Crystal Structure of a Claudin Provides Insight into the Architecture of Tight Junctions

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Science  18 Apr 2014:
Vol. 344, Issue 6181, pp. 304-307
DOI: 10.1126/science.1248571

How Tight?

In metazoans, sheets of epithelial cells separate different tissue spaces and control their composition. Tight junctions are cell-cell adhesion structures in these cell sheets that form a seal between cells but also provide some selective permeability to ions and small molecules. Claudins are the main constituents of tight junctions, and mutations in claudins cause inherited human disorders involving the disruption of ionic balance. Suzuki et al. (p. 304) report the structure of mouse claudin-15 at 2.4 angstrom resolution, which shows an extracellular β-sheet domain anchored to a transmembrane four-helix bundle. The electrostatic distribution on the claudin surface reveals a negatively charged groove in the extracellular domain that may provide a pathway for positive ions.


Tight junctions are cell-cell adhesion structures in epithelial cell sheets that surround organ compartments in multicellular organisms and regulate the permeation of ions through the intercellular space. Claudins are the major constituents of tight junctions and form strands that mediate cell adhesion and function as paracellular barriers. We report the structure of mammalian claudin-15 at a resolution of 2.4 angstroms. The structure reveals a characteristic β-sheet fold comprising two extracellular segments, which is anchored to a transmembrane four-helix bundle by a consensus motif. Our analyses suggest potential paracellular pathways with distinctive charges on the extracellular surface, providing insight into the molecular basis of ion homeostasis across tight junctions.

Multicellular organisms comprise many organ compartments that are separated from their external environments by epithelial cell sheets. The movement of ions and solutes through the epithelia is regulated by the barrier function of the sheets, and its proper regulation is important for homeostasis and organism survival (1). Ions are transported through the epithelial sheet by two routes: the transcellular pathway through membrane transporters and channels, and the paracellular pathway through the intercellular spaces. Ion transport through the paracellular pathway is restricted by an intercellular seal that is formed by specific junctional complexes, termed tight junctions (TJs), which circumscribe the apical-most part of the epithelial cells and completely occlude the intercellular space between the plasma membranes of adjacent cells (2). Physiological studies indicate that TJs not only serve as barriers, but also provide paracellular channels that are selectively permeable to ions and small molecules in a charge- and size-specific manner (3, 4).

TJs are multimolecular complexes in which various transmembrane and cytoplasmic proteins constitute a network of continuous intramembrane particle strands or fibrils, called TJ strands (5, 6). The backbone of the TJ strands is formed by a group of membrane proteins called claudins (79), a multigene family in humans comprising 27 members with tissue-specific expression patterns (10). Mutations in claudin genes cause many inherited human disorders involving the disruption of ionic balance in body compartments, such as familial hypomagnesemia (11), neonatal sclerosing cholangitis (12), and autosomal recessive deafness (13). Claudin-1 also plays a role in hepatitis C virus entry (14) and is thus a potential pharmaceutical target. Members of the claudin family share a structural topology of four putative transmembrane (TM) segments, a large extracellular loop containing a consensus sequence motif, and a second shorter extracellular loop (15). Claudins facilitate cell adhesion by head-to-head interactions between claudins in adjacent cell membranes and polymerize by side-by-side interactions within the same cell membrane. Mutational and chimeric analyses indicate that the two extracellular loops are crucial for the formation of paracellular barriers and pores for solutes and are thought to determine the permeability characteristics of TJs (16, 17). Although high-resolution structures have been determined for various membrane channels involved in transmembrane ion and solute transport (1820), structural information on claudins has remained elusive.

