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Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin
M Nagahama1, Y Okagawa, T Nakayama
1Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Japan.
Abstract:
Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.
Insights
Mutations in Clostridium perfringens alpha-toxin at H-68 or H-148 eliminated its hemolytic and lethal activities. Specific residues are crucial for toxin function and metal binding.
Area of Science:
- Microbiology
- Protein biochemistry
- Toxicology
Background:
- Clostridium perfringens alpha-toxin is a key virulence factor responsible for various pathological effects.
- Understanding the structure-function relationship of alpha-toxin is crucial for developing targeted therapeutics.
Purpose of the Study:
- To investigate the role of specific histidine residues (H-68, -148, -126, -136, -46, -207, -212, -241, and -11) in the biological activities of Clostridium perfringens alpha-toxin.
- To elucidate the involvement of metal ions, particularly zinc, in the toxin's function and membrane binding.
Main Methods:
- Site-directed mutagenesis was employed to generate variant alpha-toxins with specific histidine residue alterations.
- Hemolytic activity, phospholipase C, sphingomyelinase, and lethal activities were assessed for wild-type and mutant toxins.
- Toxin binding to erythrocytes and membranes was analyzed in the presence of various metal ions (Ca2+, Co2+, Mn2+).
- Metal content analysis (including [65Zn]2+ binding) was performed on wild-type and mutant toxins.
Main Results:
- Mutations at H-68 and H-148 completely abolished hemolytic, phospholipase C, sphingomyelinase, and lethal activities.
- Mutations at H-126 and H-136 reduced these activities by approximately 100-fold.
- H-68, -126, and -136 residues are involved in metal-dependent membrane binding, with Co2+ and Mn2+ restoring activity.
- H-148 tightly binds one zinc atom essential for the active site, while H-68, -126, and -136 bind labile metals crucial for membrane interaction.
Conclusions:
- Histidine residues H-68, -126, -136, and -148 play critical roles in the enzymatic and hemolytic activities of Clostridium perfringens alpha-toxin.
- The toxin utilizes distinct metal-binding sites: H-148 for the active site zinc and H-68, -126, -136 for labile metals facilitating membrane binding.