Cells have specialized proteins known as transporters in their surface membranes that move molecules into or out of the cell. Transporters pass their cargo through the membrane by changing shape. This process requires energy and is sometimes driven by simultaneously transporting a charged ion such as sodium. There are different classes of transporters and researchers have described a range of structural changes, and therefore transport mechanisms, that different transporters use.

Citrate transporters are found in a wide range of organisms. In bacteria, they bring the citrate substrate molecule into the cell to be used as a nutrient. In humans, citrate transporters are important in metabolism, and are of interest as targets for drugs that could potentially treat obesity and diabetes. This requires an understanding of the atomic structure and the transport mechanisms used by citrate transporters, which were not known.

Wöhlert et al. now use a technique called X-ray crystallography to uncover the structure of a citrate transporter called SeCitS in high detail. This transporter is found in a bacterium called Salmonella enterica, a well-known human pathogen that causes typhoid. The crystallized protein simultaneously showed three different structural states – one where the citrate binding site faces the outside of the cell, and two where the binding site faces the inside of the cell. The simultaneous occurrence of different functional states in one and the same crystal structure of a membrane transporter is so far unique.

Combining the detailed structures of SeCitS with biochemical studies allowed Wöhlert et al. to deduce that citrate is transported in a six-step process. Sodium ions attach to SeCitS, and then citrate binds to the transporter from outside the cell. This binding causes part of the protein to undergo a substantial rotation, shifting it to an inward-facing state and moving the citrate and sodium ions inside the cell. The release of the citrate and sodium ions then triggers the reverse rotation of the transporter, bringing the empty binding site back to the outside of the cell for a repeat of the cycle.

After working out the mechanisms of a bacterial citrate transporter, the next challenge is to extend the analysis to the structure of similar transporters in more complex organisms, including human cells. This could provide an accurate basis for drug development.

Citrate transporters are found in a wide range of bacteria, archaea and eukaryotes. Bacteria use specific transporters (Sobczak and Lolkema, 2005) to take up di- and tricarboxylates as a carbon source (Mulligan et al., 2014; Pos et al., 1998). The human citrate transporter NaCT plays a central role in fatty acid synthesis and glycolysis (Gopal et al., 2007), and is a potential drug target against obesity and diabetes (Liang et al., 2015). The Drosophila INDY gene encodes a related dicarboxylate transporter implicated in fat storage (Rogina et al., 2000). The x-ray structure of VcINDY, a homologous dicarboxylate transporter from Vibrio cholerae is known in the inward-facing state (Mancusso et al., 2012). Unexpectedly, a recent cryo-EM structure of the citrate transporter KpCitS from Klebsiella pneumoniae (Kebbel et al., 2013) revealed a similar overall domain architecture to VcINDY (Mancusso et al., 2012) and to archaeal Na⁺/H⁺ antiporters of the NhaP family (Goswami et al., 2011; Paulino et al., 2014; Wöhlert et al., 2014), in both cases without detectable sequence homology. CitS, VcINDY and the NhaP antiporters all form homodimers of two protomers, each organized in a helix bundle and a dimer contact domain (Kebbel et al., 2013; Mancusso et al., 2012; Vinothkumar et al., 2005), which suggests similar transport mechanisms.

In membrane vesicles (Lolkema et al., 1994; van der Rest et al., 1992) and proteoliposomes (Pos and Dimroth, 1996), CitS from Klebsiella pneumoniae (KpCitS) was previously shown to transport citrate as HCit^2- in a sodium-dependent manner. We observed similar transport properties for CitS from Salmonella enterica (SeCitS), which is closely related to KpCitS. The two homologues share a remarkably high sequence identity of 92% (Figure 1), indicating that their transport mechanisms must be very similar. Iso-citrate and, to a lesser extent, malate inhibit Na⁺-dependent ¹⁴C-citrate uptake by SeCitS into proteoliposomes. Succinate, α-ketoglutarate, and glutaric acid reduce uptake slightly, whereas tricarballylic acid, which lacks the citrate hydroxyl group, has no effect (Figure 2A). This demonstrates the specificity of the CitS binding site for 2-hydroxycarboxylates. Malate, which is smaller than citrate, inhibits citrate uptake by SeCitS but is not transported (Figure 2B). Citrate symport is driven by Na⁺ but not by K⁺ or Li⁺ (Figure 2C, D), demonstrating the exquisite specificity of SeCitS for Na⁺ ions. Sodium transport is cooperative with a Hill coefficient of 1.89, whereas citrate is not, suggesting that citrate transport is coupled to at least two Na⁺ ions (Figure 3). SeCitS is active between pH5 and pH8 with an optimum at pH7, resulting in a roughly bell-shaped pH profile (Figure 3—figure supplement 1). Down-regulation of transport at low pH can be attributed to a limitation in sodium binding, while at elevated pH the availability of the HCit^2- citrate species is limiting. Citrate uptake is enhanced at lower outside pH (Figure 3—figure supplement 2A). Under these conditions, transport by SeCitS is electroneutral, since valinomycin has no effect (Figure 3—figure supplement 2B). A lower outside pH would shift the citrate buffer equilibrium towards HCit^2-. Therefore, a low outside pH increases the local substrate concentration, while a high inside pH tends to deprotonate the HCit^2- substrate and thus removes it from the transport equilibrium. We conclude that protons do not participate directly in the transport mechanism. This conclusion is substantiated by the observation that an increase in the internal Na⁺ concentration does not stimulate citrate uptake (Figure 3—figure supplement 2C), which argues against a previously proposed citrate/proton symport (or citrate/hydroxide antiport) in exchange for internal sodium (Pos and Dimroth, 1996).

