAI scanner at a 7-μm step size.

Image processing

Well-ordered areas of 5000×5000 to 10,000×10,000pixels were used for the analysis. Filtering and unbendingdigitized images were performed using the MRCpackage.61 Symmetry was determined as p22121 based onthe result of the allspace program from the MRC package.p22121 symmetry was confirmed when each image wasprocessed as having p1 symmetry (without assuminghigher symmetry). Images were merged by refiningparameters for origin, scale, tilt axis, tilt angle, and beamtilt. For the lattice constant, c=200 Å was used, but thedensity was assumed to exist within a width of 120 Å.Handedness was confirmed as described previously.62

Rigid-body refinement

Residues 31–104 (helices B+C), 123–142 (helix E), 147–166 (helix F), and 214–233 (helix G) were extracted as onerigid body, and residues 251–306 (helices J+K) wereextracted as another rigid body from the coordinates of theClC transporter (Protein Data Bank code 1OTS).49 Allresidues other than G, P, and A are trimmed up to the Cβ

atom and treated as A (alanine) for later refinement. Rigid-body refinement was performed using CNS.63

Structural comparison

In Fig. 6a, the second motifs (residues 251–306) of thedimer were aligned using the program LSQMAN‡. ForFig. 6b and c, all motifs (residues 32–104 and 251–306) ofthe dimer were used for the alignment. The distancebetween loop B−C loops in the dimer was measured usingthe N73 residues of each monomer. The distance betweenloop J−K loops is measured using residue G286. The widthin the z-direction was measured using the difference in z-height between N73 and G286.

Multiple sequence alignment

Sequences were fetched from public databases by PSI-BLAST64 using the sequences of AE1 (B3AT_HUMAN)and ClC (CLCA_ECOLI) transporters to initiate thesearch. Next, sequences were aligned using CLUSTALW65 and adjusted manually if necessary. Twenty-eight

‡http://xray.bmc.uu.se/usf/

sequences from the AE family and six sequences from theClC family were selected; the sequences used are welldiverged and aligned without ambiguity within eachfamily (Supplementary Fig. 4).The characteristics of each site are assigned based on the

alignment of 28 sequences of the AE family, regardless ofsequences of the ClC family. Sites that include at least onecharged residue (R, K, D, or E) are marked as “charged”sites. Sites that include at least one typical hydrophobicresidue (V, I, L, or M), are limited only to hydrophobicresidues (V, I, L, M, G, A, and F), and are mutated twice ormore are marked as “variable hydrophobic” (variphobic)sites.56 Sites that include at least one tiny residue (G, A, orP) and are limited only to small residues (G, A, P, S, T, andC) are marked as “small residue” sites.

Accession number

The Electron Microscopy Data Bank§ accession numberfor the density map of AE1 is EMD-1645.

Acknowledgements

This work was supported by the Japan NewEnergy and Industrial Technology DevelopmentOrganization. We thank Ms. T. Ogata for technicalassistance. We also thank Drs. Y. Hiroaki and Y.Fujiyoshi for help with electron microscopy.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2010.01.027

References

1. Fairbanks, G., Steck, T. L. & Wallach, D. F. (1971).Electrophoretic analysis of the major polypeptides ofthe human erythrocyte membrane. Biochemistry, 10,2606–2617.

2. Chang, S. H. & Low, P. S. (2003). Identification of acritical ankyrin-binding loop on the cytoplasmicdomain of erythrocyte membrane band 3 by crystalstructure analysis and site-directed mutagenesis.J. Biol. Chem. 278, 6879–6884.

3. Pasternack, G. R., Anderson, R. A., Leto, T. L. &Marchesi, V. T. (1985). Interactions between protein4.1 and band 3. An alternative binding site for anelement of the membrane skeleton. J. Biol. Chem. 260,3676–3683.

4. Rybicki, A. C., Schwartz, R. S., Hustedt, E. J. & Cobb,C. E. (1996). Increased rotational mobility andextractability of band 3 from protein 4.2-deficienterythrocyte membranes: evidence of a role for protein4.2 in strengthening the band 3–cytoskeleton linkage.Blood, 88, 2745–2753.

§http//www.ebi.ac.uk/pdbe-srv/emsearch/index.html

188 EM Structure of AE1

5. Zhang, D., Kiyatkin, A., Bolin, J. T. & Low, P. S. (2000).Crystallographic structure and functional interpreta-tion of the cytoplasmic domain of erythrocytemembrane band 3. Blood, 96, 2925–2933.

6. Vince, J. W. & Reithmeier, R. A. (2000). Identificationof the carbonic anhydrase II binding site in the Cl−/HCO3

− anion exchanger AE1. Biochemistry, 39,5527–5533.

7. Hamasaki, N. & Yamamoto, M. (2000). Red blood cellfunction and blood storage. Vox Sang. 79, 191–197.

8. Romero, M. F., Hediger, M. A., Boulpaep, E. L. &Boron, W. F. (1997). Expression cloning and charac-terization of a renal electrogenic Na+/HCO3

− cotran-sporter. Nature, 387, 409–413.

9. Takano, J., Noguchi, K., Yasumori, M., Kobayashi, M.,Gajdos, Z., Miwa, K. et al. (2002). Arabidopsis borontransporter for xylem loading. Nature, 420, 337–340.

10. Passow, H. (1986). Molecular aspects of band 3protein-mediated anion transport across the redblood cell membrane. Rev. Physiol., Biochem. Pharmacol.103, 61–203.

11. Nanri, H., Hamasaki, N. & Minakami, S. (1983).Affinity labeling of erythrocyte band 3 protein withpyridoxal 5-phosphate. Involvement of the 35,000-dalton fragment in anion transport. J. Biol. Chem. 258,5985–5989.

12. Ortwein, R., Oslender-Kohnen, A. & Deuticke, B.(1994). Band 3, the anion exchanger of the erythrocytemembrane, is also a flippase. Biochim. Biophys. Acta,1191, 317–323.

13. Casey, J. R. & Reithmeier, R. A. (1991). Analysis of theoligomeric state of band 3, the anion transport proteinof the human erythrocyte membrane, by size exclu-sion high performance liquid chromatography. Olig-omeric stability and origin of heterogeneity. J. Biol.Chem. 266, 15726–15737.

14. Colfen, H., Boulter, J. M., Harding, S. E. & Watts, A.(1998). Ultracentrifugation studies on the transmem-brane domain of the human erythrocyte aniontransporter band 3 in the detergent C12E8. Eur.Biophys. J. 27, 651–655.

15. Lemieux, M. J., Reithmeier, R. A. & Wang, D. N.(2002). Importance of detergent and phospholipid inthe crystallization of the human erythrocyte anion-exchanger membrane domain. J. Struct. Biol. 137,322–332.

16. Van Dort, H. M., Moriyama, R. & Low, P. S. (1998).Effect of band 3 subunit equilibrium on the kineticsand affinity of ankyrin binding to erythrocytemembrane vesicles. J. Biol. Chem. 273, 14819–14826.

17. Wrong, O., Bruce, L. J., Unwin, R. J., Toye, A. M. &Tanner, M. J. (2002). Band 3 mutations, distal renaltubular acidosis, and Southeast Asian ovalocytosis.Kidney Int. 62, 10–19.

18. Kittanakom, S., Cordat, E. & Reithmeier, R. A.(2008). Dominant-negative effect of Southeast Asianovalocytosis anion exchanger 1 in compound het-erozygous distal renal tubular acidosis. Biochem. J.410, 271–281.

19. Shami, Y., Rothstein, A. & Knauf, P. A. (1978).Identification of the Cl− transport site of human redblood cells by a kinetic analysis of the inhibitoryeffects of a chemical probe. Biochim. Biophys. Acta, 508,357–363.

20. Okubo, K., Kang, D., Hamasaki, N. & Jennings, M. L.(1994). Red blood cell band 3. Lysine 539 and lysine851 react with the same H2DIDS (4,4′-diisothiocyano-dihydrostilbene-2,2′-disulfonic acid) molecule. J. Biol.Chem. 269, 1918–1926.

21. Jin, X. R., Abe, Y., Li, C. Y. & Hamasaki, N. (2003).Histidine-834 of human erythrocyte band 3 has anessential role in the conformational changes thatoccur during the band 3-mediated anion exchange.Biochemistry, 42, 12927–12932.

22. Izuhara, K., Okubo, K. & Hamasaki, N. (1989).Conformational change of band 3 protein inducedby diethyl pyrocarbonate modification in humanerythrocyte ghosts. Biochemistry, 28, 4725–4728.

23. Jennings, M. L., Anderson, M. P. & Monaghan, R.(1986). Monoclonal antibodies against human eryth-rocyte band 3 protein. Localization of proteolyticcleavage sites and stilbenedisulfonate-binding lysineresidues. J. Biol. Chem. 261, 9002–9010.

24. Wainwright, S. D., Tanner, M. J., Martin, G. E., Yendle,J. E. & Holmes, C. (1989). Monoclonal antibodies tothe membrane domain of the human erythrocyteanion transport protein. Localization of the C-termi-nus of the protein to the cytoplasmic side of the redcell membrane and distribution of the protein in somehuman tissues. Biochem. J. 258, 211–220.

25. Hamasaki, N., Okubo, K., Kuma, H., Kang, D. & Yae,Y. (1997). Proteolytic cleavage sites of band 3 proteinin alkali-treated membranes: fidelity of hydropathyprediction for band 3 protein. J. Biochem. 122, 577–585.

