Structural basis for the alternating access mechanismof the cation diffusion facilitator YiiPMaria Luisa Lopez-Redondoa,1, Nicolas Coudraya,1, Zhening Zhanga,2, John Alexopoulosa, and David L. Stokesa,3

aSkirball Institute, Department of Cell Biology, New York University School of Medicine, New York, NY 10016

Edited by Robert M. Stroud, University of California, San Francisco, CA, and approved January 31, 2018 (received for review August 24, 2017)

YiiP is a dimeric antiporter from the cation diffusion facilitatorfamily that uses the proton motive force to transport Zn2+ acrossbacterial membranes. Previous work defined the atomic structure ofan outward-facing conformation, the location of several Zn2+ bind-ing sites, and hydrophobic residues that appear to control access tothe transport sites from the cytoplasm. A low-resolution cryo-EMstructure revealed changes within the membrane domain that wereassociated with the alternating access mechanism for transport. Inthe current work, the resolution of this cryo-EM structure has beenextended to 4.1 Å. Comparison with the X-ray structure defines thedifferences between inward-facing and outward-facing conforma-tions at an atomic level. These differences include rocking and twist-ing of a four-helix bundle that harbors the Zn2+ transport site andcontrols its accessibility within each monomer. As previously noted,membrane domains are closely associated in the dimeric structurefrom cryo-EM but dramatically splayed apart in the X-ray structure.Cysteine crosslinking was used to constrain these membrane do-mains and to show that this large-scale splaying was not necessaryfor transport activity. Furthermore, dimer stability was not compro-mised by mutagenesis of elements in the cytoplasmic domain, sug-gesting that the extensive interface between membrane domains isa strong determinant of dimerization. As with other secondarytransporters, this interface could provide a stable scaffold for move-ments of the four-helix bundle that confers alternating access ofthese ions to opposite sides of the membrane.

zinc homeostasis | membrane transport | alternating access mechanism |cysteine crosslinking | cryo-EM

The cation diffusion facilitator (CDF) family comprises sec-ondary transporters that maintain homeostasis for transition

metal ions. The family was initially identified from operons as-sociated with the czc metal resistance determinant (1) and latercharacterized by a phylogenetic analysis that highlighted diversityin ion selectivity and cell localization (2). The CDF family is nowrecognized to comprise three broad groups (3): Group 1 containsZn2+ transporters from humans [Znt1-10 (4)], fungi [Zhf, Zrc1,and Cot1 (5)], plants [metal transport proteins (MTPs) (6)], andbacteria [e.g., ZitB (7)]. Group 2 includes the well-characterizedYiiP (FieF) from Escherichia coli (8–10) as well as transportersthat have been reported to transport or mediate tolerance to awide range of ions, including Zn2+, Cd2+, Fe2+, Co2+, and Ni2+.Group 3 is dominated by plant and fungal transporters that pro-vide tolerance to Mn2+ (11). All CDF transporters are thought tobe antiporters that use the proton motive force to export theirrespective transition metal ions from the cytoplasm (12, 13).The alternating access mechanism represents a paradigm for

secondary transporters, in which substrates are carried across themembrane as the protein cycles between inward-facing (IF) andoutward-facing (OF) conformations (14). Definition of theseconformations is a first step toward characterizing the transportmechanism, which ultimately also requires understanding of stableintermediate states as well as the energy landscape that governstransitions between these states. In the case of the major facili-tator superfamily (MFS) and amino acid-polyamine-organocation(APC) superfamily, structural studies have gone a long way to-ward providing the relevant structural information for individual

representatives. Both superfamilies are characterized by internalsequence repeats which fold into two distinct domains. The con-formational changes from IF to OF states are described as eitherrocking-bundle or elevator-like movements of one domain relativeto the other, and these movements typically involve coordinated,symmetric structural changes in the respective repeats (15–17).CDF transporters, however, do not have an obvious sequencerepeat, and although many are reported to form homodimers, anX-ray structure of YiiP shows that independent transport sites arepresent within each monomer (18, 19).More specifically, the X-ray structure of E. coli YiiP showed a

dimer that is stabilized by a conserved salt bridge at the cyto-plasmic membrane surface and interactions between C-terminal,cytoplasmic domains (CTDs). The transmembrane (TM) do-mains were splayed apart with no intermolecular interactions.Zn2+ ions were bound at the transport sites within each TMdomain, and their accessibility suggested that this structure rep-resented an OF conformation. A subsequent structure of a closelyrelated YiiP from Shewanella oneidensis (45% identity) was pro-duced by our laboratory using cryo-EM images of tubular, 2Dcrystals in a lipid environment (20). Although this cryo-EMstructure was at low resolution (13 Å), it was clear that the TMdomains adopted a closely apposed conformation, whereas thecytoplasmic domain and its dimer interface were very similar tothe X-ray structure. Constrained fitting of the X-ray structure tothe cryo-EM map suggested that it represented an IF conforma-tion, brought about by rocking movements of a four-helix bundlein the TM domain of each monomer. Thus, comparison of the twostructures led to a transport model that involved large-scale scis-soring of the TM domains accompanied by smaller-scale rocking

Significance

Zn2+ is a micronutrient that plays important roles throughoutthe body. We are interested in molecular mechanisms by whichappropriate levels of Zn2+ are maintained in cells. We havecombined structural and functional studies to deduce the phys-ical changes that a bacterial transporter uses to carry Zn2+ acrosscell membranes. We have identified parts of the molecule thatremain static and characterized the movements of other partsthat bind Zn2+ ions and allow them to cross the membrane.

Author contributions: M.L.L.-R., N.C., and D.L.S. designed research; M.L.L.-R., N.C., Z.Z.,J.A., and D.L.S. performed research; M.L.L.-R., N.C., Z.Z., J.A., and D.L.S. analyzed data; andM.L.L.-R., N.C., and D.L.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: Crystallography, atomic coordinates, and structure factors have beendeposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 5VRF), and in theEM Data Bank (EMDB code EMD-8728).1M.L.L.-R. and N.C. contributed equally to this work.2Present address: New York Structural Biology Center, New York, NY 10027.3To whom correspondence should be addressed. Email: stokes@nyu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1715051115/-/DCSupplemental.

Published online March 5, 2018.

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of this four-helix bundle to achieve alternating access of the Zn2+

transport sites. A more recent study of hydroxyl radical footprintingidentified specific residues along the M5 helix; differing reactivitiesin the presence and absence of Zn2+ suggested that rigid-bodymovements of M5 restricted access to the transport sites in theOF state (21).For this report, we sought to further characterize conforma-

tional changes underlying alternating access and Zn2+ transportby YiiP. To start, we obtained a high-resolution structure of theIF state using cryo-EM images of the membrane-bound, helicalcrystals. This structure allowed us to build an atomic model andto evaluate detailed structural differences relative to the OFstate from X-ray crystallography. This comparison indicatesnonrigid-body movements among the four-helix bundle thatsurrounds the Zn2+ transport site. To assess the functional im-plications of the apparent scissoring motion of the TM domain,we generated cysteine substitutions and made intermolecularcrosslinks in conjunction with in vitro analysis of transport ac-tivity. Finally, we made mutations to elements that appear tostabilize the homodimer, to elucidate the structural basis for thisconserved feature of the CDF superfamily. Our results indicatethat scissoring of TM domains is not essential for transport. Wehave developed a hybrid model for the OF state that illustratesconformational changes that we believe underlie the alternatingaccess mechanism of YiiP.