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To understand the mechanism in detail we determined the structure of CitS from the human pathogen Salmonella enterica (Figure 4) by single-wavelength anomalous dispersion with crystals of seleno-methionine derivatized protein (Figure 4—figure supplement 1). Phases were extended to the 2.5 Å diffraction limit of native crystals (Table 1). The asymmetric unit contains two homodimers of two protomers in different conformations (Figure 4A, B). Each protomer has 13 helix elements (H1–H13), including eleven transmembrane helices (TMH) and two helix hairpins (H6, H12), with the N-terminus on the cytoplasmic side and the C-terminus on the outside. Helices H2-–7 and H8–13 are organized in two repeats with inverted topology (Figure 4C), connected by a flexible cytoplasmic loop (Figure 4B). Together, helices H5–7 of repeat 1 and H11–13 of repeat 2 form a bundle on either side of the central contact domains, which hold the dimer together through extensive hydrophobic interactions of H2, 4, 8 and 10. A 16 Å-deep hydrophobic cavity on the cytoplasmic side of the dimer interface contains the hydrophobic tail of a detergent or lipid molecule (Figure 5A).

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The two dimers in the asymmetric unit are similar, with an overall rmsd of 0.5 Å, whereas the protomers within one dimer differ substantially by an rmsd of 8.4 Å. The most conspicuous differences are manifest in the vertical positions of the two hairpins H6 and H12 in the helix bundle (Figure 5A). Comparing the two protomers of dimer 1, the C-terminal end of H6 projects 16 Å above the outer membrane surface in protomer A, while it hardly protrudes in protomer B. Conversely, the cytoplasmic H12 ends roughly at the inner membrane surface in protomer A, but extends 13 Å above it in protomer B. The relative position of helices and hairpins within each bundle is unchanged. Evidently, the whole bundle moves as a rigid body from its position in protomer A to that in protomer B, while the central dimer contact domain remains static. The crystal contacts of both dimers in the asymmetric unit are different. Since the polyptide structures of the two dimers are almost identical, the observed asymmetry cannot be attributed to crystal packing. Dimer asymmetry is equally striking with respect to surface structure and electrostatic potential distribution. Protomer A has more positive charges on the periplasmic side than protomer B (Figure 6A). On the cytoplasmic side, positive charges predominate on the surface of protomer B, while positive and negative charges are roughly evenly distributed on protomer A (Figure 6B). Overall, positive charges dominate on the cytoplasmic side of the dimer (Figure 6C, 6D), in line with the positive-inside rule for membrane proteins (Nilsson and von Heijne, 1990; von Heijne, 1992).

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Short stretches of glycine-rich unwound polypeptide link the two halves of hairpins H6 and H12. Together they define the substrate-binding site (Figure 5C–E) at the interface between the helix bundle and the dimer contact domain. On the extracellular surface, the binding site is found in a ∼6 Å deep cavity of protomer A (Figure 6A), while in protomer B it is located at the bottom of a ∼13 Å-deep channel on the cytoplasmic side (Figure 6B). We conclude that protomer A is outward-facing and that protomer B faces inward. The binding sites in both protomers are strongly positively charged (Figure 6A, B). Two detergents and one lipid molecule were identified on the periphery of the dimer. A further detergent molecule was situated in a hydrophobic cavity between the central dimer contact domain and the six-helix bundle of the outward-facing protomer A (Figure 6C–E). H5 in this bundle is straight in protomer A but kinked near its cytoplasmic end in protomer B, to accommodate the movement of the helix bundle (Figure 6E, F).

