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Structural basis for human claudin‐9 ion selectivity and tight junction dissociation by a bacterial toxin
Author(s) -
Vecchio Alex J,
Stroud Robert M
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.656.14
Subject(s) - claudin , tight junction , extracellular , microbiology and biotechnology , epithelium , enterotoxin , chemistry , intracellular , cell junction , biology , biophysics , biochemistry , cell , gene , genetics , escherichia coli
Tight junctions are macromolecular polymers involved in cell−cell adhesion and intercellular transport of solutes through epithelial or endothelial cell sheets. Claudins, a family of integral plasma membrane proteins with 27 isoforms in humans, have conserved topologies but diverge in their primary sequences and functions. Being the major structural components of tight junctions, claudins self‐associate on neighboring cells in adhesion and simultaneously form size and charge selective pores that permeate ions vital for epithelial tissue homeostasis. Clostridium perfringens enterotoxin (CpE) can disrupt gastrointestinal balance by targeting then binding claudins and subsequently dissociating tight junctions in gut epithelium. Foodborne illness from CpE is the second most common form in developed countries, with one million annual cases in the United States. The C‐terminal claudin‐binding domain of CpE (cCpE) is being investigated as a molecular probe for detection and targeted destruction of cancer cells, a tool for tuning blood‐brain barrier permeability in drug delivery, and as a competitive inhibitor to CpE‐type food poisoning. Claudin‐9 functions in reducing cation permeability in inner ear epithelium and is essential for hearing; acts as an entry co‐receptor for hepatitis C virus (HCV); and despite not being present in the gut in high abundance, an extracellular peptide of claudin‐9 can bind CpE in vitro . To better understand the structural basis for claudin‐9 anion selectivity, HCV entry, and CpE recognition, we determined structures of human claudin‐9 (hCLDN‐9) in complex with cCpE using X‐ray crystallography. We next used bio‐layer interferometry to measure hCLDN‐9:toxin kinetics and affinities, then bright‐field microscopy and cell‐based assays to visualize and measure the cytotoxic effects of CpE on hCLDN‐9‐expressing cells. Results indicate hCLDN‐9 is a high‐affinity receptor for CpE, and that cells expressing hCLDN‐9 display morphological damage and undergo cell death when treated with CpE but not cCpE, which lacks a cytotoxic essential domain. We show by structural analysis that cCpE binds both claudin extracellular segments and uses polar and non‐polar interactions to enable binding to hCLDN‐9, and side chains used by HCV for viral entry are focused at a single region of hCLDN‐9. Through combined sequence and homology‐modeling studies we hypothesize a nonconserved motif in one extracellular segment is essential for high‐affinity CpE binding to claudins and predict amino acids that form the hCLDN‐9 anion‐selective pore. Taken together, these findings elucidate a molecular basis for claudin‐9 selective ion permeability and binding to CpE, providing a mechanism for how CpE disrupts gastrointestinal homeostasis by dissociating tight junctions to affect epithelial adhesion and cellular transport. Support or Funding Information R01 GM024485This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .