Morphologic determinant of tight junctions revealed by claudin-3 structures

Abstract

Tight junction is a cell adhesion apparatus functioning as barrier and/or channel in the paracellular spaces of epithelia. Claudin is the major component of tight junction and polymerizes to form tight junction strands with various morphologies that may correlate with their functions. Here we present the crystal structure of mammalian claudin-3 at 3.6 Å resolution. The third transmembrane helix of claudin-3 is clearly bent compared with that of other subtypes. Structural analysis of additional two mutants with a single mutation representing other subtypes in the third helix indicates that this helix takes a bent or straight structure depending on the residue. The presence or absence of the helix bending changes the positions of residues related to claudin-claudin interactions and affects the morphology and adhesiveness of the tight junction strands. These results evoke a model for tight junction strand formation with different morphologies - straight or curvy strands - observed in native epithelia.

Fig. 1

Structure of mCldn3 in complex with C-CPE. a Overall structure of the mCldn3_cryst/C-CPE complex in ribbon representation viewed parallel to the cell membrane. mCldn3_cryst in rainbow colors from the N terminus (blue) to the C-terminus (red). C-CPE is colored magenta. Gray bars suggest boundaries of outer (Ext.) and inner (Int.) leaflets of the lipid bilayer. b Left-hand model of the mCldn3 structure

Fig. 2

Comparison with structure-determined subtypes. a Structure-determined Cldn subtypes. mCldn3_cryst, hCldn4, mCldn19, and mCldn15 are colored blue, green, yellow, and orange, respectively. C-CPEs paired with mCldn3_cryst, hCldn4, and mCldn19 are omitted for clear display of the Cldns. Pro134 of mCldn3 is colored cyan. b Superposition of mCldn3_cryst and the other structure-determined subtypes

Fig. 3

Structures of Pro134 mutants. a Superposition of mCldn3_cryst (blue), mCldn3_cryst P134G (violet), and mCldn3_cryst P134A (red). C-CPEs paired with mCldn3_cryst, mCldn3_cryst P134G, and mCldn3_cryst P134A are omitted. b Determination of the TM3 structure with proline, glycine, or alanine. TM3 is bent in the case of proline (blue). TM3 is straight in the case of glycine, with probable swaying (violet). TM3 is straight in the case of alanine (red). c TM3 thenar in the left-hand model. The TM3 thenar is highlighted by the colored arc. When the TM3 thenar is proline, the bent TM3 makes the hand of the ECD lean toward the back of the hand (left). When the TM3 thenar is alanine, the straightened TM3 leads the hand of the ECD to align with the forearm of the TMD (right)

Fig. 4

Structural shift by TM3 bending. a Superposition of mCldn3_cryst and mCldn3_cryst P134A. The Cα strands of mCldn3_cryst and mCldn3_cryst P134A are colored blue and red, respectively. Distances arising from the TM3 bending are shown in green. b Enlarged view of the cis-interaction pocket periphery. The residues constituting a cis-interaction pocket are shown in stick representation. Double-headed arrows indicate the size of the cis-interaction pocket in the direction perpendicular to the cell membrane plane. c Cis-interaction observed in mCldn15 crystal (PDB ID: 4p79). Two neighboring molecules of mCldn15 are shown in ribbon representation, and key residues in the cis-interaction are shown in stick representation

Fig. 5

Morphology and adhesivity of TJ strands formed by mCldn3 with a different TM3 structure. a Adhesive property of TJs formed by a stable SF7-derived cell line expressing mCldn3. Dissociation assay showed that TJs formed by mCldn3 P134G or P134A had stronger adhesivity than TJs of mCldn3 WT (n = 18/group). Results from three distinct clones are shown as means ± SEM. P-values were calculated using a two-tailed independent t-test, and P < 0.05 was considered significant. n.s., not significant; ***p < 0.001. b–d Representative electron microscopic images of freeze-fracture replicas of TJ strands of mCldn3. b TJ strands consisting of wild-type mCldn3 appear as straight strands (arrow). c TJ strands consisting of mCldn3 P134G appear as curvy strands with many hairpin curves (arrowhead). d TJ strands consisting of mCldn3 P134A appear as curvy strands with many hairpin curves. PF = protoplasmic face. EF = exoplasmic face. Scale bar = 500 nm. Source data are provided as a Source Data file (a)

Fig. 6

Mechanism for determining the flexibility of TJ strands by TM3 bending. a Proline renders TM3 bent, as seen in the mCldn3_cryst structure, with the formation of a narrow cis-interaction pocket. The narrow pocket would restrict the orientation of the cis-interaction, resulting in linear TJ strands having little flexibility. b The alanine or glycine renders TM3 straight, as seen in mCldn3_cryst P134A or P134G structure, with the formation of a wide cis-interaction pocket. The wide pocket would enable wide orientation range, resulting in curvy TJ strands with high flexibility. Hydrophobic interactions in cis-interaction are represented in green. TJ strands are depicted according to the anti-parallel double-row model