Structural Basis for the Counter-Transport Mechanism of a H+/Ca2+ Exchanger

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Science  12 Jul 2013:
Vol. 341, Issue 6142, pp. 168-172
DOI: 10.1126/science.1239002

Inward-Facing Antiporter

Calcium/cation antiporters play a role in regulating the cytosolic calcium concentration by using the electrochemical gradient of other cations to catalyze Ca2+ transport across cell membranes. The structure of a Na+/Ca2+ exchanger in an outward-facing conformation was recently determined. Nishizawa et al. (p. 168, published online 23 May) now report the crystal structure of a H+/Ca2+ exchanger in an inward-facing conformation. Comparison of the structures shows how structural changes create hydrophilic cavities to alternate between the intra- and extracellular sides of the protein, facilitating cation transport.


Ca2+/cation antiporters catalyze the exchange of Ca2+ with various cations across biological membranes to regulate cytosolic calcium levels. The recently reported structure of a prokaryotic Na+/Ca2+ exchanger (NCX_Mj) revealed its overall architecture in an outward-facing state. Here, we report the crystal structure of a H+/Ca2+ exchanger from Archaeoglobus fulgidus (CAX_Af) in the two representatives of the inward-facing conformation at 2.3 Å resolution. The structures suggested Ca2+ or H+ binds to the cation-binding site mutually exclusively. Structural comparison of CAX_Af with NCX_Mj revealed that the first and sixth transmembrane helices alternately create hydrophilic cavities on the intra- and extracellular sides. The structures and functional analyses provide insight into the mechanism of how the inward- to outward-facing state transition is triggered by the Ca2+ and H+ binding.

Calcium ions are involved in diverse physiological processes, such as muscle contraction, cell proliferation, exocytosis, and apotosis (13). The Ca2+/cation antiporter (CaCA) superfamily members are important regulators of cytosolic Ca2+ levels (46). They use the electrochemical gradient of other cations—such as Na+, H+, or K+—to catalyze Ca2+ transport across biological membranes (46). The CaCA superfamily comprises five major groups: Na+/Ca2+ exchangers (NCX), K+-dependent Na+/Ca2+ exchangers (NCKX), H+/Ca2+ exchangers (CAX), cation/Ca2+ (other cation than Na+, H+, or K+) exchangers (CCX), and the bacterial homologous gene (YrbG) (6, 7). All CaCA proteins contain two highly conserved α-repeat regions (α-1 and α-2), which are thought to have arisen from an ancient gene duplication event (6, 8) and are reportedly important for cation binding and transport (810).

Recently, the crystal structure of the Na+/Ca2+ exchanger from Methanococcus jannaschii (NCX_Mj) in an outward-facing state was reported, revealing a pseudosymmetric architecture formed by two structural repeats of five transmembrane (TM) helices with opposite orientations (10). Three Na+ sites and one Ca2+-specific site were observed within the cation binding pocket, leading to an exchange model in which the sequential binding of three Na+ ions causes the release of Ca2+ during the exchange cycle (10). However, the mechanism by which Ca2+ and the counter-transported cations stimulate the structural transition between the inward- and outward-facing states remains elusive (4, 5, 11) because of the lack of the structural information of its inward-facing state. Furthermore, the molecular basis for the cation recognition in other members of the CaCA superfamily remains unclear. We performed the structural analysis of a CaCA homolog from Archaeoglobus fulgidus (CAX_Af). Liposome- and Escherichia coli–based transport assays confirmed that the homolog has H+/Ca2+ exchange activity (Fig. 1A and figs. S1 to S3). The purified CAX_Af protein was crystallized by the lipidic cubic phase (LCP) method (12) under low-pH (6.0 to 6.5) conditions. The structure was determined with the multiple anomalous diffraction method by using mercury derivatives and refined to 2.3 Å resolution (table S1). The crystallographic asymmetric unit contained two molecules (mol A and mol B) (fig. S4). Because the overall architectures of these two molecules were essentially identical (root mean square deviation of 1.04 Å over all Cα atoms), we focused on the mol A structure.

Fig. 1 The overall structure and function of CAX_Af.