26. Abe, Y., Chaen, T., Jin, X. R., Hamasaki, T. &Hamasaki, N. (2004). Mass spectrometric analyses oftransmembrane proteins in human erythrocyte mem-brane. J. Biochem. 136, 97–106.

27. Popov, M., Tam, L. Y., Li, J. & Reithmeier, R. A. (1997).Mapping the ends of transmembrane segments in apolytopic membrane protein. Scanning N-glycosyla-tion mutagenesis of extracytosolic loops in the anionexchanger, band 3. J. Biol. Chem. 272, 18325–18332.

28. Popov, M., Li, J. & Reithmeier, R. A. (1999).Transmembrane folding of the human erythrocyteanion exchanger (AE1, band 3) determined byscanning and insertional N-glycosylation mutagene-sis. Biochem. J. 339, 269–279.

29. Kanki, T., Sakaguchi, M., Kitamura, A., Sato, T.,Mihara, K. & Hamasaki, N. (2002). The tenthmembrane region of band 3 is initially exposed tothe luminal side of the endoplasmic reticulum andthen integrated into a partially folded band 3intermediate. Biochemistry, 41, 13973–13981.

30. Tang, X. B., Fujinaga, J., Kopito, R. & Casey, J. R.(1998). Topology of the region surrounding Glu681 ofhuman AE1 protein, the erythrocyte anion exchanger.J. Biol. Chem. 273, 22545–22553.

31. Fujinaga, J., Tang, X. B. & Casey, J. R. (1999). Topologyof the membrane domain of human erythrocyte anionexchange protein, AE1. J. Biol. Chem. 274, 6626–6633.

32. Zhu, Q., Lee, D. W. & Casey, J. R. (2003). Noveltopology in C-terminal region of the human plasmamembrane anion exchanger, AE1. J. Biol. Chem. 278,3112–3120.

33. Groves, J. D., Wang, L. & Tanner, M. J. (1998).Functional reassembly of the anion transport domainof human red cell band 3 (AE1) from multiple andnon-complementary fragments. FEBS Lett. 433,223–227.

34. Groves, J. D. & Tanner, M. J. (1999). Structural modelfor the organization of the transmembrane spans ofthe human red-cell anion exchanger (band 3; AE1).Biochem. J. 344, 699–711.

35. Ota, K., Sakaguchi, M., Hamasaki, N. & Mihara, K.(1998). Assessment of topogenic functions of antici-pated transmembrane segments of human band 3.J. Biol. Chem. 273, 28286–28291.

189EM Structure of AE1

36. Ota, K., Sakaguchi, M., Hamasaki, N. & Mihara, K.(2000). Membrane integration of the second trans-membrane segment of band 3 requires a closelyapposed preceding signal-anchor sequence. J. Biol.Chem. 275, 29743–29748.

37. Li, C., Takazaki, S., Jin, X., Kang, D., Abe, Y. &Hamasaki, N. (2006). Identification of oxidized me-thionine sites in erythrocyte membrane protein byliquid chromatography/electrospray ionization massspectrometry peptide mapping. Biochemistry, 45,12117–12124.

38. Jennings, M. L. & Anderson, M. P. (1987). Chemicalmodification and labeling of glutamate residues at thestilbenedisulfonate site of human red blood cell band3 protein. J. Biol. Chem. 262, 1691–1697.

39. Kalo, M. S. (1996). Topological disposition of tyrosine486 in anion exchanger from human erythrocytes.Biochemistry, 35, 999–1009.

40. Takazaki, S., Abe, Y., Kang, D., Li, C., Jin, X., Ueda, T.& Hamasaki, N. (2006). The functional role of arginine901 at the C-terminus of the human anion transporterband 3 protein. J. Biochem. 139, 903–912.

41. Kuma, H., Shinde, A. A., Howren, T. R. & Jennings,M. L. (2002). Topology of the anion exchange proteinAE1: the controversial sidedness of lysine 743.Biochemistry, 41, 3380–3388.

42. Kuma, H., Abe, Y., Askin, D., Bruce, L. J., Hamasaki,T., Tanner, M. J. & Hamasaki, N. (2002). Molecularbasis and functional consequences of the dominanteffects of the mutant band 3 on the structure of normalband 3 in Southeast Asian ovalocytosis. Biochemistry,41, 3311–3320.

43. Wang, L., Groves, J. D., Mawby, W. J. & Tanner,M. J. (1997). Complementation studies with co-expressed fragments of the human red cell aniontransporter (band 3; AE1). The role of some exo-facial loops in anion transport. J. Biol. Chem. 272,10631–10638.

44. Popov, M., Tam, L. Y., Li, J. & Reithmeier, R. A.(1997). Mapping the ends of transmembrane segmentsin a polytopic membrane protein. J. Biol. Chem. 272,18325–18332.

