Amino acid residues involved in membrane insertion and pore formation of Clostridium botulinum C2 toxin.
The actin-ADP-ribosylating Clostridium botulinum C2 toxin consists of the enzymatic component C2I and the binding component C2II. C2II forms heptameric channels involved in translocation of the enzymatic component into the target cell. On the basis of the heptameric toxin channel, we studied functional consequences of mutagenesis of amino acid residues probably lining the lumen of the toxin channel. Substitution of glutamate-399 of C2II with alanine blocked channel formation and cytotoxicity of the holotoxin. Although cytotoxicity and rounding up of cells by C2I were completely blocked by exchange of phenylalanine-428 with alanine, the mutation increased potassium conductance caused by C2II in artificial membranes by about 2-3-fold over that of wild-type toxin. In contrast to its effects on single-channel potassium conductance in artificial membranes, the F428A mutation delayed the kinetics of pore formation in lipid vesicles and inhibited the activity of C2II in promoting (86)Rb (+) release from preloaded intact cells after pH shift of the medium. Moreover, F428A C2II exhibited delayed and diminished formation of C2II aggregates at low pH, indicating major changes of the biophysical properties of the toxin. The data indicate that phenylalanine-428 of C2II plays a major role in conformational changes occurring during pore formation of the binding component of C2II.
Lang
Alexander E
AE
Institut für Experimentelle and Klinische Pharmakologie and Toxikologie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany.
Neumeyer
Tobias
T
Sun
Jianjun
J
Collier
R John
RJ
Benz
Roland
R
Aktories
Klaus
K
D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism*; D018938:Caco-2 Cells; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D002478:Cells, Cultured; D003014:Clostridium botulinum / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000737:chemistry* / Q000378:metabolism*
D018272:Porins; D001905:Botulinum Toxins; C096324:botulinum toxin type C