All four protomers show clear electron density for citrate in the binding site (Figure 7). In the outward-facing protomers, the citrate is closely coordinated by two arginines (Arg402, Arg428), two polar sidechains (Asn186, Ser405) and the protein backbone of both hairpins (Figure 5 and 7A). The only residue in the static contact domain involved in substrate coordination is Tyr348 in H10, which forms a π-π-interaction with a citrate carboxyl. One ordered water molecule participates directly in citrate binding. Its trigonal-bipyramidal coordination geometry (Figure 7) might suggest a Na⁺ ion rather than water, which would imply that the transported entity is NaCit^2- rather than HCit^2-. Because the electron density is weak and the coordination distance of >2.8 Å is longer than would be expected for Na⁺, we interpret this density as a water molecule. In both outward-facing protomers, two Na⁺ ions are clearly resolved next to the citrate (Figures 5C and 7A). In the Na1 site, four backbone carbonyls in the unwound hairpin stretches coordinate one Na⁺. In the Na2 site, the carboxyl group of Asp407, the polar sidechains of Asn401, Ser427 and the backbone carbonyls of Cys398 and Gly403 coordinate the ion. Two ordered water molecules participate in Na⁺binding, one of them suspended between the two Na⁺ ions (Figures 5C and 7A), accounting for the observed cooperativity of Na⁺ transport (Figure 3B). Asn401, which coordinates Na1 with its backbone carbonyl and Na2 via its side chain, may contribute to this effect.

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There is no difference in substrate coordination or in main-chain conformation between the two outward-facing protomers A and A’ (rmsd 0.5 Å). A and A’ can therefore be considered as identical. Interestingly, the main chain conformations of the two inward-facing protomers B and B’ are likewise practically identical (rmsd 0.6 Å), but the citrate and Na⁺ coordination in B and B’ is clearly different. In protomer B, citrate is partially hydrated and coordinated by the hydroxyl of Ser405 and the backbone carbonyl of Gly404 in the conserved GGXG motif of H12 (Figures 1, 5D and 7B). Both Na⁺ sites are occupied and take up the same position relative to the citrate as in the outward-facing state. In protomer B', the Na2 site is empty, even though the structure of the ion-coordinating hairpin hardly changes (Figures 5E and 7C). The citrate is fully hydrated and not directly attached to a sidechain, and a second citrate is present near Gln424 in H7.

The rigid-body movement of the helix bundle from its position in protomers A and A’ to that in protomers B or B' can be described as a 31° arc-like rotation around an axis roughly parallel to the membrane and perpendicular to the long dimer axis (Figures 5, 7 and Video 1). The rotation is facilitated by the greasy interface between the helix bundle and the static dimer contact domain. The greasy interface consists almost entirely of small hydrophobic sidechains and a bound detergent molecule that may take the place of a membrane lipid alkyl chain (Figures 5 and 8 A-D). During the bundle rotation the detergent molecule is displaced by H13. As the helix bundle reaches the inward-facing position, the straight, hydrophobic helix H5 kinks at Gly143, thus preventing its partial exposure to the cytoplasm, and an ion bridge forms between Asp112 and Arg205 in H7 (Figure 8E, F; Video 2). As a result of the helix bundle rotation, the binding site with the bound citrate moves by 16 Å from the external membrane surface in the outward-facing state to a position where it is accessible from the cytoplasmic membrane surface in the inward-facing state (Figure 5). Since transport is non-cooperative with respect to citrate (Figure 3A), we conclude that the two binding sites in the dimer act independently of one another.

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Unlike the Na⁺ ions, the citrate di-anion is not occluded by the hairpin loops in SeCitS. Similarly, the dicarboxylate substrate is not occluded in VcINDY (Mancusso et al., 2012), whereas the corresponding substrate is occluded within the helix bundle of GltPh (Boudker et al., 2007). SeCitS may lack a well-defined substrate-occluded state, but the citrate would effectively be occluded during the transition from the outward-facing to the inward-facing state, while the occupied binding site rotates past the hydrophobic surface of the dimer interface domain (Video 2).

