Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin

Alpha-toxin is the key determinant in gas-gangrene. The toxin, a phospholip ase C, cleaves phosphatidylcholine in eukaryotic cell membranes. Calcium io ns have been shown to be required for the specific binding of toxin to memb ranes prior to phospholipid cleavage. Reported X-ray crystallographic struc tures of the toxin show that the C-terminal domain has a fold that is analo gous to the eukaryotic calcium and membrane-binding C, domains. We report t he binding sites for three calcium ions that have been identified, by cryst allographic methods, in the C-terminal domain of the protein close to the p ostulated membrane-binding surface. The position of these ions at the tip o f the domain, and their function (to facilitate membrane binding) is simila r to that of calcium ions observed bound to C, domains. Using the optical s pectroscopic techniques of circular dichroism (CD) and fluorescence spectro scopy, pronounced changes to both near and far-UV CD and tryptophan emissio n fluorescence upon addition of calcium to the C-terminal domain of a-toxin have been observed. The changes in near-UV CD, fluorescence enhancement an d a 2 nm blue-shift in the fluorescence emission spectrum are consistent wi th tryptophan residue(s) becoming more immobilised in a hydrophobic environ ment. Calcium binding appears to be low-affinity: K-d approximate to 175-25 0 mu M at pH 8 assuming a 1:1 stoichiometry. as measured by spectroscopic m ethods. (C) 1999 Academic Press.