(A) Time courses of 45Ca2+ uptake by liposome-reconstituted CAX_Af at different pH values. (B) Schematic representation of the CAX_Af topology. The core domain and the gating bundle are colored blue and orange, respectively. The additional helices are gray. The H+/Ca2+ binding pockets are indicated by green dotted circles. (C and D) Structure of CAX_Af (mol A) as viewed from (C) the membrane plane or (D) the extracellular side. The color coding is the same as in (B).

The structure of CAX_Af contains 12 TM helices, with both N and C ends located on the intracellular side (Fig. 1, B to D). The core domain (TM2 to TM5 and TM7 to TM10) is tightly packed together, whereas the gating bundle, consisting of the long TM1 and TM6 helices, is loosely packed against the core domain (Fig. 1, C and D). The core domain shares structural similarity with NCX_Mj (10); their N and C terminal halves (TM2 to TM5 and TM7 to TM10, respectively) are related by a pseudo twofold rotational axis within the molecule. The two conserved α-repeats (6) consist of TM2 and TM3 for α-1 and TM7 and TM8 for α-2 (figs. S5 and S6). CAX_Af contains two additional TM helices (N- and C-helices) (Fig. 1, B to D). These helices are not conserved throughout the CaCA family (6), suggesting that they are not involved in the common exchange mechanism.

Although the structure of the core domain of CAX_Af is almost identical to that of NCX_Mj, the conformation of the gating bundle is different (Fig. 2, A and B). The structure of NCX_Mj represents an outward-facing conformation, with its ion binding sites accessible from the extracellular side through an outward-facing cavity (Fig. 2A). TM6 of the gating bundle adopts a straight conformation, which forms the outward-facing cavity with the extracellular halves of TM2 and TM7 (Fig. 2A). TM1 of the gating bundle adopts a bent conformation, and is tightly packed against the intracellular halves of TM2 and TM7 (Fig. 2A). Consequently, the ion binding sites are completely occluded from the intracellular space (10). Relative to the NCX_Mj structure, the conformation of the gating bundle of CAX_Af is symmetrically inverted (Fig. 2B). TM6 of the gating bundle adopts a bent conformation and is closely packed against the extracellular halves of TM2 and TM7, with hydrophobic interactions through Phe227, Leu231, and Leu232 in TM6 (Fig. 2C). TM1 of the gating bundle adopts a straight conformation, creating a cavity surrounded by the intracellular halves of TM1, TM2, TM7, and TM8 (Fig. 2D). Therefore, the present structure of CAX_Af represents an inward-facing conformation.

Fig. 2 Structural comparison of CAX_Af and NCX_Mj.

(A and B) Structures of (A) NCX_Mj and (B) CAX_Af (mol A). The common structures (TM1 to TM10) are shown by coils. TM1, -2, -6 and -7 are shown as cylinders. The cation/Ca2+ binding pocket is indicated by a green dotted circle. The ion permeation pathway is indicated by a light green triangle. The hydrophilic and hydrophobic residues are depicted with yellow and purple stick models, respectively. (C and D) The (C) extracellular and (D) intracellular sides of the H+/Ca2+ binding pocket in CAX_Af. The cytoplasmic ion permeation pathway was identified with the program CAVER (13). Water molecules are represented by red spheres.

A comparison between the inward-facing CAX_Af and outward-facing NCX_Mj suggested that the structural changes of the gating bundle (TM1 and TM6) alternately create hydrophilic cavities suitable for cation permeation on the intra- and extracellular sides. In both CAX_Af and NCX_Mj structures, the intracellular half of TM1 and the extracellular half of TM6 are amphipathic. In the inward-facing structure of CAX_Af, a hydrophilic cluster on the TM1 helix (Ser47, Ser51, Glu55, and Glu58) faces toward the inward-facing cavity (Fig. 2D), rendering the cavity hydrophilic, whereas a hydrophilic cluster on the extracellular side of the TM6 helix (Glu225, Glu229, and Glu233) faces away from the core domain (Fig. 2C). In contrast, in the outward-facing structure of NCX_Mj the hydrophilic cluster on TM6 faces toward the core domain, forming the hydrophilic outward-facing cavity (Fig. 2A). The transition to the outward-facing state may involve a sliding motion of the gating bundle that brings the extracellular hydrophilic cluster of TM1 to the outward-facing cavity while its hydrophobic side seals the inward-facing cavity (movie S1). The second ion passageway in the NCX_Mj structure, which was suggested to be involved in the Na+ permeation (10), does not exist in the CAX_Af structure. These hydrophilic clusters on the gating bundle are conserved among other CaCA proteins as well (fig. S5), suggesting that this alternate formation of the hydrophilic cavities is a common mechanism in the CaCA superfamily.

