Ce. Naylor et al., Characterisation of the calcium-binding C-terminal domain of Clostridium perfringens alpha-toxin, J MOL BIOL, 294(3), 1999, pp. 757-770
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.