45. Wang, D. N., Sarabia, V. E., Reithmeier, R. A. &Kühlbrandt, W. (1994). Three-dimensional map of thedimeric membrane domain of the human erythrocyteanion exchanger, band 3. EMBO J. 13, 3230–3235.

46. Yamaguchi, T., Fujii, T., Abe, Y., Hirai, T., Kang, D.,Namba, K. et al. (2009). Helical image reconstructionof the outward-open human erythrocyte band 3membrane domain in tubular crystals. J. Struct. Biol.,doi:10.1016/j.jsb.2009.12.009.

47. Jentsch, T. J., Friedrich, T., Schriever, A. & Yamada, H.(1999). The CLC chloride channel family. PflugersArch. 437, 783–795.

48. Dutzler, R., Campbell, E. B., Cadene, M., Chait, B. T. &MacKinnon, R. (2002). X-ray structure of a ClCchloride channel at 3.0 Å reveals the molecular basisof anion selectivity. Nature, 415, 287–294.

49. Dutzler, R., Campbell, E. B. & MacKinnon, R. (2003).Gating the selectivity filter in ClC chloride channels.Science, 300, 108–112.

50. Accardi, A. & Miller, C. (2004). Secondary activetransport mediated by a prokaryotic homologue ofClC Cl− channels. Nature, 427, 803–807.

51. Miller, C. & White, M. M. (1984). Dimeric structure ofsingle chloride channels from Torpedo electroplax.Proc. Natl Acad. Sci. USA, 81, 2772–2775.

52. Mindell, J. A., Maduke, M., Miller, C. & Grigorieff, N.(2001). Projection structure of a ClC-type chloridechannel at 6.5 Å resolution. Nature, 409, 219–223.

53. Collaborative Computational Project, Number 4.(1994). The CCP4 suite: programs for protein crystal-lography.Acta Crystallogr., Sect. D: Biol. Crystallogr. 50,760–763.

54. Potterton, L., McNicholas, S., Krissinel, E., Gruber,J., Cowtan, K., Emsley, P. et al. (2004). Develop-ments in the CCP4 molecular-graphics project. ActaCrystallogr., Sect. D: Biol. Crystallogr. 60, 2288–2294.

55. Taylor, A. M., Zhu, Q. & Casey, J. R. (2001). Cysteine-directed cross-linking localizes regions of the humanerythrocyte anion-exchange protein (AE1) relative tothe dimeric interface. Biochem. J. 359, 661–668.

56. Taylor, W. R., Jones, D. T. & Green, N. M. (1994). Amethod for alpha-helical integral membrane proteinfold prediction. Proteins, 18, 281–294.

57. Kang, D., Okubo, K., Hamasaki, N., Kuroda, N. &Shiraki, H. (1992). A structural study of the membranedomain of band 3 by tryptic digestion. Conforma-tional change of band 3 in situ induced by alkalitreatment. J. Biol. Chem. 267, 19211–19217.

58. Hirai, T., Murata, K., Mitsuoka, K., Kimura, Y. &Fujiyoshi, Y. (1999). Trehalose embedding techniquefor high-resolution electron crystallography: applica-tion to structural study on bacteriorhodopsin.J. Electron Microsc. 48, 653–658.

59. Gyobu, N., Tani, K., Hiroaki, Y., Kamegawa, A.,Mitsuoka, K. & Fujiyoshi, Y. (2004). Improvedspecimen preparation for cryo-electron microscopyusing a symmetric carbon sandwich technique.J. Struct. Biol. 146, 325–333.

60. Fujiyoshi, Y., Mizusaki, T., Morikawa, K., Yamagishi,H., Aoki, Y., Kihara, H. & Harada, Y. (1991). Develop-ment of a superfluid-helium stage for high-resolutionelectron-microscopy. Ultramicroscopy, 38, 241–251.

61. Crowther, R. A., Henderson, R. & Smith, J. M. (1996).MRC image processing programs. J. Struct. Biol. 116,9–16.

62. Hirai, T., Heymann, J. A., Shi, D., Sarker, R., Maloney,P. C. & Subramaniam, S. (2002). Three-dimensionalstructure of a bacterial oxalate transporter. Nat. Struct.Biol. 9, 597–600.

63. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano,W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998).Crystallography & NMR System: a new softwaresuite for macromolecular structure determination.Acta Crystallogr., Sect. D: Biol. Crystallogr. 54,905–921.

64. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang,J., Zhang, Z., Miller, W. & Lipman, D. J. (1997).Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs. Nucleic Acids Res.25, 3389–3402.

65. Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994). CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment throughsequence weighting, position-specific gap penaltiesand weight matrix choice. Nucleic Acids Res. 22,4673–4680.