To gain insight into the TJ architecture, we expressed various claudin subtypes in Sf9 insect cells and assessed their capacity to form TJ-like strands (21). Mouse claudin-15 (mCldn15) could be expressed in large quantities and formed prominent TJ-like strands in the plasma membrane in contact regions between adjacent cells (fig. S1). For crystallization, 33 residues at the C terminus were truncated, and the membrane-proximal cysteines were substituted with alanines to avoid heterogeneous palmitoyl modification. The modified mCldn15 construct was expressed, purified with maltose neopentyl glycol detergent, and crystallized in lipidic cubic phase (LCP). High-resolution diffraction data were obtained from the crystals with a microfocused x-ray beam (22), and the structure was determined at 2.4 Å resolution using selenomethionine (SeMet)–labeled derivatives by the multiple anomalous dispersion (MAD) method (table S1 and fig. S2, A and B). Crystals of mCldn15 belong to the C2 space group and contain one claudin monomer per asymmetric unit (fig. S3, A and B).

The TM segments of mCldn15 (TM1 to TM4) form a typical left-handed four-helix bundle, and large portions of the two extracellular segments form a prominent β-sheet structure (Fig. 1, A to C). Except for the longer TM3, the length of the TM helices is consistent with the thickness of a lipid bilayer. The TM region contains many residues with small side chains, such as Gly and Ala, ensuring tight packing of the helices. Mutations at these residues might cause human disease (11, 13) by destabilizing the helical bundle (fig. S4). The β-sheet domain extends from the membrane surface and comprises five β strands (β1 to β5), four contributed by the first extracellular segment (ECS1) and one by the second extracellular segment (ECS2). The loop region (residues 34 to 41) between β1 and β2 is structurally disordered (Fig. 1C and Fig. 2A). At the end of ECS1, the β4 strand connects to a short extracellular helix (ECH) that connects to TM2 after a sharp bend. The cytoplasmic loop (ICL) between TM2 and TM3 is clearly resolved, but the cytoplasmic C-terminal region is disordered. TM3 extends from the extracellular membrane surface and the loop that connects to TM4 forms a β strand that is part of the β-sheet domain. The five β strands adopt an antiparallel arrangement and gradually twist along the axis parallel to the membrane plane. The β-sheet structure is further stabilized by a disulfide bond between cysteine residues at β3 and β4 (Cys52 and Cys62, respectively; Fig. 2A). The two cysteine residues are conserved among all members of the claudin family as part of a consensus W-GLW-C-C motif, in which the Gly is substituted with Asn in mammalian claudin-10b and claudin-15 (figs. S5 and S6). It has been suggested that an intramolecular disulfide bridge between these cysteines is necessary for proper claudin function (4, 23).

Fig. 1 Overall structure of the mCldn15 protomer.

(A and B) Structure of monomeric mCldn15 in ribbon representation viewed parallel to the membrane. The color changes gradually from the N terminus (blue) to the C terminus (red). Gray bars suggest boundaries of outer (Ext.) and inner (Cyt.) leaflets of the lipid bilayer. (C) Secondary structure diagram of mCldn15. The orange line represents a disulfide bond; dashed lines indicate disordered regions. An arrowhead indicates the truncation site for crystallization.

Fig. 2 Residues conserved in claudin family members.

(A) Close-up view of the extracellular domains and hydrophobic anchors. Side chains of residues in the consensus sequences are labeled and shown in stick representation. (B) Close-up view around conserved residue Arg79. Main-chain carbonyl groups and side chains involved in interactions with Arg79 are shown in stick representation. (C) Close-up view of ECS2. Side chains in the loop region are shown in stick representation. Red dashed lines indicate hydrogen bonds. Amino acid abbreviations: C, Cys; F, Phe; K, Lys; L, Leu; N, Asn; P, Pro; R, Arg; W, Trp.