The Ca2+ binding site of NCX_Mj is formed by Glu and Pro residues (Pro53, Glu54, Pro212, and Glu213), which constitute the signature motifs in the α-repeat sequence (Fig. 3A and fig. S5) (6, 10). The side chain carboxylates of Glu54 and Glu213 coordinate the bound Ca2+ in a bidentate fashion (Fig. 3A). TM2 and TM7 are kinked at Pro53 and Pro212, facilitating further coordination of Ca2+ by the backbone carbonyls of Thr50 and Thr209 (Fig. 3A) (10). In CAX_Af, the Pro and Glu residues of the signature motif are also conserved (Pro77, Glu78, Pro257, and Glu258), with TM2 and TM7 kinked at Pro77 and Pro257 (Fig. 3B and fig. S6). The backbone carbonyls of Ala74 and Ser254 located at these kinks protrude into the space between TM2 and TM7 to coordinate a water molecule together with Ser47, and Glu78 and Glu258 are situated at similar positions to their counterparts in NCX_Mj. This structural similarity suggests that these conserved residues of CAX_Af are also involved in Ca2+ binding (Fig. 3B). However, in the structure of CAX_Af we observed no electron density peak corresponding to Ca2+ (fig. S7), and the side chain carboxylate groups of Glu78 and Glu258 were oriented in different directions (Fig. 3B). Instead of the Ca2+ coordination, Glu78 forms a hydrogen bond network with the side chains of Asn103 and Ser254 and the backbone carbonyl of Val246, located on the extracellular half of the membrane (Hext) (Fig. 3B). The hydrogen bonding patterns and geometries of Hext suggested that the side chain carboxylate of Glu78 is protonated (Fig. 3D). In the middle of the membrane, Glu258 forms another hydrogen bond network with the side chains of Glu255, Ser281, and Gln285 (Hmid) (Fig. 3, B and E). Mutation of Glu255 or Gln285 to alanine greatly decreased the pH-dependent Ca2+ uptake in liposomes (Fig. 3H and fig. S9), indicating the importance of the Hmid network for the H+/Ca2+ exchange activity. Within the Hmid network, the hydrogen bonding patterns and geometries also suggested that the side chain carboxylates of Glu255 and Glu258 are protonated (Fig. 3E). The protonation state assignments of Glu78, Glu255, and Glu258 are consistent with the fact that these residues are embedded in the low dielectric environment of the TM segments, and that the crystal was obtained under low-pH conditions (pH 6.0 to 6.5). The Ala or Gln mutation of either Glu78, Glu255, or Glu258 decreased the H+/Ca2+ exchange activity (Fig. 3H), indicating the importance of their protonation and deprotonation during the exchange cycle. The residues involved in the Hext and Hmid networks (Ser254, Glu255, Ser281, and Gln285) are located at equivalent positions to the Na+ coordinating residues in NCX_Mj (Fig. 3, A and B, and fig. S5). Taken together, the structural comparison with NCX_Mj suggests that the space formed between the kinks of TM2 and TM7 is the Ca2+ binding site (SCa) (Fig. 3B), whereas the hydrogen bonding networks (Hmid and Hext) (Fig. 3B) involving Glu78, Glu255, and Glu258 can function as the H+ binding sites.

Fig. 3 Cation binding pocket.

