Crystal Structure of Clostridium perfringens Enterotoxin Displays Features of β-Pore-forming Toxins

Abstract

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å . The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of ␤-pore-forming toxins such as aerolysin, C. perfringens ⑀-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of ␤-strands, two of which span its entire length. Domain II and domain III have three short ␤-strands each, by which they are distinguished. In addition, domain II has an ␣-helix lying on the ␤-strands. The sequence of amino acids composing the ␣-helix and preceding ␤-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming ␤-barrel-made pores. These structural features imply that CPE is a ␤-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long ␤-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins. Clostridium perfringens enterotoxin (CPE),2 which damages intestinal epithelia, is a causative agent of food poisoning. The toxin consists of a single chain polypeptide of 319 amino acids. The C-terminal domain of CPE (C-CPE, residues 184 -319) recognizes and binds to certain members of the claudin family, components of tight junctions, as a receptor on target cells (1-4), and the N-terminal region is believed to be involved in forming physiological pores to disrupt the selective permeability of the plasma membrane, resulting in cell death (5-7). It was also reported that the physiological pores are composed of a large complex comprising CPE and cellular components such as claudins (2,8).The binding between C-CPE and claudins has been well characterized. The 16 -17 C-terminal amino acids of C-CPE were reportedly important for the interaction (9, 10), especially, Tyr 306 , Tyr 310 , Tyr 312, and Leu 315 (11-13). According to the crystal structure of C-CPE (14), these residues organize a cleft space that is considered to interact directly with claudins. Claudins are tetratransmembrane proteins. The region of claudins responsible for binding to CPE was located on the C-terminal side of the second extracellular loop and recently designated CPE-SR for CPE sensitivity-related region (15). The bottom of the cleft space of CPE is negatively charged, whereas the CPESRs of CPE-sensitive claudins are positively charged. Therefore, it was proposed that electrostatic attraction at...

Cysteine-Scanning Mutagenesis Supports the Importance of Clostridium perfringens Enterotoxin Amino Acids 80 to 106 for Membrane Insertion and Pore Formation

“…The 50% 86 Rb release values obtained for the F87C, Q97C, and G109C variants were only slightly lower (Յϳ2 g/ml) than the 50% 86 Rb release values measured for either the C186A variant or rCPE (Fig. 3B), confirming the initial screening results indicating that these three rCPE Cys variants retained a C186-like ability to cause 86 Rb re- Analysis of the CPE primary sequence predicted that the TM1 region between amino acids 90 and 106 comprises a ␤-hairpin with alternating hydrophilic and hydrophobic amino acids (2,15,33). A ␤-hairpin is often used by ␤-PFTs for membrane insertion and pore formation (23,34).…”

“…However, the primary effect responsible for CPEinduced intestinal fluid and electrolyte secretion is considered to be enterocyte damage resulting from pore formation (32). Two independent groups recently used X-ray crystallography approaches to solve the structure of CPE (2,15). Those analyses indicated that the CPE protein consists of two distinct domains, including a C-terminal domain and an N-terminal domain comprised of two halves.…”

“…Site-directed mutagenesis studies (16,30) identified CPE residues D48 and I51 of the N-terminal cytotoxicity domain as being particularly critical for CH-1 prepore formation. Second, the CPE region comprising amino acids 81 to 106 in the cytotoxicity domain, named TM1, contains the ␤4 strand and the only ␣-helix present in the N-terminal cytotoxicity domain (2,15). This CPE region possesses two stretches of alternating hydrophobic and hydrophilic residues, which is characteristic of a ␤-hairpin motif (33).…”

Toxin Plasmids of Clostridium perfringens

“…The CPE structure was recently solved by X-ray crystallography, which assigned this toxin to the aerolysin family of small pore-forming toxins (54,55). Furthermore, those structural analyses, coupled with mutagenesis studies (56)(57)(58)(59)(60)(61), indicated that CPE contains a C-terminal domain that binds to claudin receptors on host cells and an N-terminal domain, consisting of two halves, that is critical for pore formation by mediating oligomerization and membrane insertion.…”

Clostridium perfringens Type A Enterotoxin Damages the Rabbit Colon

“…This enterotoxin, which is an ϳ35-kDa single polypeptide with a unique amino acid sequence (5), belongs structurally to the aerolysin family of pore-forming toxins (6,7). CPE action begins with its binding to receptors, which include certain members of the claudin protein family (8)(9)(10).…”