The residues of the conserved W-LW sequence (Trp29, Leu48, and Trp49) are located adjacent to each other close to the extracellular membrane surface (Fig. 2A) and are embedded in a crevice formed by the top of the four-helix bundle (Fig. 1B). The first Trp residue in the motif, Trp29, extends from β1 into the helix bundle, whereas Leu48 and Trp49 protrude from the tip of the β2-β3 loop and appear to be exposed to the lipid environment. An electron density that could be assigned as a monoolein lipid was located near Trp49 (fig. S7A). These motif residues are likely associated with the extracellular membrane surface to serve as a “hydrophobic anchor” for the β-sheet domain. This is supported by the low temperature factors of the atoms in the membrane-proximal region of the β-sheet domain and the gradual increase in the values toward the open edge (fig. S8). The β-sheet domain and the following ECH region are further stabilized by the conserved Arg79 residue that extends from the intramembrane part of TM2 (Fig. 2B). The guanidinium group of Arg79 forms hydrogen bonds with the main-chain carbonyl groups of Leu48 and Phe65, and the ECH is held over the membrane plane, running almost perpendicular to TM2, to which it connects through the Ser72-Gly73 hinge residues (fig. S7B). Although these two residues are not conserved, the corresponding hinge regions often contain a glycine or proline residue (fig. S5). The kink between the ECH and the top of TM2 is thus likely conserved in the claudin family. Homology modeling of other family members indicates that the conformation of the four-helix bundle and ECH regions is well conserved, although there are slight structural variations in the distal edges of the β sheet in ECS1 and the loop region in ECS2 (fig. S9A).

The ECS2 loop structure starts with a highly conserved proline residue (Pro149) that forms a hydrogen bond between its main-chain carbonyl and a conserved positively charged residue (Lys155 in mCldn15; Fig. 2C). The loop region in ECS2 forms an unstructured turn that connects to the β5 strand, which is part of the β-sheet domain. Such a lariat-like conformation should be relatively flexible and thus useful to keep the less-conserved residues in the loop exposed to the extracellular space. The sequence between the β1 and β2 strands in ECS1 is similarly diverse, and it includes the disordered loop in our structure. Therefore, we defined the loop regions with poorly conserved sequences as variable regions V1 and V2 in ECS1 and ECS2, respectively (figs. S5 and S9, B and C). The V1 and V2 regions located at the extracellular half could be involved in homotypic and heterotypic head-to-head interactions with subtype-specific compatibility.

In the crystal lattice, the mCldn15 protomers form a linear polymer (Fig. 3, A and B, and figs. S3 and S10). The tandem intermolecular interactions in this direction are mediated by specific regions between adjacent extracellular domains. In particular, a conserved hydrophobic residue (Met68 in mCldn15) protrudes from ECH in one protomer and snugly fits into a hydrophobic pocket formed by conserved residues in TM3 and ECS2 (Phe146, Phe147, and Leu158) of the adjacent protomer (Fig. 3C). The two contact surfaces have complementary electrostatic potentials and are on opposite sides of the protomer (Fig. 3, D and E), allowing for the formation of a linear polymer. Freeze-fracture electron microscopy of cells expressing claudin-15 showed TJ-like strands in the plasma membranes, which were not observed when the key residues (Met68, Phe146, and Phe147) were mutated to charged or small residues (fig. S11, A to C), whereas constructs with mutations of Met68 to bulky hydrophobic residues retained the ability to form TJ-like strands (fig. S11, D and E). These results support the idea that the linear alignment of mCldn15 protomers observed in the LCP crystals might be representative of linear claudin polymers that form in TJs.

Fig. 3 Linear alignment of mCldn15 protomers.

(A and B) Ribbon representation of a single row of mCldn15 protomers aligned along the b axis of the crystal, viewed from the extracellular side (A) and parallel to the membrane (B). One protomer is colored pink. (C) Close-up view of lateral interaction sites. Critical residues for the interaction are shown in stick representation (E, Glu; M, Met). (D and E) Electrostatic potential surfaces of the lateral interfaces of the pink protomer (D) and green protomer (E) shown in (C), contoured from –2 kT/e (red) to +2 kT/e (blue), viewed parallel to the b axis. Dashed circles indicate the complementary hydrophobic protrusion and pocket.