(A to C) Stereo views of (A) the Na+/Ca2+ binding pocket in NCX_Mj and [(B), mol A; (C), mol B] the H+/Ca2+ binding pocket in CAX_Af. The signature motifs in the α-repeats are highlighted by orange rectangles. In (A), The bound Na+ and Ca2+ ions are depicted with green and red spheres, respectively. In (B) and (C), coordinated water molecules are shown as red spheres. The hydrogen-bond networks, Hext and Hmid, and the putative Ca2+ binding site, SCa, are indicated. (D to G) Schematic drawings of the hydrogen bond geometry in the H+/Ca2+ binding pocket [(D) to (F), mol A; (G) mol B]. (H) Liposome-based 45Ca2+ uptake assays of mutants.

The Glu78 and Glu258 residues are involved in both Ca2+ and H+ binding, further suggesting that the binding of Ca2+ and H+ to these sites is mutually exclusive, which is consistent with the counter-transporting mechanism of the CaCA superfamily (4). The present crystal structure presumably represents the H+ bound state and thus cannot accommodate Ca2+. Supporting this notion, Ca2+ is not bound to SCa, even though the crystal was obtained in the presence of 10 mM CaCl2 (fig. S7).

As described above, the crystallographic asymmetric unit contains two molecules of CAX_Af (mol A and mol B). The mol B structure exhibited different hydrogen bonding patterns in the Hmid network. In mol B, the carboxylate group of Glu258 does not form hydrogen bonds with Ser281 and Glu255 and is exposed toward the inward-facing cavity (Fig. 3C), suggesting that Glu258 is deprotonated (Fig. 3G and fig. S8). As a result, Hmid is partially disrupted in mol B (Fig. 3, C and G). In mol A, the hydrogen bond interactions in Hmid bridge TM7 and TM8, stabilizing the arrangement of TM7 in the core domain (Fig. 3, B and E). In contrast, in mol B the fewer hydrogen bonds in Hmid resulted in the slight twisting of TM7 as compared with that in mol A (Fig. 3C and fig. S10), allowing the accommodation of a monoolein acyl chain in the inward-facing cavity. This twisting of TM7 changes the directions of its side chains; Pro257 is shifted toward the gating bundle (fig. S10). Consequently, there is a large gap between Pro77 (TM2) and Pro257 (TM7) in mol B (Fig. 4B). Altogether, these observations suggested that mol B represents a “partially” protonated state, whereas mol A represents the “fully” protonated state after binding H+. The protonation of Glu258 in Hmid tightens the interaction between TM7 and TM8, which results in the twisting of TM7 toward the H+/Ca2+ binding pocket to close the gap between Pro77 and Pro257 (Fig. 4, A and B, and fig. S10).

Fig. 4 Hydrophobic patch.

(A to C) Hydrophobic patches of (A) CAX_Af Mol A, (B) mol B, and (C) NCX_Mj are shown with green surface-rendered CPK models. (D and E) Structural comparison of (D) CAX_Af (mol A in blue and mol B in magenta) and (E) NCX_Mj. The hydrophobic patches and gating bundles are highlighted. (F) Structural comparison of TM2 and TM7 in CAX_Af mol A (blue), mol B (magenta), and NCX_Mj (yellow). The residues of NCX_Mj are represented in parentheses.

In the structure of NCX_Mj, Pro53 and Pro212 at the kinks of TM2 and TM7 and their neighboring hydrophobic residues, Leu52 and Ile55 (TM2) and Leu211 and Leu214 (TM7), form a flat hydrophobic patch at the center of the interface between the core domain and the gating bundle (Fig. 4, C and E), which probably enables the sliding motion of the gating bundle, which is important for the transition between the inward- and outward-facing states (10). In mol A of CAX_Af, the corresponding hydrophobic patch is formed by Leu76, Pro77, and Ala80 (TM2) and Ala253, Pro257, and Ile260 (TM7) (Fig. 4A). In contrast, in mol B the hydrophobic patch is split by the gap between Pro77 and Pro257 (Fig. 4B). Thus, the H+ binding to CAX_Af closes the gap in the hydrophobic patch and enables the gating bundle to slide to the outward-facing state. Consistent with this mechanism, the gating bundle in mol A is slightly shifted toward the outward-facing state (Fig. 4D).