ResultsCryo-EM Structure of YiiP.YiiP from S. oneidensis was expressed inE. coli, and after purification, detergent-solubilized preparationswere reconstituted into bilayers of dioleoylphosphatidyl glycerol(DOPG) at a lipid-to-protein ratio of 0.5 (wt/wt) by dialysis. Asdetergent was removed, YiiP assembled into extended 2D arrayswithin the lipid bilayer. These bilayers adopted a tubular mor-phology, thus twisting the 2D arrays of YiiP into a helical ar-rangement. Cryo-EM imaging revealed two distinct helicalsymmetries, denoted types 1 and 2 (Fig. 1). For 3D reconstruction,overlapping masks were applied to images of individual tubes toproduce a set of single particles for use with iterative real-spacereconstruction algorithms (22). These single particles were sub-jected to 2D classification, which revealed the presence of sub-types for both type 1 and type 2 (denoted types 1a, 1b, 1c, 2a, and2b; Fig. S1). Each of these classes represented a unique helicalsymmetry, and we used a series of complementary approaches(23) to determine the relevant geometrical parameters. Thisanalysis illustrated that the five types had the same underlying 2Dlattice, although types 1 and 2 differed in angle of the helical ar-rays relative to the longitudinal axis of the tube. The subtypes weredistinguished by differing tube diameters, which thus encom-passed different numbers of unit cells (e.g., 5, 6, or 7 for subtypes1b, 1a, and 1c, respectively; Fig. S1E). Each symmetry produced aunique set of Bessel orders for the layer lines in Fourier trans-forms (Fig. 1) and unique values for the rise and twist of thefundamental helix, the latter of which was used to enforce sym-metry during structure refinement (Table S1). Sufficient numbersof images were obtained to generate 3D reconstructions from fourof the subtypes at varying resolutions. Despite the differences inhelical symmetry, comparison of dimers from these different re-constructions indicates that YiiP adopted the same conformationin all of the tubes (Fig. S2).The data set from type 2a tubes produced the highest reso-

lution, which was estimated by Fourier shell coefficient (FSC) tobe 4.1 Å (Fig. 1D). However, evaluation of local resolution byResMap (24) indicated that the membrane domain had signifi-cantly higher resolution than the cytoplasmic domain. In par-ticular, the resolution throughout the membrane domain was3.25–3.5 Å, thus revealing clear side-chain densities (Fig. S3) andallowing us to build a de novo atomic model for residues 7–212.Due to lower resolution in the cytoplasmic domain, side-chains

were poorly resolved, and we applied molecular dynamics flexi-ble fitting (25) and constrained real-space refinement (26), usingthe X-ray structure of YiiP from E. coli (18) to define the sec-ondary structure and overall fold for residues 213–288. The finalmodel resulted from several rounds of building and real-spacerefinement and produced a final cross-correlation coefficient of0.83 (Table S2).

Organization of the Membrane Domain.Although the conformationof YiiP is generally similar to that deduced from our previousmodel (20), the explicit chain trace reveals significant differencesin the conformation of the TM helices. Specifically, YiiP adoptsthe expected dimer in which cytoplasmic domains and TM do-mains are both closely apposed and Zn2+ transport sites are

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Fig. 1. Cryo-EM of YiiP. (A) Image of type1 and type 2 crystals. (Scale bar:50 nm.) (B and C) Diffraction from type 1 and type 2 crystals shown as theincoherent sum of Fourier transforms from numerous crystals. The layer lineshave been assigned Miller indices and Bessel orders (h, k; n), the latter ofwhich characterize the helical symmetry. (D) Fourier shell correlation (FSC)shows an overall resolution of 4.1 Å for the reconstruction from type 2acrystals. (E) Local resolution for type 2a was assessed by ResMap, showing thattransmembrane regions (gray box) had resolutions of 3.25–3.5 Å, whereas theC-terminal domain had resolutions of 4–5 Å. Sites for Zn2+ binding (ZnB andZnC) had higher resolution than the surrounding region.

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accessible from the cytoplasmic side of the membrane (Fig. 2).The accessibility of these sites, which distinguishes the IF and OFstates, is governed by a four-helix bundle (M1, M2, M4, and M5)that surrounds the Zn2+ transport site (Fig. 2B); previous work in-dicated that this four-helix bundle rocks against the M3/M6 helicesthat form a coiled coil at the TM dimer interface. However, inaddition to this general rocking movement, our structure also re-veals a rotation of this bundle relative to M3/M6 (Fig. 2C) andsubstantial differences in the length and trajectory of the individualhelices (Fig. 2D). Specifically, M2 and M5 are bent near the Zn2+

binding site, thus enhancing the IF and OF character of the respectivestructures.Another finding afforded by the higher resolution of our cryo-

EM structure was the presence of strong densities at three siteswhere Zn2+ ions were observed in the X-ray structure, denotedZnA, ZnB, and ZnC (19). Density levels at these three sites wereroughly double that of surrounding protein density (Fig. S3) andwere associated with a notable enhancement in resolution esti-mated by ResMap relative to the surrounding areas (Fig. 1E).The presence of ions in our structure was unexpected given the

absence of transition metal ions (e.g., Zn2+ and Cd2+) in thecrystallization medium. Although cryo-EM is not capable ofdistinguishing different elements, four Zn2+ ions were includedduring structure refinement: one each at ZnA and ZnB and two atthe binuclear ZnC site within the CTD. In the refined structure,these ions are coordinated at an average distance of 2.2 Å, whichis consistent with general observations of Zn2+ sites in proteins(27, 28). Moreover, the specific residues and the coordinationdistances in our structure are highly consistent with the Zn2+ sitesin the X-ray structure of E. coli YiiP (Table S3) (18).

Scissoring of Membrane Domains. In addition to movements ofindividual TM helices described above, comparison of X-ray andcryo-EM structures suggests that the membrane domains un-dergo a dramatic scissoring motion during the transition betweenOF and IF states (Fig. 2A). As previously noted (20), themembrane domains in the X-ray structures are splayed apart in away that prevents intermolecular contacts, whereas these do-mains adopt a compact configuration in the cryo-EM structurewith close contact between M3 helices (Fig. S3B). To assess thefunctional implications of this difference, we used cysteinecrosslinking to lock the domains in a compact state and evalu-ated the consequences with in vitro assays of Zn2+ transport.Specifically, we made a series of cysteine substitutions alongM3 near the intermolecular interface and used Cu2+ to induceformation of an intermolecular disulfide bond. As a first step, thesingle cysteine residue in wild-type (WT) YiiP from S. oneidensiswas mutated to alanine, and the resulting construct (C190A) wasused as a template for cysteine substitution at Ala90, Gly94,Leu98, and Tyr102 (Fig. S3 A and B). After expression and pu-rification, gel filtration coupled with UV, multiple-angle lightscattering (MALS), and refractive index (RI) detectors indicatedthat these mutants behaved the same as WT by migrating asdimers in detergent solution (Fig. S4 and Table S4). The purifiedproteins were then reconstituted into proteoliposomes in eitherthe presence or absence of Cu2+. The degree of disulfide bondformation was assessed using nonreducing SDS/PAGE (Fig. 3and Table S4), which showed varying degrees of crosslinking. Inparticular, crosslinking efficiency was close to 100% for Y102Ceven in the absence of Cu2+, whereas the other mutants showedmarked increases in the presence of Cu2+, which resulted in ef-ficiencies of ∼50% for A90C and G94C and 90% for L98C. Asexpected, C190A showed no crosslinking in the presence or ab-sence of Cu2+, whereas Cu2+ caused WT to precipitate.These proteoliposomes were then used for transport assays, in

which the Zn2+-sensitive dye, Fluozin 1, was trapped inside thevesicles and a stop-flow fluorimeter was used to measure trans-port. The initial rates were plotted as a function of Zn2+ con-centration to determine K0.5 and Vmax (Fig. S5). These dataindicated that all of the mutants had activities similar to WT andthat the degree of Cu2+-induced crosslinking had no statisticallysignificant effect on transport. Although the quantitative resultsin Fig. 3 and Table S3 represent a single set of matched samples,this analysis was repeated between 4 and 7 times on differentpreparations of the various mutants, which all showed qualita-tively similar results and no discernable differences produced bycrosslinking. Each experiment included protein-free liposomes,which showed minimal leakage of Zn2+ and which providedbackground signal that was subtracted before analysis of YiiP-dependent transport activity (Fig. S5). As a negative control, wemutated residues contributing to the Zn2+ transport site (D51A)and to the conserved intermolecular salt bridge (K79D); bothmutations abolished transport activity (Fig. 3) as had been pre-viously shown for E. coli YiiP (9, 18).