Depending on the claudin subtypes that constitute the TJ strands, TJs either form a strict barrier against paracellular diffusion of solutes or include paracellular ion channels with distinct charge selectivity (4). The charge selectivity for ion permeation through TJs is determined by specific residues in the C-terminal half of the first extracellular domain (17, 24, 25). In the structure of mCldn15, a typical channel-forming claudin (26, 27), all of the charged residues extend away from the β-sheet surface (Fig. 4A). The negatively charged residues Asp55 and Asp64 (Glu64 in human claudin-15), mutations of which reverse the ion charge selectivity (28), locate to one edge of the β-sheet domain. In addition to these residues, Trp63 in mCldn15 is at the position in which claudin-2 and claudin-4 have essential charged residues for their TJ functions (28, 29). The distribution of the electrostatic potential at the extracellular surface of mCldn15 shows that half of the distal part of the β-sheet domain forms a negatively charged “palm” (Fig. 4B). Homology models indicate that the cation-selective TJ protein claudin-2 has a similarly negatively charged palm surface, whereas the anion-selective TJ protein claudin-10a has a positively charged surface (Fig. 4, C and D, and fig. S9A). Thus, the groove in the palm region between the β3-β4 strands and ECH likely lines the paracellular ion pathway, thereby contributing to its ion selectivity.

Fig. 4 Charge distribution of the extracellular surface.

(A) Ribbon diagram and surface representation (transparent) of the extracellular domains. Charged residues and Trp63 in ECS1 are shown in stick representation and labeled (D, Asp). Dashed circles indicate the positions of residues that affect the charge-selective properties of claudins. (B) Electrostatic potential surface of the extracellular domains viewed as in (A), contoured from –2 kT/e (red) to +2 kT/e (blue). Dashed oval indicates the β3-β4 region, where residues important for charge selectivity are located. (C and D) Mapping of the electrostatic potential surfaces generated from the homology models of different claudin subtypes [claudin-2 in (C); claudin-10a in (D)], viewed as in (B).

Further studies are needed to unveil how claudins assemble into strand-like polymers to restrict the intercellular spaces and how the charged residues in the extracellular surfaces line the paracellular pathways. Our crystal structure of a claudin protein, a building block of TJ strands, will advance our molecular understanding of paracellular barriers between epithelial cells in multicellular organisms.

Supplementary Materials

Materials and Methods

Table S1

Figs. S1 to S11

References (3047)

References and Notes

  1. See supplementary materials on Science Online.
  2. Acknowledgments: We thank M. Uji for technical support, T. Suzuki for mass spectrometry, T. Imasaki and Y. Takagi for sharing their unpublished protocol for SeMet incorporation and their technical advice, the beamline staff members at BL32XU of SPring-8 (Hyogo, Japan) for technical help during data collection, and T. Walz for critical reading of the manuscript. Supported by Grants-in-Aid for Scientific Research (S) (22227004, Y.F.; 24227004, O.N.) and (A) (S.T.), Grants-in-Aid for Scientific Research on Innovative Areas (S.T.), Grants-in-Aid for Young Scientists (B) (K.T.), and Platform for Drug Discovery, Information, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and the Japan New Energy and Industrial Technology Development Organization (NEDO) and the National Institute of Biomedical Innovation (Y.F.). H.S., Y.Y., and A.T. screened claudin genes; H.S. performed protein expression, purification, and electron microscopic studies; T.N. crystallized the mCldn15 protein in lipidic cubic phase, collected and processed diffraction data, and solved and refined the structure; K.T. analyzed the structure; R.I. assisted with x-ray data collection and structure determination; N.D. performed mass spectrometric analyses; H.S., T.N., K.T., S.T., O.N., and Y.F. wrote the manuscript; and S.T., O.N., and Y.F. supervised the research. The authors declare no competing financial interests. Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 4P79.
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