Claudin-9 structures reveal mechanism for toxin-induced gut barrier breakdown

Abstract

The human pathogenic bacterium Clostridium perfringens secretes an enterotoxin (CpE) that targets claudins through its C-terminal receptor-binding domain (cCpE). Isoform-specific binding by CpE causes dissociation of claudins and tight junctions (TJs), resulting in cytotoxicity and breakdown of the gut epithelial barrier. Here, we present crystal structures of human claudin-9 (hCLDN-9) in complex with cCpE at 3.2 and 3.3 Å. We show that hCLDN-9 is a high-affinity CpE receptor and that hCLDN-9-expressing cells undergo cell death when treated with CpE but not cCpE, which lacks its cytotoxic domain. Structures reveal cCpE-induced alterations to 2 epitopes known to enable claudin self-assembly and expose high-affinity interactions between hCLDN-9 and cCpE that explain isoform-specific recognition. These findings elucidate the molecular bases for hCLDN-9 selective ion permeability and binding by CpE, and provide mechanisms for how CpE disrupts gut homeostasis by dissociating claudins and TJs to affect epithelial adhesion and intercellular transport.

Keywords: X-ray structure; claudin; membrane protein; tight junction; toxin.

Fig. 1.

Structures of TJs and hCLDN-9. (A) Schematic diagram of epithelial TJs with claudins (red), TJ-associated MARVEL proteins (TAMPs; purple), scaffolding proteins (various), and actin (black). Paracellular transport is shown as a transparent arrow (black). (B) hCLDN-9 closed (blue) and open (red) in complex with cCpE, with cCpE omitted for clarity, viewed parallel to the membrane. Bars (gray) are approximate membrane boundaries. ICS, intracellular segment. (C) hCLDN-9 closed sites of ion selectivity (orange), the F35 site of deafness mutations (purple), and disulfide bonds (yellow) shown as sticks with dot surfaces. (D) hCLDN-9 closed HCV entry sites (brown) and the NPLVA¹⁵³ motif (green) that binds CpE are shown as in C.

Fig. 2.

hCLDN-9 in closed and open complex with cCpE reveals different conformations of toxin. (A) hCLDN-9 (blue) and cCpE (copper) closed form. (B) hCLDN-9 (red) and cCpE (gray) open form. (C) Superposition of hCLDN-9 closed and open structures colored as in A and B. The cCpE undergoes a 15° movement from closed to open. (D) Solvent-accessible surface volumes (Vol.) and areas between hCLDN-9 and cCpE in the closed and open forms calculated by CASTp (46). Cavity imprints are shown as translucent blobs for hCLDN-9 closed (blue) and open (red).

Fig. 3.

Cytotoxicity and binding affinity of toxins to hCLDN-9. (A) Morphology of baculovirus-transduced Sf9 cells treated with purified cCpE or CpE and expressing hCLDN-9 (blue), hCLDN-19 (orange), or nonclaudin protein (PMP, green). Sf9 cells with no baculovirus (control, black) were treated identically. (B) Average viability with SD of Sf9 cells from A. (C) cCpE (blue trace) and CpE (black trace) binding to hCLDN-9 shown with overlaid fits (red). k_off, dissociation rate constant; k_on, association rate constant.

Fig. 4.

Model of the effect of cCpE on claudin cis interactions. cCpE-bound hCLDN-9 closed (blue) and unbound mCLDN-15 (SI Appendix, Fig. S15A) were manually docked based on the technique of Zhao et al. (44). (A) Full view of homodimeric cis interacting hCLDN-9. Residues purportedly involved in cis interactions are labeled and colored accordingly. ECH1 and ECH2 are highlighted with rectangles (blue). (B) Zoomed-in view of the cis interacting epitope of mCLDN-15 (brown). Polar (red) and nonpolar (black) interactions between protomers are shown as dashed lines. (C) Zoomed-in view of the cis interacting epitope of hCLDN-9 closed. One polar bond is shown as in B. Note disruptions to ECH1, ECH2, and the NPLVA¹⁵³ motif between C and B.