Stability of the YiiP Dimer. Our structural and biochemical dataindicate that YiiP forms a constitutive homodimer, which isconsistent with similar results from E. coli YiiP (19, 29) and with

D

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Fig. 2. Comparison of Cryo-EM and X-ray structures. (A) Overlay of cryo-EM(blue) and X-ray (red; PDB code 3H90) structures after alignment of their CTDs.The transmembrane domain adopts a compact conformation in the cryo-EMstructure compared with a splayed conformation in the X-ray structure. Theloop between M6 and the CTD acts as a hinge for the implied movementbetween compact and splayed conformations. (B) Monomers taken from eachstructure and aligned based on M3 and M6. Water accessibility is shown ingreen. Different angles for the four-helix bundle (M1, M2, M4, and M5)provide access to the Zn2+ transport site (ZnA) site from the periplasm in theX-ray structure (red) and from the cytoplasm in the cryo-EM structure (blue).(C and D) Overlay of transmembrane helices after alignment of the residuescoordinating Zn2+ at the transport site (ZnA). The view from the cytoplasmicside of the membrane (C) shows that the M3/M6 pair is rotated relative to thefour-helix bundle. The orthogonal view (D) shows unraveling and bending ofM2 and M5 originating at the Zn2+ transport sites.

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functional data from related CDF transporters (30). In previouswork, Fu and colleagues highlighted the conserved salt bridge atthe cytoplasmic membrane surface (Lys79–Asp209; Fig. S6) as adeterminant of the dimer stability and have described this saltbridge as a fulcrum for conformational changes (18). Two Zn2+

ions bound at the interface between cytoplasmic domains rep-resent another element that appear to stabilize the dimer, giventhat they are coordinated by His285 and Asp287 from onemonomer and His263 from the other monomer (Fig. S3E) andwere indeed shown to affect the association of isolated CTDsfrom the related CzrB (31). To test the contribution of theseparticular elements to dimer stability, we mutated Lys79 toprevent formation of its salt bridge with Asp209 and made twodouble mutants (H263A/H285A and H263A/D287A) to disruptZn2+ binding between the cytoplasmic domains. We then usedgel filtration with MALS and RI detectors to document theoligomeric state (Fig. S4). All of these mutants eluted as dimericspecies in detergent solution, demonstrating the extreme stabilityof the YiiP dimer and suggesting that additional elements may bepresent to mediate dimerization.

DiscussionIn this work, cryo-EM was used to produce a high-resolutionstructure of YiiP from S. oneidensis from membrane-boundcrystals, which allowed us to build an atomic model of an IFconformation. Relative to our previous model based on a 13-Åresolution structure, the higher resolution from the current

work reveals significant differences in the configuration of TMhelices surrounding the Zn2+ transport site. This higher-resolutionstructure also allows more definitive comparisons with the X-raystructure of detergent-solubilized YiiP from E. coli in an OF con-formation, which suggest two different kinds of movements withinthe membrane domain that might be associated with the alternat-ing access mechanism of transport: a large-scale scissoring of themembrane domains of the homodimer and more localized tiltingand bending of the four TM helices that control access to thetransport site within each monomer. We went on to evaluate thefunctional importance of the large-scale scissoring by generatingintermolecular crosslinks that would prevent this movement. Wefound that crosslinked dimers were fully active in Zn2+ transportassays, suggesting that the scissoring movement is not a functionalnecessity and that localized conformational changes within eachmonomer are sufficient to provide the alternating access requiredfor transport.One explanation for the splaying of membrane domains in the

X-ray structure is that the detergent micelle lacks the stiffness,curvature, and lateral pressure of the lipid bilayer (32). The factthat several TM helices become unwound in the X-ray model,whereas all six TM helices extend across the entire membrane inthe cryo-EM structure, suggests that the detergent environmentmight have a destabilizing influence. Such destabilization couldalso facilitate the separation of the membrane domains in thedetergent-based crystals, perhaps by recruiting a micelle aroundeach individual monomer. Alternatively, two different sets ofcrystal contacts appear to hold these domains apart (Fig. S6).Specifically, the asymmetric unit from the X-ray crystals comprisestwo distinct dimers. Both dimers engage in intermolecular contactsinvolving the outer surface of their membrane domains: one ismediated by the periplasmic loop between M5 and M6 and theother is an Hg-stabilized interaction betweenM1 from one moleculeandM4 from a neighboring molecule. In both cases, helices involvedin these contacts become unwound on the periplasmic side of themembrane domain. Sequence homology between YiiP from E. coliand S. oneidensis is generally high (45% identity), and particularly soalong M4 and the M5–M6 loop, so these structural differences areunlikely to reflect sequence diversity.The loop connecting M6 with the CTD appears to be the hinge

for the scissoring movement of the TM domains. This loopharbors Asp209, which forms an intermolecular salt bridge withLys79, which was previously described as a possible fulcrum formovement of the membrane domains (18). The rather limitedinteractions between the M6–CTD connecting loop and otherelements of the membrane domain are consistent with this idea.However, even though this salt bridge is conserved across groups1 and 2 of the CDF family, Asp209 is not conserved in the Mn2+

transporters from group 3. Furthermore, our studies of cross-linked dimers indicate that the scissoring motion of the TMdomains is not a requisite for transport.On the other hand, water accessibility within each monomer

indicates that the smaller-scale differences within the TM do-main of each monomer in OF and IF states are sufficient foralternating access of the Zn2+ transport sites (Fig. 2B). This TMdomain can be partitioned into a four-helix bundle (M1, M2, M4,and M5) that surrounds the Zn2+ binding site and a pair of he-lices (M3/M6) that mediates the dimer interface. Although ourprevious model suggested a relative rocking motion of one sub-domain relative to the other about an axis parallel to themembrane, our higher-resolution structure reveals an additionalrotation of the four-helix bundle relative to the M3/M6 pairabout an axis perpendicular to the membrane (Fig. 4 A and Band Movie S1). These movements expose the binding site to thecytoplasm in the cryo-EM structure and to the periplasm in theX-ray structure, as expected for IF and OF states, respectively. Inaddition, the bending of M2 and M5 near the residues that

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Fig. 3. Intermolecular crosslinking does not affect transport activity. (A andB) Nonreducing SDS/PAGE documents crosslinking in the presence or ab-sence of CuCl2, which promotes disulfide bond formation. Crosslinked pro-teins run as a dimer with Mr ∼ 65 kDa (D), whereas uncrosslinked proteinsrun as monomers (M). Lanes: 1, WT; 2, C190A; 3, C190A/A90C; 4, C190A/G94C; 5, C190A/L98C; and 6, C190A/Y102C. WT protein precipitated in thepresence of CuCl2. Molecular weight markers in the center are 116, 66, 45,35, and 25 kDa. (C and D) Transport activity of representative constructsprepared in the presence and absence of CuCl2. Rates have been normalizedrelative to Vmax shown below. D51A and K79D are known to abolish trans-port, and their rates are normalized relative to C190A. (E and F) Comparisonof K0.5 and Vmax for mutants prepared in the presence and absence of CuCl2.P values for each pairwise comparison are shown, none of which reach the95% confidence level. Hill coefficients are not shown but generally variedbetween 0.9 and 1.4 with minimal effects of CuCl2.

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coordinate the Zn2+ ion serves to enhance the IF and OF char-acter of the two states.A hydrophobic gate controlling Zn2+ access to the transport

sites was recently identified on the cytoplasmic side of themembrane domain by X-ray–mediated hydroxyl radical labeling(21). According to this study, Leu152 and to a lesser extentMet197 underwent a large decrease in accessibility upon addi-tion of Zn2+, which the authors interpreted to reflect a changefrom IF to OF states. In the X-ray structure of the OF state,these residues participate in a bonding network between M3,M5, and M6 which excludes water from accessing the Zn2+

transport site (Fig. 4C). In our cryo-EM structure of the IF state,the analogous residues (Leu154 and Leu199) are ∼10 Å apartand readily accessible to the cytoplasm (Fig. 4D), thus account-ing for the hydroxyl radical labeling results.Our cryo-EM structure revealed strong densities at each of

three Zn2+ binding sites with coordination geometries that arealmost identical with Zn2+ ions in the X-ray structure (Fig. S3and Table S3). The presence of ions in our structure could reflect