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In the outward-facing state, strong polar and ionic interactions facilitate citrate binding at low ambient substrate concentrations (Figure 3A and 5C). In the inward-facing state, the binding affinity for the substrate is reduced (Pos and Dimroth, 1996), so the citrate can detach. We propose that the three citrate positions we observe in the two inward-facing protomers mark the path of the substrate during its release from the binding site past the highly conserved Arg428 (Figures 1, 5 and 7), along a trajectory that guides the negatively charged substrate towards the cytoplasm, where it is metabolized. Partial release of the citrate di-anion would weaken Na⁺ binding, which explains why only one Na site is occupied in B’. Since transport is electroneutral, both Na⁺ ions must dissociate from the inward-facing state. MD simulations suggest that in other Na-dependent transporters such as LeuT (Grouleff et al., 2015), GltPh (Zomot and Bahar, 2013), vSGLT (Watanabe et al., 2010), at least one of the Na⁺ ions is released before the main substrate. In the case of SeCitS, comparison of the inward-facing protomers B and B’ indicates unambiguously that citrate is released before Na⁺, and that Na2 is released before Na1 (Figure 5D, E). Once the citrate has left the binding site, the helix hairpins or H13 would need to rearrange to release Na1, while a minor reorientation of the Asp407 or Ser427 sidechains is sufficient to release Na2.

Comparison of the binding sites in the outward-facing protomers indicates that both Na⁺ ions have to be in place before citrate can bind. A cryo-EM structure from 2D crystals of the closely related KpCitS from Klebsiella pneumoniae found that sodium citrate induced a major conformational change in the helix bundle, whereas potassium citrate did not (Kebbel et al., 2013), supporting the proposed binding order. Therefore the complete transport mechanism entails the following six steps: (1) The Na sites are occupied by Na⁺ in the outward-facing state; (2) a citrate binds from the external medium; (3) citrate binding triggers the arc-like rotation of the helix bundle in the transition from the outward-facing to the inward-facing state; (4) in the inward-facing state, the citrate becomes hydrated and diffuses into the cytoplasm; (5) the sodium ions come off; (6) the release of all substrates enables the reverse arc-like rotation of the helix bundle to expose the empty binding site again to the cell exterior, and the cycle repeats (Figure 9; Videos 1 and 2).

Figure 9

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Notwithstanding the large domain movements associated with substrate translocation, citrate exchange rates are high, with a turnover of up to 137 s^-1 reported for the closely related KpCitS (Pos and Dimroth, 1996). Citrate uptake by SeCitS is substantially slower at 1.2 molecules per minute (Figure 3). Therefore, the arc-like rotation of the helix bundle that translocates the bound substrate across the membrane is not rate-limiting. The same seems to hold true for GltPh, which shows a slow substrate uptake rate of 0.29 molecules per minute (Ryan et al., 2009), while crosslinking experiments show that the conformational change happens within seconds (Reyes et al., 2009). Assuming that the Na⁺ concentration does not limit substrate binding or release under physiological conditions, the rate-limiting step in SeCitS is most likely the reverse rotation of the helix bundle with the binding site empty.

An influence of lipids on the conformational dynamics in GltPh by inserting a lipid molecule between both domains was recently proposed by MD simulations (Akyuz et al., 2015). The structure of SeCitS offers experimental evidence for the existence of a hydrophobic pocket at the interface of both domains and highlights the importance of the bilayer for the activity of membrane transporters. It remains to be seen whether any of the lipid-binding sites in these transporters are structurally conserved.

The arc-like rotation of the helix hairpins in SeCitS is reminiscent of the recently proposed conformational change for substrate translocation in GltPh (Crisman et al., 2009; Reyes et al., 2009; Verdon et al., 2014). In the Na⁺/H⁺ antiporters (Lee et al., 2013; Paulino et al., 2014) or the bile acid transporter ASBT (Zhou et al., 2014), where the binding site is defined by unwound stretches of two trans-membrane helices in a structurally homologous bundle, this process also involves a rotation of the bundle around a similar axis as in SeCitS, although the movement is significantly smaller. The domain structure of the unrelated transporter YdaH (Bolla et al., 2015) bears a striking resemblance to that of SeCitS, suggesting that it may work in the same way. The rotating arc mechanism described here for SeCitS thus seems to apply to a large class of secondary membrane transporters with unwound helix elements or hairpins that were previously thought to be unrelated.

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Author details

1. David Wöhlert

   Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany

   Contribution

   DW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

2. Maria J Grötzinger

   Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany

   Contribution

   MJG, Acquisition of data, Analysis and interpretation of data

   Competing interests

   No competing interests declared.

3. Werner Kühlbrandt

   Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany

   Contribution

   WK, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   werner.kuehlbrandt@biophys.mpg.de

   Competing interests

   WK: Reviewing editor, eLife.

4. Özkan Yildiz

   Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany

   Contribution

   ÖY, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   Oezkan.Yildiz@biophys.mpg.de

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0003-3659-2805

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