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J. Biol. Chem., Vol. 283, Issue 1, 268-274, January 4, 2008

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Structure of the Claudin-binding Domain of Clostridium perfringens Enterotoxin^*

Christina M. Van Itallie¹, Laurie Betts¹, James G. Smedley, III^¶, Bruce A. McClane^¶, and James M. Anderson^||2

From the Departments of Medicine, Pharmacology, and ^||Cell and Molecular Physiology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599 and the ^¶Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Clostridium perfringens enterotoxin is a common cause of food-borne and antibiotic-associated diarrhea. The toxin's receptors on intestinal epithelial cells include claudin-3 and -4, members of a large family of tight junction proteins. Toxin-induced cytolytic pore formation requires residues in the NH₂-terminal half, whereas residues near the COOH terminus are required for binding to claudins. The claudin-binding COOH-terminal domain is not toxic and is currently under investigation as a potential drug absorption enhancer. Because claudin-4 is overexpressed on some human cancers, the toxin is also being investigated for targeting chemotherapy. Our aim was to solve the structure of the claudin-binding domain to advance its therapeutic applications. The structure of a 14-kDa fragment containing residues 194 to the native COOH terminus at position 319 was solved by x-ray diffraction to a resolution of 1.75Å. The structure is a nine-strand β sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, including the collagen-binding domain of ColG from Clostridium histolyticum and the large Cry family of toxins (including Cry4Ba) of Bacillus thuringiensis. Correlations with previous studies suggest that the claudin-4 binding site is on a large surface loop between strands β8 and β9 or includes these strands. The sequence that was crystallized (residues 194-319) binds to purified human claudin-4 with a 1:1 stoichiometry and affinity in the submicromolar range similar to that observed for binding of native toxin to cells. Our results provide a structural framework to advance therapeutic applications of the toxin and suggest a common ancestor for several receptor-binding domains of bacterial toxins.

Received for publication, September 27, 2007 , and in revised form, October 16, 2007.

The atomic coordinates and structure factors (code 2QUO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from the National Institutes of Health, including a Pilot Feasibility project from P30 DK 034987 (to C. M. V. I.), R01 DK45134 (to J. M. A.), and R37 AI19844 (to B. A. M.).

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Cell and Molecular Physiology, University of North Carolina at Chapel Hill, 6314 MBRB, CB#7545, 103 Mason Farm Rd., Chapel Hill, NC 27599-7545. Fax: 919-966-6413; E-mail: jandersn{at}med.unc.edu.

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