a lack of EDTA treatment of our preparation, as has been donein other studies (10, 21), even though our purification andcrystallization buffers lacked any transition metal ions. AlthoughYiiP has also been shown to bind Cd2+ and Hg+ (9), we think it ismore likely that Zn2+ was carried through the purification andcrystallization process because it is more prevalent as a micro-nutrient and represents the physiological substrate. It also seemsunlikely that Mg2+, which was present during crystallization,would account for the observed densities given its different co-ordination geometry and strong preference for oxygen ligands. Itis worth noting that the crystal solutions used for the X-raystructure contained 100 mM citrate in addition to 5 mM Zn2+,which would result in submicromolar concentrations of free Zn2+.In this study, anomalous scattering as well as calorimetric titra-tions confirmed Zn2+ binding to all three sites, although withdifferent occupancies (19). These results, together with previousITC studies (9, 10, 33), suggest that the cytoplasmic sites areEDTA-resistant and of extremely high affinity, whereas trans-port sites are closer to micromolar affinity.These considerations indicate that binding affinity at the

transport sites is considerably higher than the values for K0.5measured by ourselves and others in transport assays (9, 20). Thisconclusion implies that YiiP transport does not conform toclassical Michaelis–Menten kinetics, i.e., that binding of Zn2+ isnot the rate limiting step in transport. Although descriptions ofthe alternating access mechanism generally focus on IF and OFstates, there are in fact many intermediates in the transport cycle,including apo states, bound states, and occluded states (15). Thepresence of ions at the transport sites in both cryo-EM and X-raystructures indicates that both are bound states, and comparisonof these structures therefore reflects the conformational equi-librium between bound IF and OF states. Given that YiiPfunctions as a Zn2+/H+ antiporter (13), such conformationalequilibrium would be expected both in the Zn2+-bound and inthe H+-bound states. At lower pHs, proton binding would pro-mote release of Zn2+, thus coupling transport to the protonmotive force and perhaps helping to reconcile the high equilib-rium binding affinity of Zn+ with the physiological transportprocess. Additional conformational changes can be expected asprotons replace Zn2+ at the transport sites, and the structuralnature of the protonated IF and OF states is well worth pursuingin future studies.Dimerization is widespread among CDF transporters and is

therefore presumed to have functional significance (30). Theconserved salt bridge and the binuclear Zn2+ site in the CTD areboth candidates for stabilizing the dimeric state. However, nei-ther of these elements are strictly conserved across the CDFfamily. As mentioned, Asp209 of the salt bridge is not conservedin group 3, and Zn2+ binding residues that bridge the CTDs(H263 and H285) are not conserved in group 1. The importanceof the intact molecule is illustrated by discrepant conclusionsfrom studies of intact E. coli YiiP and of isolated CTDs fromT. thermophilus CzrB. In the former, high occupancy and EDTAresistance suggest that cytoplasmic Zn2+ ions are constitutivelybound (19), whereas the latter characterized sites that werereadily titratable and showed a dramatic splaying of the CTDswhen Zn2+ was absent (31). In our cryo-EM structure, the trans-membrane interface provides a large contact area of 2,750 Å2 thatincludes residues along the entire length of M3 as well as resi-dues at the periplasmic end of M1 and M2. Our crosslinkingresults suggest that this interface is persistent and, in addition tostabilizing the dimer, it would help to encapsulate both the Zn2+

transport sites and a water-filled pathway toward the periplasmin the OF state, both of which appear to be exposed to the hydro-phobic core of the bilayer in the splayed conformation revealed bythe X-ray structure (20). It is possible that lipid would help tostabilize this interface, thus explaining why this conformation wasobserved in membrane crystals as well as the functional effects of

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M6

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M6L154

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Fig. 4. Model for the alternating access mechanism. The four-helix bundleundergoes complex movements relative to M3/M6 within each YiiP monomer.(A) The bundle rotates about an axis perpendicular to the membrane plane, asseen from the periplasmic side of the membrane. (B) The bundle rocks againstM3/M6 as viewed parallel to the membrane plane. (C) X-ray and (D) cryo-EMstructures viewed from the cytoplasm illustrate the change in accessibility ofL152 and M197, which are thought to form a hydrophobic gate to the Zn2+

transport sites. (E) Models for the IF and OF states. The former corresponds toour cryo-EM structure, whereas the latter corresponds to the X-ray structureafter moving the transmembrane domains into a compact configuration. Cαpositions of crosslinked residues are yellow. Zn2+ ions are pink with arrowsdepicting accessibility to opposite sides of the membrane.

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various lipid species on the transport activity of the mammalianhomolog Znt8 (34).A persistent dimer interface within the membrane implies that

YiiP’s alternating access mechanism would involve a mobiletransport domain moving against a static scaffold domain, as hasbeen observed for other families of secondary transporters.According to this mechanism, depicted in Fig. 4E, transport isdriven by movements of the four-helix bundle relative to a staticM3/M6 scaffold (Movie S2), which is fundamentally differentfrom previous models that postulate hinge-like movements eitherof the CTDs (18) or of the entire membrane domain (20) usingthe conserved salt bridge as a fulcrum. The general strategy of amobile transport domain moving relative to a stable scaffold has,in fact, been adopted by a wide variety of transporters (35). Forthose that employ the so-called elevator mechanism, the scaffolddomain is often responsible for mediating intermolecular inter-actions that stabilize a dimeric or sometimes trimeric assembly.The conformational changes in YiiP do not involve the verticalmovements that characterize the elevator mechanism and, in-stead, resemble a rocking bundle associated with the LeuT fold(35). Nevertheless, the interface between membrane domainscould be important in anchoring the protein relative to the bilayerwhile the M1, M2, M4, and M5 bundle undergoes the movementsnecessary for alternating access. It remains an open question as towhether oligomer formation facilitates cooperativity or whetherthe scaffold helps convey signals (e.g., ion binding by the CTD)that initiate the dynamic movements of the mobile domain todrive transport. Further work is clearly needed to address thesequestions and to reveal structures of other intermediates states.

Materials and MethodsAll YiiP constructs were expressed in E. coli using a vector with a C-terminaldeca-histidine tag. The membrane fraction was solubilized with n-dodecyl-

β-D-maltoside, and after elution from a Ni-NTA affinity column, the tag wasremoved using tobacco etch virus protease. Final purification employed asize-exclusion column equilibrated in n-decyl β-D-maltopyranoside (DM). Theoligomeric state was determined using an HPLC equipped with detectors forUV absorbance, MALS, and refractive index.

Tubular crystals of YiiP were generated by adding DOPG to detergent-solubilized protein solutions at a lipid-to-protein weight ratio of ∼0.5, fol-lowed by dialysis against 100 mM NaCl, 5 mM MgCl2, 5 mM NaN3, and 20 mMTES pH 7 at 27 °C for 7–14 d. After plunge freezing on perforated carbonfilms, samples were imaged in electron microscopes using direct detectors.After correcting images for motion and for the contrast transfer function,individual tubes were isolated, and alternative approaches were used tocharacterize their helical symmetry (23). The 3D reconstructions were de-termined from subsets with consistent symmetries. Local resolution was esti-mated using ResMap (24), and an atomic model was built into the highestresolution reconstruction using the real-space refinement tools of PHENIX(26). Water accessibility of transport sites was evaluated using HOLLOW (36).

For measuring Zn2+ transport, YiiP mutants were reconstituted into E. colipolar lipids at a lipid-to-protein molar ratio of 1,000:1 by dialysis for 6 d at4 °C and were loaded with the Zn-sensitive fluorophore FluoZin-1 by freeze–thaw. These proteoliposomes were mixed with various concentrations ofZnCl2 using a stop-flow apparatus, and time-dependent fluorescence wasused to monitor the influx of Zn2+ into the vesicles. After normalization andbackground substraction, the initial rates of transport were plotted againstZn2+ concentration and fitted with the Hill equation to determine values forK0.5 and Vmax (Fig. S5). Crosslinking of cysteine mutants was induced by1 mM CuCl2 during the first 4 d of the dialysis period. The extent of cross-linking was assessed by SDS/PAGE under nonreducing conditions.

ACKNOWLEDGMENTS. We gratefully acknowledge Olaf Andersen andRadda Rusinova from Weil Cornell Medical College for use of their stop-flow fluorimeter. This work was supported by funds to D.L.S. from NIHGrants R01 GM095747 and U54 GM094598. We also acknowledge use offacilities at the Simon Electron Microscopy Center, New York StructuralBiology Center, as well as the Microscopy Core and the High PerformanceComputing center at New York University Langone Medical Center.

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