A structural comparison of the Ca2+ binding sites between CAX_Af and NCX_Mj suggested that Ca2+ binding induced conformational changes in TM2 and TM7. In the structure of NCX_Mj, the distance between the backbone carbonyls at the kinks of TM2 and TM7 (Thr50 and Thr209) (Fig. 4, C and F) that coordinate Ca2+ is 4.1 Å (Fig. 4C). In contrast, in mol B of CAX_Af the distance between the corresponding carbonyl oxygen atoms is 5.6 Å (Fig. 4B). Consequently, TM2 and TM7 of CAX_Af are twisted toward the gating bundle, as compared with those of NCX_Mj (Fig. 4F and fig. S11). The twisting of TM7 is more substantial than that of TM2 (fig. S11B). The structural comparison suggests that Ca2+ binding to CAX_Af induces the twisting of TM2 and TM7 toward the H+/Ca2+ binding pocket (Fig. 4F). This twisting changes the directions of the backbone carbonyls at the kink of TM2 and TM7 and also the side chains of Glu78 and Glu258, enabling optimal coordination geometry for the Ca2+ as observed in NCX_Mj (Fig. 4F and fig. S11B). Thus, similar to the case of H+ binding, Ca2+ binding brings Pro77 and Pro257 close together (Fig. 4F and fig. S11). Last, this closing the gap enables the sliding of the gating bundle.

The crystal structure of CAX_Af revealed the inward-open conformation of the CaCA protein, which revealed the alternate formation mechanism of the hydrophilic cavities on the intracellular and extracellular sides. With the concomitant functional analyses, the present structural analysis suggested how the binding of either Ca2+ or H+ induces the structural change (fig. S12): The gap between the TM2 and TM7 fixes the gating bundle in the inward-facing apo structure to prevent ion leakage across the membrane (4), whereas the binding of either Ca2+ or H+ induces the gap closure, which enables the sliding motion of the gating bundle, as predicted from the NCX_Mj structure (10). Further discussion of the structural change mechanism may require the elucidation of the CAX_Af structure in the Ca2+ bound state.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S14

Tables S1 to S2

References (1424)

Movie S1

References and Notes

  1. Acknowledgments: We thank H. Nishimasu and M. Hattori for useful discussions and critical comments on the manuscript; T. Tsukazaki for sharing beamtime and useful discussion; A. Kurabayashi for technical assistance; the beam-line staffs at BL32XU and BL41XU of SPring-8 for assistance in data collection; and the RIKEN BioResource Center (Ibaraki, Japan) for providing the Archaeoglobus fulgidus genomic DNA. The diffraction experiments were performed at SPring-8 BL32XU and BL41XU (proposals 2012A1093, 2012A1201, and 2012A1087) and with the approval of RIKEN. Part of this work was performed with the support of the Radioisotope Center, University of Tokyo. This work was supported by the Japan Society for the Promotion of Science (JSPS) through its “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST program)” to O.N., by the Core Research for Evolutional Science and Technology (CREST) Program “The Creation of Basic Medical Technologies to Clarify and Control the Mechanisms Underlying Chronic Inflammation” of Japan Science and Technology Agency (JST) to O.N., by a grant for HPCI STRATEGIC PROGRAM Computational Life Science and Application in Drug Discovery and Medical Development by MEXT to R.I., by a Grant-in-Aid for Scientific Research on Innovative Areas (23136517) from Ministry of Education, Culture, Sports, Science and Technology (MEXT) to S.K., by a Grant-in-Aid for Scientific Research (C) (23590319) from JSPS to T.I., and by a Grant-in-Aid for Scientific Research (S) (24227004) and a Grant-in-Aid for Young Scientists (A) (22687007) from the MEXT to O.N. and R.I., respectively. T.N. expressed and purified CAX_Af for crystallization, collected the diffraction data, solved the structures, and performed functional analyses in liposomes. N.F. screened CaCA genes and identified CAX_Af. S.K. and T.I. performed transport assays in E. coli cells. A.D.M. performed transport assays in E. coli spheloplasts. G.K. made mutants. K.H. assisted with data collection. S.O. supported crystallization. N.D. analyzed the purified protein by mass spectrometry. T.N., R.I., and O.N. wrote the manuscript. R.I. and O.N. directed and supervised all of the research. The coordinates and the structure factors have been deposited in the Protein Data Bank (PDB) under accession codes 4KPP.
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